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Regulation of macrophage apoptosis via Bcl-2 family members and ceramide Wang, Shih Wei 2007

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R E G U L A T I O N OF M A C R O P H A G E APOPTOSIS VIA BCL-2 F A M I L Y M E M B E R S AND C E R A M I D E by SHIH WEI WANG B.Sc, Simon Fraser University, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Experimental Medicine THE UNIVERSITY OF BRITISH COLUMBIA August 2007 © Shih Wei Wang, 2007 Abstract Apoptosis is an important mechanism involved in regulating the number of macrophages present at sites of inflammation. Several lines of evidence indicate that blocking macrophage apoptosis can increase atherosclerosis. We previously reported that oxidized LDL (oxLDL) can inhibit apoptosis in cultured bone marrow-derived macrophages in part by activating the phosphoinositide 3 kinase (PI3K)/protein kinase B (PKB) pathway and subsequent expression of pro-survival protein Bcl-X L. Here we report that oxLDL also alters the levels of the pro-apoptotic protein, Bax. This effect of oxLDL on Bax regulation was at a post-transcriptional level, mediated by accelerated degradation via the ubiquitin-proteasome pathway. However, Bax knockout macrophages were not resistant to apoptosis following cytokine withdrawal, suggesting that the downregulation of Bax is only partially responsible for the pro-survival effects mediated by oxLDL in these cells. OxLDL is also able to increase the expression of the prosurvival relative, Mcl-1. The effect of oxLDL on Bax degradation and Mcl-1 expression was blocked by inhibitors of the PI3K/PKB pathway. To investigate the upstream receptor(s) activated by oxLDL to mediate macrophage survival, we used pertussis toxin (PTX) to test whether Gj protein coupled receptors are involved. Unexpectedly, we found that PTX by itself selectively blocks macrophage apoptosis in a dose-dependent manner. PTX acts in part by inhibiting acid sphingomyelinase activity which in turn prevents generation of ceramide during apoptosis. A Gi activator peptide, mastoparan, increased ceramide levels in macrophage and induced apoptosis, but pre-treatment with PTX partially overrode mastoparan-induced apoptosis. PTX failed to prevent ASMase activation or apoptosis in ii macrophages lacking toll-like receptor 4 (TLR4). Like oxLDL, the anti-apoptotic effect of PTX also activated the PI3K/PKB pathway which led to nuclear localization of the transcription factor N F K B and up-regulation of Bcl-XL. These results indicate that G ; proteins, TLR4, ASMase and the PI3K/PKB pathway are crucial components for regulation of macrophage apoptosis. We also looked at regulation of ceramide generation in response to apoptosis. Using ASMase-/- mice, we found that ceramide is still generated. Using inhibitors to enzymes involved in the de novo ceramide synthesis pathway, we concluded that both de novo synthesis and sphingomyelin hydrolysis can contribute to ceramide generation during macrophage apoptosis. iii Table of Contents Abstract ii Table of Contents iv List of Tables viii List of Figures ix List of Abbreviation xii Acknowledgements xiv 1 Introduction 1 1.1 Atherosclerosis 1 1.1.1 The role of macrophages in the pathogenesis of atherosclerosis 1 1.1.2 Effect of oxLDL on pathogenesis of atherosclerosis 2 1.1.3 Effect of oxLDL on macrophage recruitment 3 1.1.4 Biological effect of oxLDL on macrophage proliferation 3 1.1.5 Biological effect of oxLDL on macrophage apoptosis 5 1.1.6 Biological effect of oxLDL on macrophage survival 6 1.1.7 Intracellular mechanisms employed in oxLDL-mediated macrophage survival 8 1.1.8 Controversy regarding oxLDL's effect in causing survival and apoptosis 10 1.2 Apoptosis 12 1.2.1 Cel! Death overview 12 1.2.2 Caspases 13 1.2.3 Bcl-2 family members 15 1.2.4 Mitochondrial fission 19 1.2.5 Therapies targeting Bcl-1 family members 20 1.3 Ceramide and apoptosis 22 1.3.1 Overview of ceramide 22 iv 1.3.2 Regulation of ceramide metabolism in relation to apoptosis 23 1.3.3 Ceramide as second messenger to regulate apoptosis 28 1.3.4 Ceramide as a modulator of membrane structure to regulate apoptosis 28 1.3.5 Therapeutic implications 30 1.4 Objectives 31 2 Materials and methods 32 2.1 Materials 32 2.2 Lipoprotein isolation, oxidation and acetylation 33 2.3 Cell culture '. 33 2.4 Genotyping 34 2.5 Cell Viability assay 35 2.6 Immunofluorescence microscopy 35 2.7 Flow cytometric analysis 36 2.8 Reverse transcription and Real time PCR 37 2.9 Immunoblotting and immunoprecipitation 37 2.10 Lipid labelling for ceramide and sphingomyelin level determination 38 2.11 Sphinomyelinase assay 39 2.12 Nuclear preparations and Electrophoretic Mobility Shift Assay 39 2.13 Diacylglycerol Kinase assay for ceramide mass 40 2.14 Microsome preparation for in vitro serine palmitoyltransferase and ceramide synthase assays 41 2.15 Serine palmitoyltransferase assay 41 2.16 Ceramide synthase assay 41 2.17 Statistical analysis 42 3 OxLDL promotes macrophage survival by facilitating Bax degradation and increasing Mcl-1 expression 43 3.1 Introduction 43 3.2 Results 45 v 3.2.1 Bax is involved in macrophage apoptosis following M-CSF withdrawal 45 3.2.2 OxLDL reduces Bax protein levels in Macrophages 46 3.2.3 OxLDL regulates Bax by accelerating its degradation 47 3.2.4 Down-regulation of Bax by oxLDL is proteasome mediated 47 3.2.5 The PI3K/PKB pathway is involved in accelerated Bax degradation 48 3.2.6 Bax is only partially responsible for apoptosis induced by M-CSF withdrawal 49 3.2.7 Sequesteration of Bax by Mcl-1 is disrupted by cytokine withdrawal while oxLDL enhances this association 49 3.2.8 OxLDL acts through the PI3K/PKB pathway to increase the level of Mcl-1 50 3.2.9 Bim does not displace Mcl-1 from Bax 50 3.3 Discussion 51 4 Pertussis toxin inhibits macrophage apoptosis via the PI3K/PKB pathway... 72 4.1 Introduction 72 4.2 Results 73 4.2.1 Pertussis toxin can selectively protect macrophages from apoptosis induced by cytokine withdrawal 73 4.2.2 Pertussis toxin inhibits ceramide generation in part by blocking acid sphingomyelinase activation after growth factor withdrawal 74 4.2.3 Mastoparan, a Gi agonist, induces cell death very rapidly 75 4.2.4 Mastoparan activates ASMase in B M D M 76 4.2.5 Pertussis toxin attenuates cell death induced by mastoparan 76 4.2.6 ADP-ribosylation is required for pertussis toxin to promote cell survival 76 4.2.7 Toll-like receptor 4 may be involved in the anti-apoptotic effect of PTX in macrophages 77 4.2.8 Adenylyl cyclase is unlikely to contribute to macrophage apoptosis 78 vi 4.2.9 The anti-apoptotic effect of PTX requires the activation of the PI3K/PKB pathway 78 4.2.10 Activation of N F K B is required for PTX to provide survival by regulating Bcl-X L expression 79 4.3 Discussion 80 5 Regulation of ceramide generation during macrophage apoptosis < 101 5.1 Introduction 101 5.2 Results 102 5.2.1 ASMase is only partly responsible for ceramide generated in response to M-CSF withdrawal in B M D M 102 5.2.2 Ceramide generation in ASMase-/- cells is unlikely to arise from degradation of sphingomyelin 103 5.2.3 Accumulation of ceramide mass from de novo synthesis upon M-CSF withdrawal 103 5.2.4 De novo production of ceramide is not dependent on serine palmitoyltransferase (SPT) but ceramide synthase (CS) activities 104 5.2.5 Ceramide-1-phosphate inhibits ceramide generation despite the absence of ASMase 104 5.3 Discussion 105 6 Summary..... 116 7 Bibliography 119 vii List of Tables 1 Introduction Table 1: Enzymes of ceramide metabolism and key features viii List of Figures 1 Introduction Figure 1.1 The intrinsic apoptotic pathway 14 Figure 1.2 Models for how BH3-only proteins activate Bax and Bak 19 Figure 1.3 Ceramide metabolism 23 3 OxLDL promotes macrophage survival by facilitating Bax degradation and increasing Mcl-1 expression Figure 3.1 Bax undergoes conformational changes upon cytokine withdrawal 57 Figure 3.2 Expression of Bax is down-regulated by treatment with oxidized LDL in macrophages 58 Figure 3.3 Only extensively oxidized LDL promotes a decrease in Bax protein 59 Figure 3.4 OxLDL also reduces Bax levels in human macrophages 60 Figure 3.5 OxLDL facilitates Bax protein turn-over 61 Figure 3.6 OxLDL induces Bax degradation via the proteasomal pathway 62 Figure 3.7 OxLDL signals at least partially through a PI3K dependent pathway leading to Bax degradation 63 Figure 3.8 Bax knockout does not confer resistance to cytokine withdrawal induced cell death 64 Figure 3.9 Mcl-1 but not Bcl-2 sequesters Bax and Bak 65 Figure 3.10 Mcl-1 level does not decrease in response to cytokine withdrawal but increases with oxLDL treatment 66 Figure 3.11 Mcl-1 expression is preserved by treatment of extensively oxidized LDL 67 Figure 3.12 Bim is phosphorylated in the presence of M-CSF but not oxLDL 68 Figure 3.13 Interaction of Bim with Mcl-1 or Bax does not change during apoptosis. 69 Figure 3.14 Mcl-1 association with Bim does not change during apoptosis 70 Figure 3.15 Proposed model of OxLDL regulation of Bcl-2 family members to mediate macrophage survival 71 ix 4 Pertussis toxin inhibits macrophage apoptosis via the PI3K/PKB pathway Figure 4.1 Pertussis toxin selectively protects macrophages apoptosis 84 Figure 4.2 Pertussis toxin can inhibit ASMase activation and exogenous ceramide blocks the anti-apoptotic effect of pertussis toxin 85 Figure 4.3 Mastoparan induces apoptosis in macrophages 87 Figure 4.4 Mastoparan activates ASMase and increases ceramide levels in macrophages 88 Figure 4.5 Pertussis toxin confers partial resistance to mastoparan-induced cell death 90 Figure 4.6 Enzymatic activity of PTX is required for inhibition of apoptosis 92 Figure 4.7 Pertussis toxin may signal through the TLR4 receptor to block apoptosis 95 Figure 4.8 PKB is the major pathway required for the anti-apoptotic effect of PTX 97 Figure 4.9 Pertussis toxin signals through N F K B to mediate macrophage survival 99 Figure 4.10 A working model of PTX induced macrophage survival 100 5 Regulation of ceramide generation during macrophage apoptosis Figure 5.1 ASM deficiency confers partial resistance to cytokine withdrawal-induced apoptosis and ceramide increase 108 Figure 5.2 A S M deficiency confers partial resistance to cytokine withdrawal induced DNA fragmentation and caspase 9 activation 109 Figure 5.3 Ceramide generated in ASMase -/- B M D M is not due to SM hydrolysis.... 110 Figure 5.4 Time course for the change in ceramide mass after M-CSF withdrawal I l l Figure 5.5 Inhibitors of the de novo ceramide synthesis pathway are able to block ceramide production in ASMase-/- B M D M 112 Figure 5.6 SPT is unlikely to be the enzyme responsible for the de novo synthesized ceramide during macrophage apoptosis 113 Figure 5.7 C1P can inhibit the ceramide generation and promote cell survival independent of ASMase 114 x Figure 5.8 A working model of ceramide generation pathways in response to cytokine withdrawal 11 xi List of Abbreviation -/- knockout +/+ wildtype Ab antibody AcLDL acetylated low density lipoproteins AIM apoptosis inhibitor expressed by macrophages ANT adenine-nucleotide translocator ASMase acid sphingomyelinase BMDM bone marrow derived macrophages BSA bovine serum albumin C1P ceramide-1 -phosphate CAMP cyclic AMP CARD caspase recruitment domain Cdase ceiamidase CK cermamide kinase CS ceramide synthase CTRL control CVD cardiovascular disease DAG diacylglycerol EMSA electrophoretic mobility shift assay ER endoplasmic reticulum ERK extracellular signal-regulated protein kinase FBS fetal bovine serum GCS glucosylceramide synthase GM-CSF granulocyte-macrophage colony stimulating factor IB immunoblot IP immunoprecipitation KSR kinase suppressor of Ras LDL low density lipoproteins LPC lysophosphatidylcoline LPS lipopolysaccharide MCP-1 monocyte chemoattractant protein-1 M-CSF macrophage-colony stimulating factor MEF murine embryonic fibroblast MMP matrix metalloproteinases N F K B nuclear factor kappa B NPD Niemann-Pick disease NSMase neutral sphingomyelinase OxLDL oxidized low density lipoproteins xii PA phosphatidic acid PARP poly (ADP-ribose) polymerase PI propidium iodide PI3K phosphatidylinositol 3 kinase PKB protein kinase B PLD phospholipase D PM plasma membrane PP1 serine/threonine protein phosphatases 1 PP2A serine/threonine protein phosphatases 2A PT peimeabilitity transition pores PTX pertussis toxin PUFA polyunsaturated fatty acid SIP sphingosine-1 -phosphate SK sphingosine kinase SM sphingomyeline SMS sphingomyeline synthase SPT serine palmitoyltransferase tBid truncated Bid TLR4 toll-like receptor 4 VCAM-1 vascular cell adhesion molecule-1 VDAC voltage-depdendent anion channel xiii Acknowledgements At the end of this long journey in completing the Ph.D. degree, I would like to express my gratitude to those who have supported me along the way. First and foremost, I would like to offer my enduring gratitude to my supervisors, Drs. Vincent Duronio and Urs Steinbrecher, who have supported me throughout the program with their patience and knowledge, whilst allowing me the freedom to work in my own way. Their insightful questioning has challenged me to think more critically. I would like to express my deepest appreciation to Dr. Steinbrecher, who read my numerous thesis revisions and helped make sense where there was confusion. I also thank Dr. Antonio Gomez-Munoz, whose collaboration has made my research more interesting and motivating. I would also like to thank my other supervisory committee members, Drs Isabella Tai and Gerald Krystal, for their helpful discussions and guidance. In my daily work I have been blessed with a friendly and cheerful group of colleagues, Ivan Waissbluth, Joseph Anthony, John Chen, Maziar Riazy and Payman Hojabrpour. Without their support and humour, the completion of this work would not have been possible. I especially would like to thank Dr. Sarwat Jamil, whose work ethic, scientific spirit and kind-heartedness have been a great support and inspiration over the years and will continue to be an inspiration for years to come. Special thanks are owed to my parents, Wen Yue Wang and Mei Chin Lin, and my brother, Chun Chih Wang. Their unconditional love and support have buoyed me throughout years of education and the inevitable periods of uncertainty. Lastly, I would like to make special mention of my fiance, Thomas Zeng, as he has been incredibly supportive of me, particularly through the personal challenges of completing this degree. xiv 1 Introduction 1.1 Atherosclerosis 1.1.1 The role of macrophages in the pathogenesis of atherosclerosis Atherosclerosis remains the leading cause of death in Western societies. Early atherosclerotic lesions are characterized by intimal thickening and by expansion of the intima of arteries by lipid-laden foam cells '. Advanced lesions have a lipid core covered by a cap of fibrous tissue 2 . The rupture of advanced lesions can lead to thrombus formation that occludes the vessel lumen and results in acute myocardial infarction or stroke 3 . It is now recognized that increased cholesterol and inflammation work together to contribute to the pathogenesis of atherosclerosis 4 - 6 . Macrophages are believed to play a central role in all stages of atherosclerosis because of their role in cholesterol accumulation and because they are an essential constituent of innate immunity and inflammation 1 . The key in vivo evidence implicating macrophages in atherogenesis came from animal studies where plaque formation in the atherosclerosis prone apoE deficient mouse was almost abolished in the absence of macrophages 8 . In the initial stage, the up-regulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) on lipid activated endothelium9, is thought to promote adhesion of blood monocytes, and facilitate their movement into the intima10" n . Secretion of chemokines like monocyte chemoattractant protein-1 (MCP-1), can also promote recruitment of monocytes into intima. Deficiency of MCP-1 or its receptor, CCR2 1 4 decreases macrophage numbers in the intima and also decreases lesion size. Conversely, over-expression of MCP-1 accelerates atherosclerosis development 1 5 . Bursill et al. have recently shown that inactivation of almost all CC-class chemokines 1 strongly inhibited atherosclerosis in ApoE-/- mice 1 6 . After migrating into the intima, monocytes differentiate into macrophages, which have high levels of expression of scavenger receptors. These receptors have many functions including the recognition and internalization of pathogens and apoptotic cells. However, they also recognize modified lipoproteins and contribute to the massive accumulation of cholesterol in macrophage foam cells 1 7 ' 1 8 . Activated macrophages may intensify and perpetuate the inflammatory process by the secretion of cytokines and chemokines. In addition to its role in initial stages of atherosclerosis, the macrophage is a contributing factor to complications associated with later stages of the disease ' ' . Macrophage foam cells are mechanically much weaker than fibrous regions of plaque, and they secrete matrix metalloproteinases (MMP) which digest the stabilizing connective-tissue elements of plaque 1 1 . Both of these effects make plaques more vulnerable to rupture. Not surprisingly, ruptured plaques commonly contain high numbers of macrophages in the shoulder region 2 3 . Macrophages also secrete pro-thrombotic tissue factor, which accelerates thrombus formation following rupture or erosion 2 4 . A study in cholesterol-fed rabbits showed that 6 months of feeding a cholesterol free diet resulted in disappearance of macrophages from plaque, and stabilization of lesions by connective tissue25. 1.1.2 Effect of oxLDL on pathogenesis of atherosclerosis In humans, most of the cholesterol in plasma is carried by low density lipoproteins (LDL). Increased LDL is found to correlate with increased risk of cardiovascular disease (CVD) 2 6 . However, it is thought that LDL must be somehow modified before it can induce foam-cell formation because the uptake of native LDL by 2 the LDL receptor is subject to feedback regulation by cell cholesterol levels 1 1 . Brown and Goldstein's group showed that chemical modifications of LDL that removed the positive charge of lysine amino groups generated ligands for scavenger receptors, and such modified LDLs could cause cultured macrophages to transform into foam cells n . Subsequent studies showed that oxidation of LDL also modified lysine amino groups, and caused rapid uptake of oxidized LDL (oxLDL) by scavenger receptors 2 9 . While LDL is well protected from oxidation in the plasma compartment, it is thought to be susceptible to enzymatic and non-enzymatic modifications in the arterial intima where antioxidant defenses are less prevalent, and oxidant stress from macrophages, endothelial cells, and smooth muscle cells is quite intense. . In addition to its interaction with scavenger receptors, oxLDL has been found to exhibit a number of other pro-atherogenic properties through its effects on macrophage recruitment, proliferation, apoptosis and survival. 1.1.3 Effect of oxLDL on macrophage recruitment Recruitment of monocytes and their differentiation into macrophages are important determinants of macrophage numbers in plaque 3 1 and oxidized LDL (oxLDL) is believed to play a role in these processes 1 2 . It increases the expression of adhesion molecules and monocytes binding to endothelial cells 3 2 ' 3 3 , induces the expression of MCP-1 on endothelial cells, and can act as a direct chemoattractant34'35. 1.1.4 Biological effect of oxLDL on macrophage proliferation Another factor that controls the number of macrophages in plaques is the balance between macrophage proliferation, and macrophage death. Although cytotoxicity was 3 one of the first biological activities of oxLDL to be observed 3 6 , more recent studies have shown that at least under some conditions, oxLDL can stimulate macrophage growth and inhibit apoptosis37"40. Traditionally viewed as terminally differentiated, macrophages have now been shown to be the predominant cell type in plaque that expresses proliferation markers 4 1 ' 4 2 . Evidence showing that macrophage proliferation is closely associated with progression of atherosclerosis comes from deletion of the retinoblastoma (Rb) tumor suppressor gene from macrophages in a murine model of atherosclerosis where mice without Rb showed increased macrophage proliferation and atherosclerotic lesion 4 3 . It has also been reported that in addition to their cholesterol-lowering effect, statins significantly inhibit macrophage proliferation 4 4 . Yui et al. first reported that oxLDL has the ability to promote macrophage proliferation 4 5 . They reported that internalization of lysophosphatidylcholine (lysoPC), a major phospholipid component in oxLDL, via macrophage scavenger receptors was essential for this growth response 4 6 . Internalization of lysoPC in macrophages caused activation of protein kinase C and autocrine release of GM-CSF, which this group believed was responsible for the macrophage proliferation40'47. It was later suggested by Hamilton et al. that oxLDL "primes" macrophage proliferation in response to other ^7 factors, such as M-CSF or GM-CSF . However, results from our laboratory showed that oxLDL stimulates macrophage growth without the involvement of lysoPC or GM-CSF 3 9 ' 4 8 . Instead, we demonstrated that oxLDL induces activation of phosphatidylinositol 3 kinase (PI3K) and that this activation plays a role in oxLDL-mediated macrophage proliferation 3 9 . 4 1.1.5 Biological effect of oxLDL on macrophage apoptosis Several reports in mouse models have shown an inverse correlation between macrophage apoptosis and rate of progression of early atherosclerotic lesions (reviewed by Tabas et al 4 9). Apoptosis and necrosis of macrophages are observed in advanced stages of atherosclerosis, but high cellularity and proliferation are evident in early atherosclerotic lesions 5 0 " 5 2 . Whether macrophage death is advantageous or deleterious in atherosclerosis is not fully understood at present. Apoptosis of macrophages can be beneficial as removal of inflammatory cells from the plaque could attenuate the inflammatory response and decrease the synthesis of MMP. Loss of macrophages, however, also decreases the uptake of apoptotic bodies so that secondary necrosis occurs which contributes to the formation of an acellular lipid core, the hallmark of an advanced atherosclerotic lesion5 3. In addition to its pro-inflammatory effects, oxLDL has also been implicated in the induction of apoptosis in macrophages 5 4 . It has been shown that the mitochondrial pathway plays an important role in macrophage apoptosis induced by oxysterols, which are a major component of oxLDL 5 5 ' 5 6 . The balance between apoptotic and anti-apoptotic Bcl-2 family members is a major determinant of mitochondrial membrane integrity and a strong body of evidence implicates these proteins in apoptosis induced by oxLDL and oxysterols in macrophages. Harada et al. suggested a role for Bcl-2 in protection against oxysterol-induced apoptosis in macrophages 5 S . More recently, Rusinol et al. have shown that increased degradation of protein kinase B (PKB) in response to oxysterols leads to increased activity of the pro-apoptotic Bcl-2 family members Bim and Bad, and down-regulation of BC1-XL followed by Bax mediated release of mitochondrial cytochrome C 5 7 . Berthier et al. have suggested 5 dephosphorylation of Bad by calcineurin and Bim displacement with subsequent association with Bcl-2 as additional mechanisms 5 8 ' 5 9 . There is also evidence pointing towards lysosomes as targets of oxLDL and oxysterols in induction of macrophage apoptosis. Yuan et al. reported compromise of macrophage lysosomal membrane integrity which accompanied apoptotic transformation 6 0 . The same group later showed that endocytosed oxLDL can destabilize the acidic vacuolar compartment but also cause the upregulation and translocation of lysosomal cathepsins 6 1 ' 6 2 . The role of lysosomes in oxysterol-mediated apoptosis was further emphasized by the finding that lysosomal dysfunction precedes apoptosis and that macrophage apoptosis was reduced by inhibitors of cathepsins B and L 6 3 . Although there is evidence that lysosome/cathepsin-triggered apoptosis merges with caspase activation and the mitochondrial apoptosis pathway 6 4 ' 6 5 , the specific interactions of the two above-mentioned apoptotic arms (lysosomal and mitochondrial) have not been studied. For more detailed review on the mechanism employed by oxLDL to induce apoptosis in atherosclerosis, please refer to Martinet and Kockx 6 6 . 1.1.6 Biological effect of oxLDL on macrophage survival ApoE-/- mice lacking the pro-apoptotic protein p53 had less apoptosis of macrophages, and an increase in the size of early atherosclerotic lesions 6 1 . Paradoxically, knockout of the p53 target, p21, a cyclin-dependent protein kinase inhibitor that regulates entry into the cell cycle and inhibits apoptosis, increased macrophage apoptosis and protected mice against atherosclerosis 6 8 . This suggests that not all of the effects of p53 are due to p21. The importance of macrophage apoptosis in regulating the early development of atherosclerosis is further illustrated by a recent study demonstrating that 6 reduction of apoptosis due to Bax gene inactivation resulted in the larger atherosclerotic lesion area in LDLR-/- mice 6 9 , It was reported that oxLDL can induce expression of a macrophage survival protein, AIM (apoptosis inhibitor expressed by macrophages) which is abundant in lesions 7 0 . Furthermore, the absence of AIM dramatically reduced early atherosclerotic lesions in LDLR-/- mice 7 0 . These experiments indicate that the survival of macrophages, particularly during the early stages of atherosclerosis, may play a key role in determining whether lesions form and how quickly they progress. Hamilton and his colleagues reported a pro-survival effect of oxLDL on bone marrow derived macrophages (BMDM) 3 7 . This effect of oxLDL was not reduced in mice with both M-CSF and GM-CSF genes inactivated, thus suggesting a direct role of oxLDL rather than an indirect process mediated through these cytokines. This group also showed that doses of oxLDL < 50 u,g/ml generally promoted survival in murine and human macrophages, whereas at higher concentrations, cell numbers declined 7 1 . We confirmed that oxLDL blocks apoptosis in B M D M and found that native LDL or acetylated LDL had no effect 3 8 ' 1 1 . In those papers, we also showed that high concentrations of oxLDL were toxic, and that soluble factors in the medium (such as GM-CSF or M-CSF) were not necessary for the anti-apoptotic effect of oxLDL. 7 1.1.7 Intracellular mechanisms employed in oxLDL-mediated macrophage survival A range of signaling pathways have been implicated in the ability of oxLDL to activate vascular smooth muscle cells , endothelial cells ' and macrophages . In this section we will focus on intracellular macrophage-specific events in particular on pro-survival events in relation to disease pathogenesis. The serine/threonine protein kinase B (PKB) is activated downstream of phosphatidylinositol 3 kinase (PI3K) and this pathway presents a central point for transducing signals from oxLDL to components of the apoptotic machinery, such as IKB, and the Bcl-2 family members . Extracellular signal-regulated protein kinase (ERK)l/2 is another candidate mediator for oxLDL-induced macrophage survival as it modulates cellular processes such as proliferation, differentiation, development, stress response, and apoptosis 1 1 . Alterations of both the PKB and ERK pathway have been detected in a "7*7 "78 "7Q number of diseases ' . It is known that oxLDL activates ERK in macrophages and both ERK and PI3K have been implicated in the ability of oxLDL to promote macrophage survival 7 1 . We confirmed that oxLDL activates ERK1/2 in macrophages, but completely blocking this activation with ERK inhibitors had no effect on the pro-survival action of oxLDL. Only activation of PKB was essential for the inhibition of apoptosis in macrophages 1 1 . Minimally oxidized LDL is also reported to contribute to the survival of macrophage by activating the PI3K/PKB pathway 8 0 . As well, oxLDL immune complexes can also promote macrophage survival in a PKB-dependent manner 8 1 . Immunohistochemical analysis demonstrated PKB activation in murine atherosclerotic lesions, most of which was associated with macrophages 8 0 . 8 Nuclear factor kappaB (NFKB ) is another important signaling protein implicated in oxLDL mediated macrophage survival. Members of the NFtcB/Rel family of transcription factors regulate many genes involved in atherogenesis such as those mediating inflammatory, anti-apoptotic and proliferative responses of cells 8 2 . Physiological evidence supporting the involvement of N F K B in atherosclerosis was suggested by the demonstration of active N F K B in macrophages, smooth muscle cells, and endothelial cells in human atherosclerotic lesions, but not in healthy vessels 8 3 . Our laboratory and others showed that concentrations of oxLDL below 75 ug/ml enhance N F K B activation in macrophages and promote cell survival 7 2 ' 8 4 , but other groups have demonstrated inhibition of N F K B with high concentrations of oxLDL . Sphingolipids also play a part in the development of atherosclerotic lesions 8 6 . Ceramide is a key sphingolipid that is implicated as a facilitator of apoptosis and its generation by sphingomyelinase is thought to cause aggregation of LDL trapped in arterial intima . It has been found that secretory sphingomyelinase is more active at hydrolysing sphingomyelin in oxLDL compared to native LDL 8 8 . There are reports showing that oxLDL stimulates ' as well as inhibits generation of ceramide in macrophages. Our group found that treatment of macrophages with oxLDL inhibited ceramide generation and that this was due to inhibition of acid sphingomyelinase (ASMase) by oxLDL 7 2 . Furthermore, it was recently demonstrated that inhibition of biosynthesis of ceramide significantly decreased atherosclerotic lesion area 9 1 " 9 4 . As well, the ceramide metabolite sphingosine-1-phosphate (SIP) was shown to have mitogenic and anti-apoptotic functions 9 5 " 9 7 . Hammad et a l 9 8 recently reported that oxLDL immune complexes induced release of sphingosine kinase in U937 cells, which 9 increased the level of SIP in the medium and thereby enhanced macrophage survival. Another ceramide metabolite, ceramide-1-phosphate (C1P), was also found to be mitogenic and anti-apoptotic in macrophages " . Our laboratory showed that SIP as well as C1P, signal through similar pathways as oxLDL to promote macrophage survival 5 1' 1 0 0 , 101 1.1.8 Controversy regarding oxLDL's effect in causing survival and apoptosis Cytotoxicity was one of the first properties of oxLDL to be reported, and there are numerous articles reporting the pro-apoptotic effects of oxLDL. However, several groups have also reported mitogenic as well as pro-survival properties for oxLDL. The reasons for the discrepant results are not fully known. One key difference is the concentration of oxLDL used for in vitro studies. High concentrations of oxLDL (>100ug/ml) are toxic for most cell types. However; several groups including our own have noticed that lower concentrations of oxLDL (5-75ug/ml) promote growth or survival in macrophages 3 7 " 4 0 - 9 8 - 1 0 2 ' 1 0 3 . The method used for preparation of oxLDL also affects its biologic properties. The techniques used to oxidize LDL include metal ion oxidation, enzymatic modification, UV irradiation, incubation of L D L with cells, or addition of aldehydes or products of polyunsaturated fatty acid (PUFA) autoxidation. Although a high level of apoptosis was observed in cells exposed to "minimally modified" LDL made with metal ions 1 0 4 , incubation of LDL with cells over-expressing lipoxygenase produces a minimally modified LDL that actually promotes survival80. The degree of oxidation appears to affect the cytotoxicity of oxidized LDL, although this matter has not been fully characterized especially in relation to different 10 methods of oxidizing LDL 1 0 5 . Siow et a l 1 0 6 have reported that moderately oxidized LDL was more toxic to vascular smooth muscle cells than minimally or extensively oxidized LDL. This was attributed to a higher content of lipid hydroperoxides found in moderately modified LDL. Similar results were observed with macrophages 1 0 4 . Using UV irradiation, Yuan et al have shown that increasing exposure time will result in a more cytotoxic/apoptotic oxLDL and this corresponded with increasing concentrations of hydroperoxides 6 0 . It is possible that both the pro-survival and the cytotoxic effects of oxLDL may be involved in the development of atherosclerotic plaques. In early lesions where oxLDL may exist in low concentration, it could promote macrophage survival and thereby increase the inflammatory response. On the other hand, in advanced lesions, there may be higher concentrations of more heavily oxidized LDL, which then results in macrophage apoptosis and subsequent plaque destabilization. Clearly, an understanding of the mechanisms by which oxLDL regulates macrophage survival and apoptosis is important for understanding the pathogenesis of atherosclerosis. 11 1.2 Apoptosis 1.2.1 Cell Death overview Multi-cellular organisms require tight regulation of proliferation, differentiation and cell death to maintain proper development and tissue homeostasis 1 0 7 . In humans, dysregulation of these processes is involved in various diseases. For example, excessive cell death is thought to lead to neurodegenerative diseases while inhibition of apoptosis is implicated in many types of cancer or autoimmunity 1 0 8 ' 1 0 9 . There are two fundamentally distinct death pathways found in eukaryotes: necrosis and apoptosis. Necrosis occurs when the cells are killed by extreme trauma or injurious agents. Cells then swell and disrupt due to the inability of the plasma membrane to control ion flux. The uncontrolled release of cell contents to the surrounding causes local inflammation of the surrounding tissue "°. As a pathological endpoint, this type of cell death would be expected to have some obvious adverse consequences for an organism. In contrast, apoptosis is the type of cell death that occurs when cells commit suicide in a controlled fashion, for example during development. During apoptosis, dying cells are quickly engulfed by neighboring phagocytes, which prevents the release of intracellular contents and minimizes the inflammatory response 1 1 ' . Apoptosis is characterized by chromatin condensation, DNA fragmentation, cell shrinkage, plasma membrane blebbing and exposure of phosphatidylserine in the outer 1 1 7 leaflet of the plasma membrane , which is the signal for engulfment of apoptotic bodies by phagocytes " 3 . The apoptotic pathway was initially defined in C. elegans and D. melanogaster 1 1 4 ' 1 1 5 , but the molecular machinery of apoptosis is remarkably well conserved throughout evolution. The generation of transgenic and gene knockout mice 12 has facilitated our understanding of the apoptotic pathway in mammals, as reviewed by Ranger et al " 6 . 1.2.2 Caspases Caspases are key effector components of apoptosis 1 1 3 . They belong to a family of cysteine proteases that use cysteine as the nucleophilic group and typically cleave peptide bonds C-terminal to aspartic acid residues in the substrate 1 1 7 " 1 1 9 . The caspases normally exist as inactive zymogens. Activation of caspases requires the proteolytic cleavage of the regulatory pro-domain and assembly into a hetero-tetramer " 8 . While caspases 1 and 11 are involved in the processing of pro-inflammatory cytokines 1 2 0 , the remaining caspases can be divided into the initiator caspases that include caspase 8 and 9 and the effector caspases, such as caspase 3, 6, and 7, that are activated as a result of the proteolytic activity of the initiator caspases. The effector caspases are the executioners of apoptosis as their processing of substrates such as poly (ADP-ribose) polymerase (PARP) or DNase inhibitor proteins 1 2 1 can subsequently lead to morphological changes associated with apoptosis I 1 3 . Two major pathways can lead to caspase activation, the extrinsic and the intrinsic pathways 1 2 2 . The extrinsic apoptosis pathway is initiated at the cell surface through the binding of ligands such as FasL or TNFa to the corresponding death receptors to induce a conformational change 1 2 3 . This leads to interaction with adaptor proteins and is followed by the recruitment of pro-caspase 8. This undergoes auto-proteolytic activation and subsequent activation of effector caspases 1 2 4 ' 1 2 5 . Active caspase 8 can also amplify the apoptosis process by the cleavage of Bid, a Bcl-2 family member. Truncated Bid, tBid, is able to translocate to mitochondria and trigger the intrinsic apoptosis pathway 1 2 6 . 13 Therefore, both pathways can lead to a central control and an execution stage where activation of caspases cascade occurs ( Figure 1.1). The intrinsic pathway involves a permeability change in mitochondria, beginning with the release of apoptotic protease activating factors (Apafs) from mitochondria. Apaf-1 contains a caspase recruitment domain (CARD) that can interact with pro-caspase 9. By association with cytochrome c and ATP/dATP, Apaf-1 undergoes conformational changes that allow pro-caspase 9 to self-process and become active 1 2 7 . Together, Apaf-1, cytochrome c, ATP/dATP and caspase 9 form the apoptosome that then activates effector caspases . Recent reports indicate there are mechanisms other than the two pathways mentioned above for the activation of caspase 2 1 2 9 , 4 1 3 0 , and 1 2 1 3 U 3 2 . MAMMALS Figure 1.1 The intrinsic apoptotic pathway. Adapted from Danial et al 14 1.2.3 Bcl-2 family members Bcl-2 family members play a pivotal role in the intrinsic (mitochondrial) pathway of apoptosis. The "founding" protein, Bcl-2, was first discovered in human B cell follicular lymphoma cells carrying the t(14;18) chromosomal translocation 1 3 3 . Most lymphomas of this type have the breakpoint located in the Bcl-2 gene. Over-expression of this protein was later discovered to prolong cell survival by blocking apoptosis 1 3 4 . Bcl-2 members possess at least one of four conserved ot-helical regions known as Bcl-2 homology domains (BH1-4) 1 3 5 . Based on their function, the members can be divided into two groups, the pro-survival and pro-apoptotic members. The pro-survival members can inhibit apoptosis triggered by a wide variety of stimuli and they mostly contain all four BH domains. The pro-apoptotic members can be divided into "multi-domain" and "BH3 only" members. The multi-domain proteins resemble Bcl-2, and contain BH1-3 domains. In contrast, the other pro-apoptotic members contain only the BH3 domain that is essential for killing1 3 6"1 3 9. Structural analysis of BcI-X L revealed that BH1-3 domains assemble with a hydrophobic groove that can accommodate the BH3 domain of pro-apoptotic members 1 4 0 . Commitment to life or death often is determined by opposing members of the Bcl-2 family. Pro- and anti-apoptotic proteins can heterodimerize and compromise one another's action 1 4 1 ' 1 4 2 , and the balance of their corresponding concentration may be the key in determining whether cell death occurs. The anti-apoptotic members include proteins such as Bcl-2, BC1-XL , and Mcl-1. Bcl-2 protects against diverse cytotoxic insults that can trigger apoptosis such as starvation of growth factors, loss of cell attachment to extracellular matrix, Fas-stimulation and cytotoxic T-cell killing 1 4 3 , 1 4 4 . Over-expression of Bcl-2 protein, 15 conferring a survival advantage, is frequently found in human cancers such as B-cell lymphomas 1 4 5 and breast cancer 1 4 6 . Although Bcl-2 -/- mice develop normally, accelerated lymphocyte death in thymus and spleen, distorted small intestine and neuronal disease have been observed 1 4 7'. BH3-only members can engage pro-survival proteins by the interaction of BH3 domains. The pro-apoptotic activity of BH3-only members is kept in check by either transcriptional control or post-translational modification 1 4 8 . They serve as sensors for initiating the intrinsic apoptotic pathway in response to selective stimuli. For example, Bid is engaged through the activation of death receptors 1 2 6 , while Noxa and Puma respond to DNA damage 1 4 9 ' 1 5 ° . Bim and Bad can be activated by multiple stimuli including growth factor deprivation, detachment from the cell matrix 1 5 1 , chemotherapeutic agents or UV treatments 5 5 ' 1 5 2 . Studies using knockout mice of BH3-only proteins in certain types of cells confer resistance to selective apoptotic stimuli. For example, loss of Bim renders lymphocytes resistant to paclitaxel, ionomycin and cytokine deprivation induced apoptosis 1 5 3 while loss of Bad in mammary epithelial cells confer some resistance to withdrawal of epidermal growth factor 1 5 4 . Moreover, Noxa-deficient cells are partially resistant to DNA-damaged induced apoptosis 1 5 5 . Overall, the redundancy of BH3-only proteins creates a robust control system that integrates responses to different stimuli. After BH3-only proteins sense death stimuli, they need to activate Bax and Bak to initiate commitment to apoptosis 7 8 ' 1 5 6 . The first pro-apoptotic homolog, Bax, was first described as a protein that counteracted the pro-survival function of Bcl-2 1 4 1.Over-expression of Bax or the addition of purified recombinant Bax accelerates apoptosis141. 16 In healthy cells, Bax exists as a monomer in the cytosol or loosely associated with 157 membranes . Death stimuli cause a conformational change, allowing exposure of the hydrophobic grove that is otherwise hindered by the C-terminal helix of Bax 1 5 8 . This is immediately followed by the translocation and insertion of Bax proteins into the mitochondrial outer membrane as oligomers 1 5 9 . Inactive Bak exists as an integral membrane protein in mitochondria 1 6 0 . Bak is also induced to undergo conformational changes and oligomerization in response to apoptotic signals 1 6 1 . Bax-deficient mice display hyperplasia of thymocytes and B cells as well as abnormalities in the development of the male reproductive system162. Bak null mice show no developmental defects. However, when Bak- and Bax-deficient mice are intercrossed, a more marked phenotype is seen in the double-knockout163. Fewer than 10% of the animals survive into adulthood, and those that do display multiple developmental defects. Furthermore, the double knockout cells are resistant to multiple death stimuli 1 6 3 " 1 6 5 . It is evident from this that the combined pro-apoptotic functions of Bax and Bak are crucial for normal tissue development. Bax and Bak in concert are an essential gateway for activation of caspases in the 78 intrinsic apoptotic pathway , but there are debates as to how BH3-only proteins lead to the activation of Bax and Bak (Figure 1.2). The direct binding model suggests that the "activators" of BH3-only proteins such as Bid and Bim can directly activate Bax and Bak. Bad or Bik act as "sensitizers," that sequester the pro-survival proteins and allow unbound Bax and Bak to oligomerize 1 6 6 ' 1 6 1 . Inconsistent observations for binding of endogenous Bid and Bim to Bax or Bak lead to the suggestion that they operate at a "hit-and-run" fashion 1 5 9 ' 1 6 1 . The displacement model suggests that BH3-only proteins can 17 displace the binding of Bax or Bak to pro-survival proteins that sequester their active forms 1 6 8 . Chen et al 1 6 9 recently demonstrated the differential affinity of BH3-only proteins for pro-survival proteins. Using peptides mapped to BH3 sequence of BH3-only proteins, it was shown that certain molecules such as Puma and Bim can bind to all pro-survival proteins. Bad can counteract Bcl-2 and Bcl-XL, but not Mcl-1 while Noxa complements only by interacting with Mcl-1. By engineering Noxa to enhance promiscuous binding to all pro-survival proteins, effective killing occurred 1 6 9 . This was further supported by the observation of Willies et al 1 7 0 showing that both Noxa and Bad are required to neutralize Mcl-1 and Bel- X L respectively to drive efficient apoptosis mediated by Bak. A recent report showed that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma 1 7 1 while others showed mitochondrial permeabilization relies on BH3-only proteins engaging pro-survival Bcl-2 relatives and not Bak 1 1 2 . These results further support the notion that BH3-only proteins induce apoptosis at least primarily by engaging the multiple pro-survival relatives guarding Bax and Bak. 18 (a} Direct binding (b) Displacement v u « « l H L^IIHOtl H i v w l l tk*-*J>-Figure 1.2 Models for how BH3-only proteins activate Bax and Bak. (a) Direct binding model, (b) Displacement model. Adapted from Willis et al 1 6 8 . 1.2.4 Mitochondrial fission The precise mechanism leading to mitochondrial permeabilization is still under debate. One hypothesis is that opening of permeability transition (PT) pores at contact sites between the inner and outer mitochondrial membranes results in the rupture of mitochondria. Voltage-dependent anion channel (VDAC), the adenine-nucleotide translocator (ANT), and the matrix chaperone cyclophilin D were initially proposed to participate in the formation of PT pores 1 7 3 ' 1 7 4 and Bcl-2 family members are shown to regulate this process 1 7 5 . However, this model has been challenged by the recent observation that the ANT-deficient mitochondria can still form PT and cells can die by 19 apoptosis without ANT 1 7 6 . Similar results were also reported recently where the basic properties of the PT formation in mitochondria were not affected by the absence of VDAC 1 7 7 . An alternative model suggests that Bax can directly modulate V D A C to control mitochondria permeability 1 7 8 . Based on the structural similarity between BC1-XL and pore-forming bacterial toxins 1 4 0 , it is speculated that Bcl-2 family members themselves can form pores on the mitochondrial membrane that allows the leakage of cytochrome c. Bax, Bcl-2 and BC1-XL were shown to form a channel in liposomal membranes 6 5 ' 1 7 9 ' 1 8 1 . Regardless of the exact mechanism, it is clear that Bcl-2 family proteins play an essential role in controlling mitochondrial membrane integrity, and that mitochondrial permeabilization results in the release of cytochrome c, which leads to caspase-mediated cell destruction 1 5 8 . 1.2.5 Therapies targeting Bcl-1 family members Defects in apoptosis resulting from mutations in Bcl-2 members are often observed in tumors 1 8 2 ~ I 8 4 5 and this has prompted studies of anti-cancer drugs directly targeting pro-survival members. Given the functional importance of the BH3 domain in regulating apoptosis, two BH3 mimetics were designed. One, a 24-mer Bid BH3 peptide that is protease resistant and cell permeable, was shown to induce apoptosis in Jurkat 185 cells. It also suppressed the growth of a transplanted leukemia in vivo . The second is ABT-737, a molecule that tightly binds to the hydrophobic groove of a pro-survival protein. Behaving like Bad, it antagonizes the anti-apoptotic function of Bcl-2, Bcl-X L ; and Bcl-w but not Mcl-1. ABT-737 caused regression of certain tumors in mice 1 8 6 . Certo et al. showed that priming of Bcl-2 with Bim or Bid enhanced killing of ABT-737, but recent reports argued that the level of Mcl-1 determines the sensitivity of certain types of 20 tumors toward ABT-737 1 8 8 ' 1 8 9 . Nonetheless, ABT-737 has shown impressive single-agent toxicity in cell lines ex vivo and in vivo, and against primary human malignant cells 190 These results encourage therapeutic designs for treating non-malignant diseases that also have defective Bcl-2 family members. Another strategy could involve targeting transcriptional and/or translational control of BH3-only proteins l 4 8 . 21 1.3 Ceramide and apoptosis 1.3.1 Overview of ceramide Sphingolipids were originally considered to be just structural components of cell membranes, but some members of this family, especially ceramide, ceramide-1-phosphate, and sphingosine-1-phosphate (SIP), are now established as lipid second messengers that have important functions in cell proliferation, survival and apoptosis "' 191. 192 Sphingolipids are comprised of a long-chain sphingoid base, an amide-linked long-chain fatty acid and one of several head groups, which define the classes of sphingolipids. A hydroxyl head group gives rise to ceramide, phosphorylcholine to sphingomyelin (SM) and carbohydrates to glycosphingolipids I 9 2 . Ceramide can be generated by hydrolysis of sphingomyelin (SM), by de novo synthesis, or by recycling sphingoid bases or breakdown of complex glycosphingolipids 1 9 3 . The acyl portion of ceramide is typically saturated, although mono-unsaturated forms exist, particularly in very-long-chain fatty acid species. A schematic representation of pathways for ceramide generation is presented in Figure 1.3. 22 coo-H3N—^—H + . Serine Palmitoyl Transferase NH 2 "•CoA-C H * 0 H Palmitoyl-CoA Serine 3-Ketosphinganine OH Ketosphinganine Reductase Dihydroceramide Synthase 9 H Sphinganine Ceramide jp H> Syniliase s^yS 9H 4r Sphingosine Dihydroceramidi Desaturase (Dihydro)ceramide Sphingosine Kinase Sphingosine-1-phosphate Sphingomyelin Synthase ^ *~ ! Sphingomyelins Sphingomyelinase Ceramide Kinase Glucosyl Ceramide Synthase Glucosylceramides Ceramide-1 -phosphate 1-O-Acylceramides Figure 1.3 Ceramide metabolism. Adapted from Reynolds et al 192 1.3.2 Regulation of ceramide metabolism in relation to apoptosis Sphingomyelinase (SMase) hydrolyzes sphingomyelin to ceramide and phosphorylcholine 1 9 4'. There are three types of SMase and they are classified by the optimal pH for their activities. Alkaline SMase activity is found in intestinal mucosa and bile and may be involved in lipid digestion. As yet it has no defined role in apoptosis. Neutral and acid SMase have been found to respond to apoptotic stimuli 1 9 2 ' 1 9 5 . Acid SMase (ASMase) was originally considered an endosomal/lysosomal enzyme because of its pH optimum at 4.5-5.5 1 9 6 . However, secretory sphingomyelinase is a product of the same gene and has been shown to act on extracellular substrates such as modified LDL 8 8 ' 1 9 7'. The outer leaflet of plasma membranes has a third form of ASMase that was initially detected in cells stimulated with CD95 or CD40 1 9 8 ' 1 9 9 . Despite the neutral pH for the latter two locations, ASMase retains some activity, since an increase of the pH only changes the Km value of the enzyme 2 0 0 . All three types of ASMase require Z n 2 + for activity 1 9 7 . Ceramide accumulation in the lysosomal compartment can exert positive 23 feedback on ASMase activity, and this can lead to enhanced apoptosis in human macrophages and fibroblasts 2 0 1 . Neutral SMase (NSMase) has a pH optimum at 7.4 and requires Mg 2 + . To date, there are three mammalian isoforms identified and cloned 2 0 2 although NSMase 1 is thought to function more as a lyso-platelet activating factor-specific phospholipase C than as a bona fide NSMase 2 0 3 . NSMase can be activated in response to various apoptotic stimuli, including TNFa, Fas ligand, IL-1, IFNy and chemotherapeutic agents 204, 205 activity of NSMase is postulated to be positively regulated by cytosolic phospholipase A2 and negatively by glutathione levels in the cell 2 0 6 . Additionally, it has been observed that ceramide generation in macrophages results in the stimulation of NSMase activity 2 0 1 . The de novo pathway of ceramide synthesis has emerged as another key pathway of apoptosis regulation that is responsive to agonist stimulation. Two key enzymes in this pathway are serine palmitoyltransferase (SPT) and (dihydro)ceramide synthase (CS). SPT initiates the rate-limiting step in the pathway by condensing serine and palmitoyl CoA ' . Ceramide synthase is responsible for acylating sphinganine to generate dihydroceramide. Both enzymes in the pathway of de novo ceramide synthesis can be stimulated by chemotherapeutic agents and ionizing radiation " . This pathway can also be activated by the addition of free palmitoyl CoA and this has been proposed to play a role in diabetes and obesity, which result from increased levels of free fatty acids 212 Under normal physiological conditions, ceramide is not a major end-product in this pathway; rather, it is a precursor for the synthesis of complex sphingolipids. Several 24 enzymes involved in synthesis of complex sphingolipids have been implicated in regulating apoptosis. SM synthase is responsible for transferring phosphorylcholine from phosphatidylcholine to ceramide and generating SM and diacylglycerol (DAG). A survival advantage has been attributed to increased SM synthase activity in several reports 2 1 3 ' 2 1 4 whereas apoptosis is induced when conversion of de novo ceramide to complex sphingolipids is inhibited 2 1 5 . Glucosylceramide synthase (GCS) glycosylates ceramide to glucosylceramide in the Golgi, and this can be further modified into more complex glycosphingolipids. GCS can increase cellular resistance to apoptosis induced by TNFa 2 1 6 as well as anticancer agents 2 1 7 by reducing ceramide levels. Interestingly, there is evidence of compartmentalization in that only de novo generated ceramide was found to be efficiently converted to glucosylceramide whereas ceramide accumulation induced by SM hydrolysis was not 2 1 8 . Ceramidase (CDase) catalyzes the formation of sphingosine by cleaving ceramide at the amide bond to remove the fatty acid. Over-expression of CDase has been shown to lower ceramide levels and reduce apoptosis 2 X 9 . Sphingosine generated by CDase can then be phosphorylated into sphingosine-1-phosphate (SIP) by sphingosine kinase (SK) 2 2 0 . Thus, in addition to ceramide clearance, CDase can also confer protection to apoptosis by shunting ceramide into SIP, which is known to promote cell survival and proliferation 1 0 0 ' 2 2 1 " 2 2 3 . Ceramide kinase is also implicated in shunting ceramide into a metabolite with very different biologic properties. It catalyzes the phosphorylation of ceramide to ceramide-1-phosphate (C1P), which has pro-survival effects 9 9 5 1 ' 1 0 1 and also plays a role in inflammation 2 2 4> 2 2 5 ; cell proliferation 2 2 6 , 2 2 7 and phagocytosis 2 2 8 . 25 Table 1 summarizes the subcellular localization of key enzymes of ceramide metabolism and lists some inhibitors. Many of the enzymes of ceramide metabolism are emerging as regulated switches controlling the levels of ceramide relative to those of other bioactive lipids such as DAG, SIP and C1P that oppose ceramide's actions in 229 231 apoptosis " . The multiple pathways involved in ceramide metabolism have been shown to interact to regulate ceramide levels . 26 Enzyme Sphingomyelinase ASMase 1 9 6 Topology Inhibitor NSMase 2 4 0 Lysosomes/ Endosomes Secreted88'197 PM in Caveoli 2 3 8' 2 3 9 NSMase 1 244 202 NSMase2 NSMase3 AlkSMase 2 4 9 Enzymes involved in de Serine palmitoyltransferase 2 0 8 Ceramide Synthase Dihydroceramide Desaturase Others SM Synthase Ceramidase ACDase NCDase AlkCDase Glucosylceramide Synthase Ceramide Kinase Sphigosine Kinase ER/Golgi PM/ Golgi 245 246 (Gastrointestinal tract) Novo Synthesis ER ER ER Status Golgi, PM, Nucleus, / Mitochondria Lysosomal Mitochondria/endosomes ER/Golgi Glogi Microsomal fraction Cytoplasm/PM Desipramine 72 Scyphostatin241'242 GW4869 243 Myriocin/ Cycloserine Fumonisin B l 253 D609 NOE C6UreaCER DMAPP PDMP N,N-dimethylSph B5334C, F12509A Cloned 2 3 5 " 2 3 7 Cloned 2 4 5 Cloned 2 4 7 Cloned 2 4 8 Cloned 2 5 0 Cloned Cloned 251 252 Cloned 2 5 4' 2 5 5 Cloned 2 5 6 Cloned Cloned Cloned Cloned Cloned 257 258 259, 260 261 262-264 Table 1: Enzymes of ceramide metabolism and key features. 27 1.3.3 Ceramide as second messenger to regulate apoptosis Intracellular mediators for ceramide-induced apoptosis include protein kinases, phosphatases and cathepsin D 1 9 3 ' 2 6 5 . Cathepsin D is a lysosomal protease that was originally identified as a ceramide-binding protein that was activated by ceramide 2 6 6 . Activated cathepsin D is proposed to cleave Bid in response to TNFa-mediated ASMase activation and results in activation of caspases 2 6 7 . Kinase suppressor of Ras (KSR) 2 6 8 - 2 6 9 and Raf-1 have been suggested to be downstream targets of ceramide. Ceramide can mediate apoptosis through KSR to activate Raf-1 ' ' . Another ceramide activated kinase is PKCi^. Several groups have shown that natural ceramide binds directly to and activates PKCi^ 2 7 2 whose apoptotic effect might be exerted by negatively regulating PKB 2 7 3 . Ceramide accumulation can also activate serine/threonine protein phosphatases 1 (PP1) and PP2A 2 7 4 ' 2 7 5 . Ceramide-activated PP2A can counteract the anti-apoptotic effect of Bcl-2 by dephosphorylating Bcl-2, leading to its inactivation 2 7 6 . Ceramide activation of PP2A can also result in the inactivation of PKB 2 7 7 ' 2 7 8 . Ceramide can activate PP1 which in turn dephosphorylates Rb and causes cell cycle arrest 2 7 9 . Ceramide-activated PP1 can dephosphorylate serine/arginine-rich (SR) proteins 2 8 ° , whose function is to regulate constitutive and alternative splicing, including that of the key apoptotic mediators caspase 9 and Bc l -X 2 8 1 . 1.3.4 Ceramide as a modulator of membrane structure to regulate apoptosis The functions of ceramide depend on its sub-cellular location and site of formation. Ceramide, SM and glycosphingolipids are highly enriched in caveolae and membrane lipid rafts 2 8 2 . ASMase translocates to the outer leaflet of the cell membrane T i n O'JQ where it is in close proximity to the bulk of cellular SM ' . Ceramide can be 28 generated there, and associate with membrane rafts where it acts to facilitate clustering and activation of TNF family receptors, such as Fas ' . In ASMase-deficient cells, it has been shown that this enzyme is required for death receptor clustering, and this in turn is essential for induction of apoptosis by the extrinsic pathway 2 8 3 - 2 8 4 . ASMase activation and ceramide generation have also been reported to inactivate the PI3K/PKB survival signaling cascade 7 2> 2 8 5. Inhibition of PI3K by ceramide is associated with recruitment of caveolin-1 to PI3K-associated receptor complexes in rafts. Antisense knockdown of caveolin dramatically reduces ceramide-induced PI3K deregulation in fibroblasts, suggesting that caveolin-1 is required for the inhibition of PI3K by ceramide 2 3 8 . Only 10-20% of cellular SM resides in the inner leaflet of the plasma membrane (PM). Relatively rapid SM hydrolysis at the cytosolic side of the PM following TNFa and CD40L is believed to be caused by NSMase via the adaptor protein FAN 2 8 6 ' 2 8 7 . The generation of ceramide from this pool is speculated to lead to alteration of cell surface morphology concomitant with the last phases of apoptosis 2 8 8 . ASMase overexpression in mitochondria causes ceramide generation there but not in other cellular compartments, and induces apoptosis 2 8 9 . Furthermore, the addition of exogenous ceramide to purified mitochondria inhibites oxidative phosphorylation and promotes cytochrome c release 2 9 0 ' 2 9 1 . Mitochondria contain enzymes regulating ceramide level, such as ceramide synthase 2 9 2 ' 2 9 3 and CDase 2 9 4 . Several apoptotic stimuli have been shown to induce apoptosis correlating with an increase in mitochondrial ceramide levels ' . Ceramide accumulation in mitochondria can induce changes in the electron transport chain leading to generation of reactive oxygen species that precede 7Q7 9QR membrane permeability increases . Siskind et al have proposed that ceramide is 29 capable of directly forming a channel on mitochondria at the concentration observed during apoptosis. Phospholipid interactions after hydrolysis of SM to ceramide can have profound effects on membrane structure, including membrane blebbing, vesicle shedding, and apoptotic body formation 2 8 8 . 1.3.5 Therapeutic implications There is evidence implicating sphingolipid pathways in the pathogenesis of many diseases. For example, the neurodegenerative disorder Niemann-Pick Disease (NPD) types A and B, is the result of inherited deficiency of ASMase activity 2 9 9 ' 3 0 0 . Type A NPD is a severe infantile neuronopathic form that is usually fatal by age 3. Type B manifests as hepatosplenomegaly with minimal neurological involvement and patients often survive into adulthood 3 0 1 . Both disorders are due to mutations in the ASMase gene, but type B is associated with a small amount of residual ASMase activity. Drug resistance in some cancer cells was attributed to their failure to sustain high levels of ceramide in response to chemotherapy due to either the increased clearance or decreased degradation of complex sphingolipids ' ' . Recently, apoptosis of P-islet cells induced by ceramide, whose synthesis is enhanced by free fatty acid overload, has been implicated in the pathogenesis of diabetes in obesity 3 0 4 " 3 0 6 . These examples illustrate how knowledge of sphingolipid metabolism can potentially provide better understanding of disease pathogenesis and offer a novel approach to pharmacological intervention. 30 1.4 Objectives Macrophages are cells that are critical to the body's ability to repel pathogens and to remove damaged tissue and dying cells caused by normal growth and development. Apoptosis is an important mechanism involved in regulating the number of macrophages. The dysregulation of macrophage function is implicated in several human diseases such as rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Understanding how macrophage apoptosis is regulated can help develop novel therapeutic approaches to J diseases. We recently showed that oxLDL inhibits apoptosis in macrophages through the PI3K/PKB pathway and subsequent level of pro-survival protein BC1-XL 11. We are interested in exploring if other Bcl-2 family members also play a role in macrophage apoptosis and if oxLDL is able to exert its anti-apoptotic function by regulating these proteins. In previous studies, we showed that tyrosine phosphorylation as well as PI3K activation increase in response to oxLDL treatment in THP-1 cells 3 9 . To investigate the upstream receptor(s) activated by oxLDL to mediate macrophage survival, we propose using pertussis toxin (PTX) to test whether G protein coupled receptors are involved. We also observed that during macrophage apoptosis the ASMase activity increases in parallel with ceramide generation . Using mice deficient in ASMase, we seek to further elucidate the metabolic pathways that are responsible for ceramide generation in these cells. 31 2 Materials and methods 2.1 Materials Antibodies to mouse Bak, Bax, vinculin and p85a isoform of PI3K were purchased from Upstate. Anti-cytochrome c, anti-PARP and annexin V-FITC conjugated antibody were from BD Pharmingen. Atni-Mcl-1 antibody was from Rockland. Antibody to the mitochondrial outer membrane receptor TOM20 was a kind gift from Dr. G.C. Shore, McGill University. Anti-ubiquitin was from Cell Signaling Technology. Anti-Bim was from Affinity BioReagent. Antibody recognizing active Bax (6A7) was purchased from Travegen. Antibodies against phospho-MB, phospho-ERK and phospho-Ser473PKB were from Cell Signaling Technology. Lambda protein phosphatase was purchased from New England Biolab. Anti-Bcl-XL and -actin were from Santa Cruz Biotechnology Inc. Escherichia coli diacylglycerol kinase, P-octyl glucoside, mastoparan, mastoparan 17, and all other inhibitors were supplied by Calbiochem. Pertussis toxin, P-oligomer, propidium iodide, glutathione, MG132, protease inhibitor cocktail, non-hydroxyl fatty acid ceramide, ceramideTl -phosphate, and RPMI 1640 medium were purchased from Sigma-Aldrich. Caspases FLICA kit was from Immunochemistry Technologies. Fetal bovine serum (FBS), random primer, Superscript RNaseH-free reverse transcriptase, RNaseH and RNase-out were obtained from Invitrogen. Reagents required for 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell viability assays were purchased from Promega. Protein-G agarose beads were from Amersham Biosciences. QuantiTect SYBR green PCR kit and RNeasy mini kit were from Qiagen. Goat anti-mouse and anti-rabbit IgG, horseradish peroxidase secondary antibodies were from DAKO. Nitrocellulose 32 membranes and protein markers were purchased from Bio-Rad. BSA protein standards and BCA assay reagents were from Pierce. C2-ceramide, C2-dihydroceramide, cardiolipin were purchased from Avanti Polar. [y-32P]ATP, [3H]serine, [14C]palmitoyl-CoA, [3H]palmitate and [N-methyl-HC] bovine sphingomyelin were purchased from Perkin-Elmer NEN. 2.2 Lipoprotein isolation, oxidation and acetylation Low density lipoprotein (LDL, d= 1.019-1.063) was isolated by sequential ultracentrifugation of EDTA-anticoagulated fasting plasma obtained from healthy normolipidemic volunteers as described in 3 0 1 . Oxidation was performed by incubating 200 jig/ml LDL with 5uM CuS0 4 in Dulbecco's PBS for 2, 5, or 24 hours at 37°C. The reaction was stopped by addition of 40 uM butylated hydroxytoluene and 300 uM EDTA. The modified LDL was then washed and concentrated using Centricon Plus-20 ultrafilters (Millipore, Bedford, MA) 3 0 7'. The protein concentrations of oxidized LDL were then determined using BCA protein assay. Unless otherwise stated, extensively oxidized LDL (incubated with copper for 24 hours) was used throughout the study. Acetylation of LDL was performed by the sequential addition of acetic anhydride 2 8 . 2.3 Cell culture CD1 and C57BL/6 mice were obtained from the UBC animal facility. Bax knockout mice were from the Jackson Laboratory. C57BL/10ScCr mice are a strain with a naturally occurring deletion of the Tlr4 gene 3 0 8 , and were purchased from the Jackson Laboratory (Bar Harbor, ME). ASMase knockout mice were obtained from Dr. R. Kolesnick. 33 Bone marrow was harvested from the femurs of 6-8 weeks old female CD1 mice as described 1 2 . Cells were plated overnight in RPMI 1640 supplemented with 10% FBS, ImM sodium pyruvate, 2mM L-glutamine, 100 units/ml penicillin/streptomycin and 10% L-cell conditioned medium as the source of M-CSF. Non-adherent cells were removed and cultured in the above medium until 80% confluence was reached (4-6 days). Peripheral blood mononuclear cells (PBMCs) from consenting normal donors were isolated on Ficoll-Paque™Plus (Pharmacia Biotech) gradients according to the manufacturer's protocol. PBMCs were resuspended in media (DMEM, 10%> FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 u.g/ml streptomycin) and dispensed onto tissue culture plates at a concentration of 1.5 x 106 cells per cm2 and incubated for two hours in a humidified atmosphere containing 5% CO2. Non-adherent cells were removed by washing three times with warm Dulbecco's phosphate-buffered saline (DPBS). The adherent cell fractions were differentiated into macrophages by culturing for 5 to 6 days in media supplemented with 50 ng/ml recombinant human macrophage colony stimulating factor (R&D Systems). The macrophages were lifted using a rubber cell scraper and then seeded in 100 mm dishes at 1.0 x 10s cells per cm2 and grown for 24 hours before treatment with oxLDL. FDCP-1 cells, a murine factor dependent hematopoietic precursor cell line, were cultured in the same medium as macrophages, except that M-CSF was substituted with 2.5% conditioned medium from WEHI-3 cells as a source of IL-3. 2.4 Genotyping ASMase-/- mice were genotyped by PCR 3 0 9 . Genomic DNA was mixed with an ASMase sense primer (PS; 5- AGCCGTGTCCTCTTCCTTAC-3') and two antisense 34 primers, one from within exon 2 of the ASM gene (PA 1; 5'-CGAGACTGTTGCCAGACATC-3) and one from within the neo cassette (PA2; 5'-CGCTACCCGTGATATTGCTG-3') . Thirty cycles of PCR amplification, each consisting of 1 min at 93 °C , 1 min at 58 °C , and 1 min at 72 °C , were performed. In wild-type mice, a single band of 269 bp corresponding to the undisrupted A S M gene was amplified, while in ASM-/- mice a single band of 523 bp was amplified from the sense and neo primers. 2.5 Cell Viability assay When 80% confluence was reached, B M D M were harvested using a rubber cell scraper. 5x104 cells per well were seeded into 96-well plates and incubated overnight. Cells were then washed with PBS, and drugs in the absence or presence of PTX were added in 100 (J.1 of the same medium except without M-CSF. At the end of the 24 hour incubation, 20 ul of MTS/PMS solution (prepared according to the manufacturer's instructions) was added to each microwell. The plate was incubated for 1-4 hours at 37°C and was read using an ELISA plate reader at 490 nm. We previously showed that the bioreduction rate of MTS is linearly correlated with the number of viable macrophages 3 9 . 2.6 Immunofluorescence microscopy Macrophages were plated on sterile glass coverslips and incubated for 18 h in RPMI 1640 supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 units/ml penicillin/streptomycin and 10% L-cell conditioned medium. Cells were washed, and then cultured in the absence or presence of cytokine for 24 hours. The pan-caspase inhibitor Z-VAD was added at 100 uM to block later stages of apoptosis and 35 minimize detachment of cells. The coverslips were then washed twice with cold PBS and then fixed with 4% paraformaldehyde for 30 minutes at room temperature. Cell membranes were permeabilized with 0.2% Triton X-100 in PBS for 20 minutes and blocked with blocking solution (10% FBS with 0.1% Triton X-100 in PBS) for 30 minutes at room temperature. Coverslips were then incubated with primary antibody in blocking solution for 30 minutes and then with AlexaFluor conjugated goat anti-mouse or goat anti-rabbit secondary antibody (Molecular Probes) for 30 minutes in the dark. Cells were then examined on a Zeiss Axiophot fluorescence microscope equipped with a digital imaging system. 2.7 Flow cytometric analysis One million cells were seeded in 6 well plates, incubated under the indicated conditions for 24 hours, harvested with a rubber scraper, and pelleted by centrifugation. To assess DNA fragmentation, cells were fixed with cold 70% ethanol for 10 minutes, and resuspended in propidium iodide staining solution (10 ug/ml RNaseA, 20 ug/ml PI, in PBS + 0.1% glucose). To quantify phosphatidylserine externalization, macrophages were incubated with annexin V-FITC according to the manufacturer's instructions. Measurement of caspase activation was carried out with a fluorescent-labeled indicator kit, FLICA (Immunochemistry), and assayed by flow cytometry according to manufacturer's instructions. Cells were analyzed by Beckman Coulter flow cytometer (EPICS XL-MCL) on the FL3 channel for DNA content, on the FL1 channel for FITC fluorescence with ten thousand events counted for each analysis. 36 2.8 Reverse transcription and Real time PCR Total RNA was isolated from B M D M using RNeasy kit from Qiagen and reverse-transcribed using Superscript II® according to manufacturer's directions. Using the cDNA generated, Bax and actin were amplified by PCR. The amplification generates a 270-bp fragment for Bax (forward primer, 5'-AGATGAACTGGATAGCAATATGGA-3' ; reverse primer, 5'-CCACCCTGGTCTTGGATCCAGACA-3') and a 138-bp fragment for actin (forward primer, 5' -AGAGGGAAATCGTGCGTGAC; reverse primer, 5'-CAATAGTGATGACCTGGCCGT). The amplification conditions were as follows: hold at 95°C for 10 minutes, then 40 cycles at 94°C for 30s, 55°C for 30s, 72°C for 30s. Final extension was performed at 72°C for 5 minutes. The PCR products were separated by electrophoresis in a 1.2% agarose gel, and stained with ethidium bromide. The cDNA was also used for real time PCR by using QuantiTect SYBR green PCR kit using the same amplification conditions as above. 2.9 Immunoblotting and immunoprecipitation For immunoblotting whole cell lysates, 1.5 million cells were washed with PBS and lysed in 50 ul of ice-cold 20 mM Tris HCL pH 8.0, 1% NP40, 10% glycerol, 137 mM NaCl, 10 mM NaF (solubilization buffer A) supplemented with protease inhibitor cocktail and 200uM sodium vanadate. The cells were then sonicated for 5 seconds and centrifuged at 23,000 x g for 5 min. The supernatant was collected and assayed for protein concentration. The extracted proteins were adjusted to equal concentration and were boiled in SDS sample buffer for 5 min. 50 \ig of cell iysate was loaded in each lane of a 12% SDS-polyacrylamide gel. For detection of active Bax, macrophages were lysed 37 with solubilization buffer B (10 m M HEPES, pH 7.4, 150 m M NaCl, and 1% Chaps). One microgram of 6A7 anti-Bax antibody was added to 500 ug of cell lysate for and incubated overnight at 4°C on an oscillating stage. Protein-G agarose beads (Amersham Bioscience) were added to the mixture for 1 hour at 4°C. The beads were spun down and washed 4 times with solubilization buffer B and immunoprecipitates were then dissolved in 2x sample buffer and loaded onto a 12% SDS-polyacrylamide gel. Transfers were done by semi-dry blotting onto nitrocellulose membranes. The membranes were blocked for one hour in 5% low fat dry milk in Tris-buffered saline with 0.05 % Tween 20 followed by overnight incubation at 4°C with appropriate antibody. Bound antibody was visualized with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies and enhanced chemiluminescence. 2.10 Lipid labelling for determination of ceramide levels Radioactivity in ceramide was assayed after labeling of B M D M with 5 uGi/ml of [3H]palmitate for 24 h in RPMI 1640 without or withl0% FBS and 10% M-CSF. The cells were washed twice with PBS and scraped into 1 ml of methanol, which was then mixed with 1 ml of chloroform and 0.9 ml of 2M KC1, 0.2M H 3 P 0 4 3 1 ° . The aqueous phase was discarded, and the chloroform phase was dried under nitrogen. Ceramides were isolated by TLC by using Silica Gel 60-coated glass plates developed with chloroform/methanol/acetic acid (9:1:1 by volume) for half their length and then with petroleum ether/diethyl ether/acetic acid (60:40:1 by volume). Lipids were visualized by iodine and identified by co-chromatography with authentic standards. Radioactivity was measured by scraping the corresponding bands from TLC plates and liquid scintillation counting. 38 2.11 Sphinomyelinase assay For assay of sphingomyelinase (SMase) activity cell lysates were prepared by three cycles of freeze/thawing in TE buffer (20 mMTris/HCl, pH 7.5, and 1 mM EDTA) containing protease inhibitor cocktail. DTT (1 mM) was also added for lysates to be used for NSMase assay, while 50 mM sodium acetate, pH 5.0 was included in the buffer for ASMase assay. The homogenate was centrifuged at 1,000 * g for 10 minutes and the supernatants were collected for assay of SMase activities. The activities of neutral and acid sphingomyelinases were determined exactly as described by Liu and Hannun 3 1 1 using [N-methyl-H-C] sphingomyelin as the substrate. The final reaction buffer for NSMase was 0.1% Triton X-100, 5 mM MgCl 2 , 5 mM DTT and 0.1 M Tris/HCl,pH 7.4, and that for ASMase was 100 mM sodium acetate, pH 5.0 and 0.2% TritonX-100. After incubating for 1.5-2 h, the reaction was stopped by the addition of 0.5 ml of chloroform:methanol (2:1). The samples were vortexedand then centrifuged to separate the two phases. The upper phase, containing labeled phosphorylcholine released from sphingomyelin, was transferred to scintillation vials and counted by liquid scintillation counting. Negative controls containing no enzyme were run concomitantly. 2.12 Nuclear preparations and Electrophoretic Mobility Shift Assay Cells were grown in 100 mm tissue culture plates until 80% confluent. Isolation of nuclei and radioactive labeling of an N F - K B EMSA probe was performed as previously described 3 1 2 . Nuclear extracts (10 ug) were preincubated for 15 min in binding buffer (20 mM HEPES, pH 7-9, 100 mM KC1, 10% glycerol, 1 mM DTT) containing 1 ug of poly dldC (Amersham). The [ P]-labeled probe (20,000 counts per minute) was then added and the reaction mixture incubated at room temperature for 30 39 min before electrophoresis in a 5% non-denaturing polyacrylamide gel in 0.25 X TBE (Tris 89 mM, boric acid 89 mM, EDTA 2 mM) at 200 V for 1.5 h. The gel was subsequently dried for 45 min and imaged using a Bio-Rad FX phospho-imager. 2.13 Diacylglycerol Kinase assay for ceramide mass Ceramide levels were measured using the diacylglycerol kinase method as described previously ' . In brief, total cellular lipids were extracted with chloroform/methanol/2 M KC1, 0.2 M HC1, resuspended in a micellar solution of 1 mM cardiolipin, 1.5% N-octyl- P -D-glucopyranoside, 0.2 mM DETAPAC, 5 ug of diacylglycerol kinase and lOmM DTT. The reaction was initiated with 1 uGi of [7-3 2P]ATP diluted with unlabelled ATP to give a final concentration of 1 mM. After incubation for 45 min at 30°C, lipids were extracted and separated on Silica Gel 60 TLC plates with choloroform/methanol/2N NH 4 OH (65:35:7.5). The plates were dried and redeveloped with chloroform/acetone/ methanol/acetic acid/water (50:20:10:10:5). Ceramide-1-phosphate spots were scraped from the plates and quantitated by scintillation counting. The assay was calibrated with a standard curve of authentic ceramide. Results were normalized to total lipid phosphate. To measure lipid phosphate, the chloroform phase of the cell extract was evaporated under N 2 , and incubated at 180 °C for 30 min in 50ul of 70% HC104. Then 278 ul of H 2 0 , 55 uf of 2.5% ammonium molybdate, and 55 ul of 10% ascorbic acid were added and incubated for a further 15 min at 95 °C. Inorganic phosphate was detected by absorbance at 700 nm and quantified based upon a standard curve of glycerol phosphate. 40 2.14 Microsome preparation for in vitro serine palmitoyltransferase and ceramide synthase assays. Microsomes were prepared by sonication of cell pellets in 50 mM HEPES, pH 7.4, 10 mM EDTA, 5 mM DTT, and 0.25 M sucrose supplemented with protease inhibitor cocktail from Sigma-Aldrich. The preparation was centrifuged at 1000 x g, and the resulting supernatant was then ultracentrifuged at 100,000 x g. The resultant pellet was suspended by homogenization in 50 mM HEPES, pH 7.4,5 mM DTT buffer containing 20% glycerol to form microsomes for the assays. Protein concentration was determined using the Bio-Rad dye protein assay reagents with a standard curve of bovine serum albumin. 2.15 Serine palmitoyltransferase assay Serine palmitoyltransferase was assayed as described previously 3 1 4 . Briefly, enzyme activity in 50-100 ug of microsomal membranes was determined in 100 mM HEPES (pH 8.3), 5 mM DTT, 2.5 mM EDTA (pH 7.0), and 50 uM pyridoxal 5'-phosphate. The reaction was initiated by the addition of 200 \xM palmitoyl CoA and 3 uCi of L-[3H] serine, with a final serine concentration of 1 mM. Reactions were incubated for 20 min at 37 °C prior to termination with 1.5 ml of chloroform/methanol (1:2) followed by 1 ml of chloroform and 1.8 ml of 0.5 N N H 4 O H with sphinganine as carrier. Lipids were extracted as described previously and quantified by liquid scintillation counting 3 1 4 . 2.16 Ceramide synthase assay (Dihydro)ceramide synthase was assayed according to Bose et al. 2 1 0 . Briefly, 50-200 ug of microsomal membranes were incubated in 20 mM HEPES (pH 7.4), 2 mM 41 MgCb, 20 uM fatty acid-free bovine serum albumin, 2.5 uM DTT and 20 uM sphinganine. The reaction was then initiated by the addition of 2 |aCi of [1-14C]palmitoyl-CoA in the presence of 100 uM palmitoyl-CoA and allowed to incubate for 60 min at 37 °C. Lipids were extracted and ceramide was isolated and counted as described above. 2.17 Statistical analysis Results were expressed as means ± SD. Statistical analysis was done by Student's /-test as appropriate. A P value of less than 0.05 was taken as significant. 42 3 OxLDL promotes macrophage survival by facilitating Bax degradation and increasing Mcl-1 expression3 3.1 Introduction Accumulation of lipid laden macrophages (foam cells) in the intima of susceptible arteries is thought to play an important role in the development of atherosclerosis " ]. Foam cells are the hallmark of early and intermediate atherosclerotic lesions, pointing to a role in lesion formation and progression. In addition, macrophage-rich plaques are weaker than fibrous plaques because of the lipid deposits and because macrophages secrete matrix degrading metalloproteases (MMP) 3 1 5 . Several studies have demonstrated that macrophage-rich plaques have a significantly increased risk of rupture and thrombosis. Oxidized LDL (oxLDL) has many biological properties which could promote atherogenesis, including the recruitment and retention of macrophages into the arterial intima. OxLDL can also induce proliferation of macrophages, and block macrophage apoptosis 2 0 ' 2 6 . OxLDL is present in atherosclerotic lesions 2 6 , and could play a key role in expanding macrophage populations in the arterial intima. The effect of oxLDL on macrophage apoptosis is mediated in part through Bcl-X L , a member of the Bcl-2 family of regulators of apoptosis. This family is composed of proteins with an anti-apoptotic effect such as Bcl-2, B C I - X L , Mcl-1 and proteins with a pro-apoptotic effect, such as Bax, Bak, Bid, and Bad 1 0 7 . Bax was first shown to exert its pro-apoptotic effect by counteracting the pro-survival functions of Bcl-2 1 4 2 ' 1 7 9> 3 1 6. Bax 316 can form oligomers on mitochondria which allow cytochrome c release . Both ectopic a A version of this chapter will be submitted for publication. Wang, S.W., Duronio, V. and Steinbrecher, U.P., Effect of oxLDL on Mcl-1 and Bax in macrophages. 43 expression of Bax and the addition of purified recombinant Bax can accelerate apoptosis 1 4 1 . The anti-apoptotic members on the other hand can protect cells from apoptosis by sequestering Bax. Bax is able to heterodimerize with Bcl-2 and mutagenesis studies have shown that this ability of Bcl-2 to bind Bax is required for its pro-survival effect142. Taken together, these observations suggest that Bax plays an important role in cell death. The ubiquitin proteasome system is a conserved mechanism for controlling degradation of cellular proteins 3 l 7 . It is involved in many cellular processes, including lift "X 1 Q apoptosis, since it targets proteins of the apoptotic machinery such as Bid , Bik , Bax 3 2 0 , Bcl-2 3 2 1 , Bim 3 2 2 , Mcl-1 3 2 3and XIAP 3 2 4 . Abnormalities of proteasome function have been implicated in the pathogenesis of several diseases, including atherosclerosis 325 326 Mcl-1, a pro-survival Bcl-2 family protein, is essential for the homeostasis of early hematopoietic progenitors by ensuring cell survival 3 2 7 . Over-expression of Mcl-1 inhibits cell death induced by various apoptotic stimuli 3 2 8 , 3 2 9 while elimination of Mcl-1 induces apoptosis . Its association with Bim is implicated in apoptosis in multiple myeloma cells Although it is widely agreed that Bax and Bak are essential in executing apoptosis 1 6 3 , the mechanism by which Bcl-2 family members interact and allow the activation of Bax and Bak still remain poorly understood 1 6 8 . One proposed model suggests that a subclass of BH3-only pro-apoptotic proteins, including Bim and truncated Bid (tBid), can bind directly not only to pro-survival proteins but also to Bax and Bak to activate them. This model further proposes that the other pro-apoptotic members bind to pro-survival proteins, and simply lower their capacity to sequester the activators 1 6 6 ' 1 8 7 . 44 An alternative view supported by recent findings is that the BH3-only proteins exclusively displace the pro-survival proteins, overcoming their sequestration of Bax/Bak 1 6 9 " 1 7 2 , Thus, whether the BH3-only proteins activate Bax/Bak directly or indirectly (or both) remains to be established. We have previously shown that oxLDL promotes survival of bone marrow derived macrophages by blocking apoptosis through the PI3K/PKB signaling pathway, which leads to activation of N F K B and subsequent upregulation of Bcl-X L 3 8 ' 7 2 . The aim of the current study was to determine whether other Bcl-2 family members such as Bax, Mcl-1 and Bim levels are altered in macrophages upon treatment with oxLDL, and to define whether their interactions are required for the anti-apoptotic effects of oxLDL. 3.2 Results 3.2.1 Bax is involved in macrophage apoptosis following M-CSF withdrawal Various apoptotic stimuli activate Bax by inducing a conformational change at its • 157 N-terminus, which then leads to its translocation to mitochondria to initiate apoptosis 170,332 j o determine whether Bax plays a role in macrophage apoptosis induced by M -CSF withdrawal, we used the antibody, 6A7, which is specific for the active conformation of Bax. B M D M were incubated without M-CSF for various times and were then lysed using CHAPS buffer to preserve Bax conformation 2 9 5 , immunoprecipitated with 6A7, and then blotted with a conformation-independent anti-Bax antibody. Results in Figure 3.1 A indicate that Bax is activated in BMDMs after 24 hours of cytokine withdrawal, coinciding with the significant number of cells undergoing apoptosis at that stage 3 3 3 ' 3 3 4 . 45 Our next aim was to determine cellular localization of active Bax protein in the macrophages. In non-apoptotic cells, Bax is a soluble monomeric protein diffusely distributed in the cytoplasm 2 9 5'. There was no detectable staining for active Bax with 6A7 antibody in healthy control cells. However, when the macrophages were cultured without cytokines to induce apoptosis, active Bax was found in a pattern that colocalized with the mitochondrial outer membrane protein TOM 20 as seen in Figure 3.IB. These results confirm that there is a conformational change of Bax and redistribution to the mitochondria during apoptosis in bone marrow derived macrophages. 3.2.2 OxLDL reduces Bax protein levels in Macrophages We have previously shown that oxLDL blocks apoptosis induced by growth factor withdrawal in BMDM, and that it does so partly by up-regulating expression of the pro-survival protein, Bcl-X L 1 2 . In view of the key role of Bax in apoptosis, we hypothesized that oxLDL may also regulate Bax to promote survival. To test this, we incubated macrophages with oxLDL for 24 hours, and measured the level of Bax by immunoblotting. Figure 3.2A shows that there was a decrease in Bax after addition of oxLDL in B M D M while no significant change was observed in Bak. This was also observed in differentiated THP-1 cells (Figure 3.2B). The only significant down-regulation of Bax was observed in cells treated with heavily oxidized LDL (Figure 3.3). Native LDL, mildly oxidized LDL, and acetylated LDL had no significant effect. The increase in Bax relative to actin acetyl LDL may be an artifact due to the low-intensity actin band. Viability of cells incubated with acLDL was poor and although the amount of protein loaded was the same, the amount of actin per unit cell protein was evidently low. These results correlate with our previous findings that acetyl LDL and native LDL do not 46 confer resistance to macrophage apoptosis 3 8 . Bax was rapidly downregulated by oxLDL in mouse B M D M as well as in human monocyte-derived macrophages (Figure 3.4). 3.2.3 OxLDL regulates Bax by accelerating its degradation To determine if oxLDL controls Bax expression at the level of transcription, mRNA was extracted from macrophages after 24 hours of treatment with or without M -CSF or oxLDL, and amplified by real-time PCR. Instead of decreasing, Bax mRNA in oxLDL treated cells increased 1.9 ± 0.2 fold compared to healthy control cells (n=3, p<0.005). This suggests that oxLDL promotes the decrease of Bax by a post-transcriptional mechanism. To determine if the rate of degradation of Bax is increased by oxLDL in macrophages, cells were pre-treated with 10 ug/ml cycloheximide for 1 hour to stop new protein synthesis and then oxLDL was added. As shown in Figure 3.5A, oxLDL induced a rapid drop in Bax levels and by 30 minutes it was roughly half of the starting level (Figure 3.5B). Cytokine withdrawal alone did not alter Bax levels during the 8-hour time course (Figure 3.5B). 3.2.4 Down-regulation of Bax by oxLDL is proteasome mediated Bax has been reported to be degraded by the ubiquitin/proteasome-dependent pathway ' , and oxLDL has been previously shown to affect this pathway ' ' To determine if the accelerated degradation of Bax involves proteasomes, 3 different proteasome inhibitors, A L L N , MG132 or lactacystin were added to B M D M in addition to oxLDL. Treatment with these inhibitors blocked the effect of oxLDL on Bax degradation (Figure 3.6A). Furthermore, Bax levels increased with increasing concentration of MG132 and A L L N in the presence of oxLDL (Figure 3.6B). As in 3.6A 47 also shows a second Bax band migrating at a slightly higher molecular weight, only in lysates from cells treated with oxLDL. It has been reported that Bax can be serine phosphorylated by PKB to promote its heterodimerization with pro-survival counterparts, Mcl-1 and Bcl-X L 3 3 7 . Treatment of the samples with X phosphatase did not eliminate the slower migrating band (data not shown). This negative result suggests that the band shift may not be due to phosphorylation. To further elucidate the role of the proteasome pathway in the degradation of Bax, we treated the cells in the presence of oxLDL with A L L M , an inhibitor of calpains and cathepsins that is said to have no effect on proteasomal degradation. A L L M had no effect on cell viability in the presence of oxLDL (Figure 3.6C). 3.2.5 The PI3K/PKB pathway is involved in accelerated Bax degradation Previous results from our laboratory showed that although oxLDL can cause activation of both ERK and PI3K/PKB pathway, only the latter pathway is vital for oxLDL-mediated B M D M survival1 2. PKB has been shown to regulate survival of pre-B hematopoietic cells by inhibiting the conformational change of Bax 3 3 8 . To examine whether the effect of oxLDL on Bax was mediated through the PI3K/PKB pathway, macrophages were treated with the selective PI3K inhibitor LY294002 in the presence of oxLDL. This drug partially restored the expression of Bax (Figure 3.7). In contrast, treatment with U0126 or SB203850, selective inhibitors of M E K kinase and p38 MAPK respectively, had no effect on reversing oxLDL mediated Bax down-regulation. This suggests that the PI3K/PKB pathway activated by oxLDL plays a role in down-regulating Bax expression. 48 3.2.6 Bax is only partially responsible for apoptosis induced by M-CSF withdrawal. To assess the importance of Bax down-regulation in oxLDL-mediated survival events we compared macrophages obtained from Bax knockout and wild-type mice. We confirmed that Bax was undetectable by immunoblotting of B M D M lysates from knockout mice (Figure 3.8A). B M D M from these mice had an identical extent of apoptosis as B M D M from wild-type mice following cytokine withdrawal, and oxLDL was fully capable of blocking apoptosis (Figure 3.8B). This indicates that the pro-survival effect of oxLDL is not entirely due to its ability to down-regulate Bax. 3.2.7 Sequesteration of Bax by Mcl-1 is disrupted by cytokine withdrawal while oxLDL enhances this association In many types of cells, Bax and Bak are thought to be kept in check by binding to pro-survival family members such as Bcl-2 and Mcl-1. However, as shown in Figure 3.9A, Bcl-2 does not sequester Bax or Bak in healthy BMDM. On the other hand, Mcl-1 associated with Bax in healthy BMDM, but when cells were deprived of M-CSF there was a substantial decrease in the association of Bax with Mcl-1 (Figure 3.9B). As shown in Figure 3.9C , the association of Mcl-1 with Bax rapidly decreases following cytokine withdrawal. However, despite the decreased level of Bax in the presence of oxLDL, Mcl-1 is still found to associate with Bax (Figure 3.9C). 49 3.2.8 OxLDL acts through the PI3K/PKB pathway to increase the level of Mcl-1 Mcl-1 is an important survival factor for multiple myeloma where its down-regulation has been shown to induce apoptosis 3 2 3 . Mcl-1 is a short-lived protein with a half-life between 30min and a few hours depending on the cell type 3 3 9 , 3 4 ° and is reported to be regulated by proteasomal degradation during apoptosis 1 7 0 . Although Mcl-1 was not rapidly degraded in apoptotic macrophages (Figure 3.10A), its expression increased in response to oxLDL treatment (Figure 3.10B). Furthermore, this increase of Mcl-1 correlated with increased oxidation of LDL, while acetylated LDL (AcLDL) had no effect (Figure 3.11). Interestingly, like Bax, this effect of oxLDL is also under the control of PI3K/PKB pathway since inhibition of PI3K diminished oxLDL mediated Mcl-1 increase (Figure 3.10B last lane). This suggests that oxLDL regulates Bax and Mcl-1 in opposite directions through PI3K to promote cell survival. 3.2.9 Bim does not displace Mcl-1 from Bax In order to induce apoptosis, BH3-only proteins are believed to activate Bax/Bak proteins and/or dissociate pro-survival Bcl-2 family members from Bax/Bak 1 6 8 . We chose the BH3-only protein Bim to investigate its role in macrophage apoptosis. Phosphorylation of Bim is reported to disrupt its interaction with Bax and contribute to cell survival 3 4 1 . We found that Bim was phosphorylated in healthy cells and this phosphorylation disappeared upon cytokine withdrawal. However, phosphorylation of Bim was not observed in oxLDL treated cells (Figure 3.12). Furthermore, we could not demonstrate Bax binding to Bim in macrophages incubated with or without M-CSF 50 (Figure 3.13). Mcl-1 co-immunoprecipitated with Bim but we found no change in association between Bim and Mcl-1 when cells were deprived of M-CSF (Figure 3.14). 3.3 Discussion Dysregulation of apoptosis by Bcl-2 family members plays an important role in the pathogenesis of many diseases. Bax mutation in particular has been implicated in leukemias and colorectal cancer 3 4 2 - 3 4 3 . it has been shown that advanced atherosclerotic lesions show higher Bax immunoreactivity and more TUNEL-positive cells 3 4 4 while IL-10 is able to enhance oxLDL-induced foam cell formation by up-regulating prosurvival members of the Bcl-2 family 3 4 5 . The importance of Bax regulation in atherosclerosis is further illustrated by a recent study demonstrating that reducing macrophage apoptosis by selective knockout of Bax resulted in accelerated lesion progression 6 9 . In this study, we show that oxLDL promotes Bax degradation via the proteasome pathway and increases levels of a pro-survival member of the Bcl-2 family, Mcl-1. In contrast, several previous studies have reported that oxLDL promotes apoptosis of cells 54,346-348 a n t j g a x j e v e j s a r e e i e v a t e d j n the presence of oxLDL 3 4 4 ' 3 4 9 . We believe these discrepancies come from methods of oxidation of LDL, concentration of oxLDL and the cell types used in those studies. For example, we and others have shown that high concentrations of oxLDL 3 8 ' 3 5 0 or peroxide-rich oxLDL 7 2 ' 8 0 can be cytotoxic to cells. Our observation that oxLDL can cause Bax degradation by proteasomes is in agreement with our previous findings that oxLDL treatment results in phosphorylation and proteasomal degradation of I K B , leading to activation of N F K B . Active N F K B has been shown to be present in atherosclerotic lesions 8 3 . Several reports also showed that 51 oxLDL was able to alter the activity of proteasome and HDL was able to counteract this effect 8 5' 3 3 5' 3 5 1. The PI3K/PKB pathway has been shown to mediate macrophage survival by M -CSF 7 l ' 3 5 2 . PKB can promote cell survival by directly phosphorylating regulatory components of the apoptotic machinery, such as forkhead transcription factors 3 5 3 , I K B 3 5 4 , or indirectly by changing the levels of expression of the genes that encode components of cell death, such as the Bcl-2 family members 1 6 . While both p38 MAPK 3 5 5 and PI3K 3 3 8 have been reported to regulate cell survival by preventing Bax translocation to mitochondria, oxLDL signals only through the latter pathway in primary macrophages (Figure 3.7). PI3K/PKB has been reported to be responsible for targeting various proteins for proteasomal degradation, such as p27Kipl 3 5 6 , and Bim 3 5 7 . It is possible that Bax is selectively marked for proteasomal degradation by PKB in response to oxLDL signalling, similar to I K B . The observed reduction in the electrophoretic mobility of Bax in response to oxLDL may reflect a modification necessary for targeting to proteasomes. Although there are reports that calpain and/or cathepsin can cleave Bax to generate a shorter fragment 3 5 8 " 3 6 0 5 this is unlikely to occur in the case of oxLDL-mediated cell survival. We tested the ability of the oxLDL to generate the 18KDa fragment of Bax by using an antibody directed against the C terminus of Bax. Our results (data not shown) did not reveal the presence of any such fragment suggesting the effect of oxLDL on Bax does not involve cathepsins or calpain. Furthermore, the short form of Bax was reported to enhance apoptosis 3 6 0 by acting like a BH3-only protein that binds strongly to BC1-XL thus leaving full-length Bax to translocate to mitochondria 3 6 1 . While inhibition of calpain and cathepsin with A L L M did not alter cell viability, the additional 52 inhibition of proteasome with A L L N prevented oxLDL's ability to promote cell survival (Figure 3 . 6 C ) . Hence it is unlikely either calpain or cathepsin-mediated Bax degradation plays a role in the ability of oxLDL to promote cell survival. Interestingly, survival of macrophages in the presence of M-CSF was not dependent upon degradation of Bax, indicating that there are likely multiple pathways that can promote survival. The fact that cells from Bax knockout mice were able to undergo apoptosis following cytokine withdrawal also supports the conclusion that other means of inducing apoptosis, e.g. via activation of Bak, can likely compensate for the complete lack of Bax. This is in accordance with the observation that Bax/Bak double knockout cells were resistant to many apoptotic stimuli (such as growth factor withdrawal, UV irradiation, drug treatment, TNFa), while single knockout of either of these two multidomain pro-apoptotic family members did not offer protection 1 6 3 because the deficiency in one is often compensated by an increase in the expression in the other protein Mcl-1, a pro-survival Bcl-2 family member, is critical to embryonic development since deletion of this gene results in peri-implantation embryonic lethality . Over-expression of Mcl-1 is shown to inhibit cell death induced by various apoptotic stimuli 3 2 8 , no Tin while elimination of Mcl-1 induces apoptosis . Furthermore, lymphocytes and hematopoietic stem cells lacking Mcl-1 expression undergo apoptosis and exhibit defective differentiation 3 2 7 ' 3 6 4 . We showed that extensively oxidized LDL upregulates Mcl-1 levels and that this is mediated through P I 3 K / P K B pathway. This is in accordance with reports that demonstrated that the P I 3 K / P K B pathway controlled the expression of Mcl-1 in primary human macrophages and that Mcl-1 was essential for macrophage 53 survival i b i ' i ( : i 7 . Our lab previously showed that PKB can increase Mcl-1 translation following cytokine stimulation to promote cell survival 2 1 1 . Hence it is possible this increase in Mcl-1 by oxLDL treatment may be due to increased mRNA translation and/or posttranslational stabilization of the protein. It is commonly accepted that the pro-survival proteins sequester Bak/Bax before apoptosis is induced 1 4 1 ' 1 7 2 . The anti-Mcl-1 immunoprecipitate shown in Figure 3.9B indicates that in macrophages undergoing apoptosis, less Bax is bound to Mcl-1. Immunoprecipitation with anti-Bax (Figure 3.9C) shows a similar pattern, with less Mcl-1 associated with Bax at 12 hr or 24 hr without oxLDL. OxLDL maintains a strong binding between Mcl-1 and Bax. It has been shown that along with B C 1 - X L , Mcl-1 keeps Bak in check in healthy cells 1 7 0 . We postulate that oxLDL promotes downregulation of Bax but not Bak, but that there is no change in cell viability because more Mcl-1 or Bcl-X L is available to sequester Bak. This is the first report linking the expression of Mcl-1 to oxLDL's ability to promote macrophage survival. Phosphorylation of Bim by cytokines such as M-CSF or IL3 is shown to regulate its apoptotic function by promoting its proteasomal degradation of the protein or its interaction with Bax 3 2 2 ' 3 4 3 6 8 . Certain stresses, such as UV irradiation or cytokine withdrawal, induce the release of Bim from the dynein motor complex, allowing Bim to neutralize pro-survival Bcl-2 members 1 5 1 or directly activate other pro-apoptotic proteins 3 4 1 . Although we found Bim is phosphorylated in the presence of M-CSF (Figure 3.12), we did not observe Bim interacting with Bax (Figure 3.13). Others have proposed that BH3-only proteins displace the pro-survival relatives that constrain Bax/Bak rather than directly activate them to induce apoptosis 1 7 1 . However, with the finding that the 54 association between Bim and Mcl-1 not changing during apoptosis (Figure 3.14), we concluded that Bim is unlikely to mediate macrophage apoptosis through Bax, no matter which mechanism it employs to initiate apoptosis. There are other BH3-only proteins that can induce apoptosis by neutralizing the protective function of Mcl-1 or by activating Bax/Bak. For example, besides truncated Bid (tBid) and Puma, Noxa is reported to selectively displace Mcl-1 from binding to Bak 1 6 9 ' 1 7 0 . Further studies are required to determine the exact role of Mcl-1 in oxLDL-mediated macrophage survival. In conclusion, these results support our previous findings showing that PI3K/PKB play an important role in oxLDL-mediated survival 3 8 ' 7 2 . Besides the previously shown role for PI3K/PKB in maintaining BC1-XL expression, oxLDL selectively promotes the proteasomal degradation of Bax and an increase in Mcl-1 expression, both mediated via the PI3K/PKB pathway (Figure 3.15). 55 A . 56 Figure 3.1 Bax undergoes conformational changes upon cytokine withdrawal. (A) Macrophages were cultured in the absence of cytokine for the indicated time. Cell lysates were generated with CHAPS buffer. Bax was immunoprecipitated with monoclonal antibody 6A7 which is specific for the active conformation of Bax, and immunoblotted with a polyclonal Bax antibody as described in Methods. Whole cell lysates were blotted for Bax to indicate the level of total Bax in each sample prior to immunoprecipitation. Data are representative of three independent experiments. (B) Macrophages were cultured in the absence or presence of cytokine for 24 hours. Z-VAD (lOOuM) was included to prevent caspase activation and the consequent shrinkage and detachment of cells. Cells were then immunostained for anti-active Bax 6A7 and TOM20. Data are representative of three independent experiments. 57 A. OxLDL M-CSF Bax Bak p85 B. OxLDL Bax p85 Figure 3.2 Expression of Bax is down-regulated by treatment with oxidized L D L in macrophages. (A) B M D M were cultured in the presence or absence of cytokine with or without oxLDL for 24 hours. Cell lysates were probed for total Bax, Bak and p85. Data are representative of five independent experiments. (B) THP-1 cells treated with 40nM PMA for 24 hours to induce differentiation. Then FBS was withdrawn and cells incubated with or without 25ug/ml oxLDL for 24 hour. Cells lysates were blotted for total Bax and for p85 as loading control. Data are representative of two similar experiments. 58 /"V. _ . OxidationTime (hr) - " " 2 5 2 4 Modification - Nat. Ac. Ox. Ox. Ox. LDL - + + + + + B . Ctrl Nat. Ac. Ox2 Ox5 Ox24 + LDL Figure 3.3 Only extensively oxidized L D L promotes a decrease in Bax protein. (A) BMDM were incubated for 24 h in the absence of M-CSF alone (control) or treated with native LDL (Nat.), acetyl LDL (Ac), or LDL oxidized for the indicated times. Lysates were immunoblotted for total Bax and for actin as loading control. (B) Densitometric result of immunoblots that were normalized to the loading control and expressed as a ratio of the control. The data represent means ± S.D. of at least two independent experiments. 59 Time (hr) 0 Bax Actin B. Time (min) 0 Bax Vinculin 7.5 15 Figure 3.4 OxLDL also reduces Bax levels in human macrophages. (A) Murine BMDMs were incubated with 25ug/ml of oxLDL for the indicated time. Cell lysates were immunoblotted for total Bax and actin as loading control. Data are representative of five independent experiments. (B) Macrophages derived from human peripheral blood mononuclear cells were cultured in the absence of M-CSF for 4 hours before addition of oxLDL for the indicated time. Cell lysates were immunoblotted for total Bax and for vinculin as loading control. 60 A . • OxLDL + OxLDL Time (min) Bax p85 B. - O x L D L •+OxLDL Figure 3.5 OxLDL facilitates Bax protein turn-over. (A) B M D M were pre-treated for lh with 10 ug/rnl of cyclohexamide before addition of oxLDL for the indicated time. Cell lysates were immunoblotted for total Bax and p85. Data are representative of three independent experiments. (B) Densitometric results of immunoblots expressed as a ratio of the time zero control, corrected for loading with p85. The data represent means ± S.D. of at least three experiments, except the 15 min time point is from one experiment only. *p<0.05,**p<0.005 vs. time zero. 61 A . B. Ctrl — A M L Bax U^^HWNBMW' p85 MG132 ALLN 0.1 1 3 10 Ctrl 1 5 Bax p85 4MHMI 4MMfc 4MMfe 4fcpBfc ^ HBl^ H^BP^  160% =5 140% 4 k. *-> o 120% o 100% £ 80% | 60% > > 40% H ,2 20% 0% O-OxLDL • +OxLDL No Inhibitor A L L N ALLM Figure 3.6 OxLDL induces Bax degradation via the proteasomal pathway. (A) BMDM were pre-treated with various inhibitors (A: 5 uM A L L N , M : 10 uM MG132, L: 10 uM Lactacystin) for 1 hour in the absence of M-CSF and then incubated with oxLDL for 24 hours. B M D M cultured in the absence of M-CSF served as control. Cell lysates were blotted with anti-Bax antibody and anti-p85. * shifted band indicating possible post-translational modification. Data are representative of three independent experiments. (B) Effects of MG132 and A L L N on Bax level in the presence of oxLDL. B M D M were pre-treated with varying concentrations of MG132 or A L L N for 30 minutes and then 25 \xg/m\ of oxLDL was added for 2 hours. Cell lysates were immunoblotted with anti-Bax and anti-p85 antibody. Data are representative of two independent experiments. (C) BMDM were treated without inhibitors or with A L L N or A L L M for 24 hours in the absence of M-CSF with or without the addition of oxLDL. Cell viability was expressed relative to cells cultured in the presence of M-CSF. The data represent means ±S.D. of at least three experiments done in triplicate. *p<0.005 vs. ox with no inhibitor. 62 A. +OxLDL Ctrl — ALLN LY SB UP B. Ctrl — ALLN LY SB UO + OxLDL Figure 3.7 OxLDL signals at least partially through a PI3K dependent pathway leading to Bax degradation. (A) BMDM were pre-treated with various inhibitors for one hour in the absence of M-CSF (control) before addition of oxLDL for 24 hours. 5 uM A L L N ; 20 uM LY294002; 30 uM SB203580; 10 uM U0126. Whole cell lysates were then blotted for Bax and for p85 as a loading control. Data are representative of three independent experiments. (B) Densitometric result of immunoblots expressed as a ratio of the control, corrected for loading as monitored by p85. The data represent means ± S.D. of at least three experiments. *p<0.05 vs. oxLDL alone, **p<0.01 vs. control. 63 A. • -OxLDL • +OxLDL Bax+/+ Bax-/-Figure 3.8 Bax knockout does not confer resistance to cytokine withdrawal induced cell death. (A) B M D M from Bax+/+ and Bax-/- mice were cultured in the presence of M-CSF. Cell lysates were harvested and immunoblotted for Bax, Bak and p85. Data is representative of three independent experiments. (B) B M D M were seeded at 5x10 cells/well in 96-well plates. Cells were cultured in the absence of M-CSF with or without the addition of 25 ug/ml of oxLDL for 24 hours. Cell viability was then measured with the MTS assay, and is expressed relative to cells cultured in the presence of M-CSF. The data represent means ± S.D. of five experiments done in triplicate. *p<0.005 vs. control; **p<0.001 vs. no oxLDL. 64 IP: Bcl-2 IB: CM . o 9= £ Bax Bak Bel B. IP: Mcl-1 IB: Ab Ctrl +MCSF -MCSF Bax Mcl-1 WCL Bax +MCSF -MCSF C . + OxLDL Figure 3.9 Mcl-1 but not Bcl-2 sequesters Bax and Bak. (A) B M D M were cultured in the presence of M - C S F and whole cell lysates were immunoprecipitated with anti-Bcl-2 antibodies and immunoblotted for Bax, Bak and Bcl-2. Data shown are representative of three independent experiments. (B) BMDM were cultured in the absence or presence of M - C S F for 24 hours. Whole cell lysates were blotted for Bax to check for imput and immunoprecipitated with anti-Mcl-1 antibodies and immunoblotted for Bax, and Mcl-1. Data shown are representative of three independent experiments. (C) BMDM were cultured in the absence of M - C S F with or without oxLDL for the indicated time. Whole cell lysates were immunoprecipitated with anti-Bax antibodies and immunoblotted for Mcl-1. Antibody control (ab Ctrl) represents a sample of beads incubated with the respective antibodies, in the absence of cell lysates. 65 A. Time (hr) 0 3 6 9 Mcl-1 Vinculin B. Time (min) 0 Mcl-1 P-PKB Vinculin 15 +LY 30 60 120 120 Figure 3.10 Mcl-1 level does not decrease in response to cytokine withdrawal but increases with oxLDL treatment. (A) BMDM were starved of M-CSF for the indicated time. The whole cell lysates were immunoblotted for Mcl-1 and vinculin, the loading control. Data shown are representative of three independent experiments. (B) BMDM were incubated for 2.5 hours without M-CSF. 20 uM of LY294002 was then added for 30 min, and finally oxLDL was added. After incubating with oxLDL for the indicated time, cells were lysed and blotted for Mcl-1, phospho-PKB (P-PKB) and vinculin. Data shown are representative of three independent experiments. 66 A . OxidationTime (hr) Modification LDL Mcl-1 0 2 5 24 24' -Nat. Ox. Ox. Ox. Ox. Ac. + + + + + + B. 1.8 1.6 £ 1.4 w | *3H 13 1 x £ 0.8 | °'6"I £ 0-4 0.2 0 OxLDL Ac L D L 0 2 5 24 LDL Modification Time (hr) Figure 3.11 Mcl-1 expression is preserved by treatment of extensively oxidized L D L . (A) BMDM were incubated for 4h in the absence of M-CSF alone (control) or treated with LDL modified by oxidation or acetylation for the indicated times. Two different batches of 24hr-oxidized LDL were used. Lysates were immunoblotted for total Mcl-1. Data shown are representative of two independent experiments. (B) Densitometric result of immunoblot expressed as a ratio of the control. The data represents means ± S.D. of two experiments. 67 -Appt + A ppt OxLDL M-CSF - + Figure 3.12 Bim is phosphorylated in the presence of M - C S F but not oxLDL. B M D M were cultured in the presence or absence of M-CSF +/- oxLDL for 24 hours. Cell lysates were divided into two sets. One is treated with X, phosphatase according to manufacture's instruction. Cell lysates were then run on the 10% low-bis SDS-PAGE gel before immunoblotted with anti-Bim antibodies. Data are representative of three experiments. * denotes phosphorylation of Bim. 68 IP: Bim IB: Mcl-1 +MCSF -MCSF L j ^ ^ M ^ j ^ ^ ^ — j r ^ ^ J ^ ^ ^ ^ ^ ^ ^ ^ M i ^ Bax Bim Figure 3.13 Interaction of Bim with Mcl-1 or Bax does not change during apoptosis. BMDM were cultured in the absence or presence of M-CSF for 24 hours before immunoprecipitation with anti-Bim antibodies. Then it was immunoblotted with anti-Mcl-1, Bax, Bak and Bim antibodies. Data are representative of three experiments. * denotes antibody light chains. Antibody control (ab Ctrl) represents a sample of beads incubated with the respective antibodies, in the absence of cell lysates. 69 IP: Mcl-1 Time (hr) IB: Bim Mcl-1 Figure 3.14 Mcl-1 association with Bim does not change during apoptosis. B M D M were cultured in the absence of M-CSF for the indicated time. Whole cell lysates were immunoprecipitated using anti-Mcl-1 antibodies and checked for association with Bim. Immunoprecipitation without cell lysates was used as the control (Ab C t r l ) . 70 OxLDL Y PI3K PKB Bak Figure 3.15 Proposed model of OxLDL regulation of Bcl-2 family members to mediate macrophage survival. OxLDL prevents macrophage apoptosis following M -C S F withdrawal by activating the PI3K/PKB pathway. PKB then mediates phosphorylation of IKB which marks the protein for its degradation by the proteasome and release N F K B . The freed N F K B is then available to maintain the expression of Bcl-X L . PKB is also responsible for the proteasomal degradation of Bax and the upregulation of Mcl-1. Meanwhile, oxLDL also promotes Bax sequestration by Mcl-1. The dashed lines denote the postulated effect of oxLDL. 71 4 Pertussis toxin inhibits macrophage apoptosis via the PI3K /PKB pathwayb 4.1 Introduction In previous studies using mouse peritoneal macrophages we showed that oxidized low density lipoproteins (oxLDL), or lysophosphatidylcholine (LPC), stimulated the activity of phospholipase D (PLD). This enzyme activity generates phosphatidic acid (PA) from glycerophospholipids. PA is a potent mitogenic agent and second messenger that has been implicated in various pathophysiological processes, including atherosclerosis 3 6 9 . We found that PLD activation by oxLDL or LPC was inhibited by pertussis toxin, abbreviated PTX 4 8 ' 3 7 0 ' . PTX is a protein that is secreted by Bordetella pertussis and consists of five different subunits, designated SI to S5. There are two S4 subunits, and so the complete toxin molecule is a hexamer 3 7 1 . PTX consists of an A subunit that carries the biologic activity and a B subunit that binds the complex to the cell membrane 3 7 1 . The SI subunit constitutes the A protomer and the B oligomer is formed by the remaining five subunits 3 7 1 . PTX is a member of the family of ADP-ribosylating bacterial toxins. The SI subunit of PTX ADP-ribosylates Cys352 of Gi. This modification of cysteine prevents G protein heterotrimers from interacting with receptors to block their coupling and activation. PTX has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in cell signaling processes. The inhibitory effect of PTX on PLD activation by oxLDL or LPC suggested that Gi proteins were involved in this process. b A version of this chapter has been accepted for publication. Wang, S. W. et al (2007) Pertussis toxin promotes macrophage survival through inhibition of acid sphingomyelinase and activation of the phosphoinositide 3-kinase/protein kinase B pathway. Cellular Signaling 19(8):1772-83. 72 Some of the effects of PTX can be explained through actions other than G-protein inhibition. For example, PTX has been shown to use Toll-like receptor (TLR) 4 signaling to mediate some of its pathologic effects. It facilitates the break-down of the blood-brain barrier and is widely used as an adjuvant in experimental autoimmune encephalomyelitis 372 373 . TLRs are expressed on several cell types, including cells of the immune system One action of TLR4 signaling results in translocation of NF-kB, which induces transcription of a variety of genes, including those for pro-inflammatory cytokines. Therefore, some of the effects of PTX can be explained through actions other than G-protein inhibition. In the present study, we show that PTX inhibits acid sphingomyelinase activation and the resulting accumulation of ceramides in bone marrow-derived macrophages. This is associated with inhibition of apoptosis that normally results from withdrawal of M -CSF from these cells. PTX caused phosphorylation of protein kinase B (PKB), activation of the transcription factor N F K B and up-regulation of Bcl-XL, an anti-apoptotic Bcl-2 protein. These are the same mechanisms involved in the inhibition of apoptosis by oxLDL 1 1 . 4.2 Results 4.2.1 Pertussis toxin can selectively protect macrophages from apoptosis induced by cytokine withdrawal We have previously shown that B M D M undergo apoptosis if incubated in the absence of M-CSF for 24 hours or more, and that this is blocked by incubation with oxLDL 1 1 . To test if a G-protein-coupled membrane receptor(s) might be involved in oxLDL mediated cell survival, the G-protein inhibitor PTX, was used. Unexpectedly, we 73 found that control incubations done with PTX in the absence of cytokine or oxLDL also promoted cell viability in a dose-dependent manner in macrophages (Figure 4.1 A). The concentration range of PTX used is in the same range as that reported previously for specifically inhibiting Gj/0 function in intact macrophages 3 7 4 . Furthermore, PTX treatment prevented DNA fragmentation (Figure 4. IB). This anti-apoptotic effect of PTX was cell type specific because FDCP-1, a mouse progenitor myeloid leukemia cell line whose survival is dependent on interleukin (IL)-3, did not respond to PTX (Figure 4.1). 4.2.2 Pertussis toxin inhibits ceramide generation in part by blocking acid sphingomyelinase activation after growth factor withdrawal We previously showed that when bone marrow derived macrophages were cultured in the absence of M-CSF, acid sphingomyelinase (ASMase) activity and the level of ceramide increased and the cells became apoptotic 1 2 . We therefore tested the possibility that PTX promoted cell survival by inhibiting ASMase and ceramide generation. As shown in Figure 4.2A, ASMase activity was inhibited in a dose dependent manner by PTX. Ceramide levels increased by about 2.3 ± 0.4 fold (mean ± SD, n=4) in cytokine-deprived cells, and this was also blocked by PTX. The ability of ceramide to induce cell death was examined by treating the macrophages with exogenous C 2 -ceramide. Figure 4.2B shows that C2-ceramide caused a substantial decrease in macrophage viability. In addition, C2-ceramide, but not its inactive analogue dihydro-C2-ceramide obliterated the cytoprotective effect of PTX (Figure 4.2B). This supports the notion that ASMase-derived ceramides are likely to be responsible for the induction of apoptosis in B M D M incubated in the absence of M-CSF and that inhibition of ASMase is at least part of the mechanism whereby PTX exerts its anti-apoptotic action. 74 4.2.3 Mastoparan, a Gi agonist, induces cell death very rapidly. Mastoparan, an activator of G i a subunits 3 7 5 that is found in wasp venom 3 7 6 , acts directly on PTX-sensitive G protein 3 7 7 to stimulate guanine nucleotide exchange and GTP hydrolysis by a mechanism similar to that used by surface G protein-coupled 378 receptors . We employed mastoparan to determine if G protein activation induced macrophage apoptosis. Macrophages were treated with various concentrations of mastoparan in the presence of M-CSF. As seen in Figure 4.3A, mastoparan caused a sharp decrease in macrophage viability whereas the inactive analogue, mastoparan 17, had no effect. Flow cytometry results shown in Figure 4.3B demonstrate that DNA fragmentation and annexin V binding paralleled the changes in cell viability associated with the addition of mastoparan. Furthermore, as we have previously observed with oxLDL, caspases 3/7, 8, and 9 were all activated by treatment with mastoparan (Figure 4.3C). Finally, poly(ADP-ribose) polymerase (PARP), a substrate for activated caspase 3, was cleaved in response to mastoparan treatment but not in response to the inactive analogue, mastoparan 17 (Figure 4.3D). Although mastoparan has been reported to stimulate mitogenesis in the presence of growth factors including insulin-like growth 370 factor-1, and platelet-derived growth factor , our results show that it induces apoptosis in macrophages. 75 4.2.4 Mastoparan activates ASMase in BMDM Since apoptosis in macrophages incubated in the absence of M-CSF has been shown to be closely associated with increased ceramide levels and activation of ASMase 72 , we tested to see whether apoptosis induced by mastoparan occurred through a similar mechanism. As shown in Figure 4.4A, ceramide accumulation was accompanied by ASMase activation, which was detected as early as 5 minutes after treatment with mastoparan. Ceramide levels increased over time after treatment with mastoparan but not with the inactive analogue mastoparan 17 (Figure 4.4B). 4.2.5 Pertussis toxin attenuates cell death induced by mastoparan PTX-catalyzed ADP-ribosylation of Gi alpha can markedly inhibit mastoparan-stimulated GTPase activity and some of its cellular and molecular effects ' . To examine whether PTX can overcome the pro-apoptotic effect of mastoparan, macrophages were pre-treated with PTX for 24 hours before the addition of mastoparan. ASMase activation was delayed by the addition of PTX (Figure 4.5A) and the increase in ceramide was attenuated (Figure 4.5B). Cell death after 4 or 8 hours of mastoparan treatment was prevented by PTX, as measured by DNA fragmentation. (Figure 4.5C). This further demonstrates that PTX-sensitive G-proteins play a role in macrophage apoptosis. 4.2.6 ADP-ribosylation is required for pertussis toxin to promote cell survival The A subunit of the PTX holotoxin possesses the catalytic domain responsible for ADP-ribosylation of the a subunit of the heterotrimeric Gj/0 proteins. The B-oligomer 76 subunit is thought to mediate the binding of the toxin to cells . To test if the enzymatic activity is required for PTX to provide cell survival, we compared the ability of the B-oligomer of PTX with that of the PTX holotoxin to promote survival of BMDM. As shown in Figure 4.6A, B-oligomer was unable to prevent the activation of ASMase or the increase in ceramide induced by growth factor withdrawal in macrophages. B-oligomer also did not prevent macrophage death (Figure 4.6B). These data support the conclusion that Gj/o inhibition by ADP-ribosylation is required for the anti-apoptotic effects of PTX. 4.2.7 Toll-like receptor 4 may be involved in the anti-apoptotic effect of PTX in macrophages It has been reported that TLR4 may be the receptor responsible for the effect of PTX on leukocyte recruitment 3 7 2 . We used macrophages from TLR4 deficient mice to test if TLR4 was responsible for transducing the pro-survival signal from PTX. After 24 hours of cytokine withdrawal, PTX failed to prevent apoptosis in B M D M lacking TLR4 (Figure 4.7A). Similar effects were observed after 48 hours (data not shown). PTX also did not prevent phosphatidylserine exposure, DNA fragmentation, ASMase activation or ceramide accumulation in TLR4 deficient cells (Figure 4.7B-D). This suggests that PTX acts at least partly through TLR4 to regulate ceramide production by ASMase and mediate cell survival. 77 4.2.8 Adenylyl cyclase is unlikely to contribute to macrophage apoptosis PTX inhibits the activation of Gj a ,which normally prevents the generation of cAMP by adenylyl cyclase 3 8 2 . We and others have previously demonstrated that cAMP can inhibit apoptosis induced by various stimuli 3 8 3 " 3 8 5 . To test this possibility, 8-bromp-cAMP (a membrane-permeable analog of cAMP), or forskolin (a stimulator of adenylyl cyclase), were added to B M D M incubated in the absence of M-CSF. As shown in Figure 4.8A, modulating cAMP levels did not affect B M D M survival. This indicates that the effect of PTX on apoptosis is not through its effect on adenylyl cyclase. 4.2.9 The anti-apoptotic effect of PTX requires the activation of the PI3K/PKB pathway Activation of ERK and PKB pathways has been reported to reduce apoptosis in several cell types 7 6 ' 7 7 . We previously reported that oxLDL stimulates both pathways in macrophages, but only the PI3K/PKB pathway was important for oxLDL-mediated survival ' ' . Similarly, the PI3K pathway was important for sphingosine 1-phosphate-, or ceramide 1-phosphate-mediated survival in B M D M 1 0 0 ) 5 1 . In the present study we tested to see if PTX could signal through these pathways to block apoptosis. As shown in Figure 4.8B, an increase in ERK1/2 phosphorylation was observed as early as 15 minutes after PTX treatment. However, the M E K inhibitors, U0126 and PD98095, did not significantly inhibit PTX-mediated macrophage survival (Figure 4.8C). PTX treatment caused phosphorylation of PKB at Ser473, which is a direct indication of its activation, after about 30 min of incubation (Figure 4.8D). The ability of PTX to prevent apoptosis was blocked by incubation of cells with the PI3K inhibitors, LY294002 and 78 wortmannin (Figure 4.8E). These results indicate that although PTX treatment results in the activation of both ERK1/2 and PKB, the pro-survival effect of PTX is mediated through activation of the PI3K/PKB pathway, similar to oxLDL 1 1 . 4.2 .10 Activation of N F K B is required for PTX to provide survival by regulating Bcl-XL expression The N F K B transcription factor is an important target of PKB 5 1>3 8 6. We previously demonstrated that M-CSF withdrawal results in N F K B inactivation and this is associated with decreased mRNA and protein levels of Bcl-X L 5 1 ' 1 1 . We therefore determined whether PTX could regulate macrophage survival through activation of N F K B . First, we found that PTX stimulated phosphorylation of IKB (Figure 4.9A), which precedes the activation of N F K B 3 8 7 . We then examined the ability of PTX to induce the DNA binding activity of stimulated N F K B . This was performed using nuclear extracts and electrophoretic mobility shift assay (EMSA). Figure 4.9B shows that there is minimal basal binding of N F K B to DNA in apoptotic macrophages. In contrast, N F K B binding to DNA was significantly increased after exposure of the cells to PTX. There are reports indicating that N F K B is the transcription factor for Bcl-X L 3 8 8 . Over-expression of Bcl-X L has been shown to provide protection toward apoptosis by % 8Q 79 preserving mitochondrial integrity . We previously showed that oxLDL , sphingosine 1-phosphate 1 0 0 or ceramide 1-phosphate 5 1 can all enhance BC1-XL expression. As shown in Figure 4.9C, PTX restored Bcl-X L expression to the same level as that in cells cultured in the presence of M-CSF. Taken together, these findings demonstrate that PTX is able to regulate the N F K B pathway, and the subsequent expression of anti-apoptotic BCI -XL - T O evaluate whether N F K B was required for the inhibition of apoptosis by PTX, we tested 79 the effects of selective inhibitors on cell survival in the presence of PTX. As shown in Figure 4.9D, the N F K B inhibitors caffeic acid phenylethyl ester (CAPE) or SC-514 blocked the pro-survival effect of PTX in BMDM, suggesting that activation of this transcription factor is required for the antiapoptotic effect of PTX. 4.3 Discussion There are several reports in the literature showing that some of the biological effects of oxLDL are sensitive to PTX. Whitman et al. demonstrated that uptake of acetylated LDL by macrophages is regulated by PTX-sensitive G proteins 3 9 0 . It was also reported that oxLDL-induced macrophage proliferation is mediated through a PTX-sensitive G-protein-coupled receptor 3 9 1 while another group documented that oxLDL induced cytotoxicity in vascular smooth muscle cells is mediated through PTX-sensitive G proteins 1 0 2 . Recent evidence showed that loss of a G-protein coupled receptor for lysophosphatidylcholine (LPC) resulted in macrophage accumulation in atherosclerotic lesions and that this may be mediated through the regulation of apoptosis 3 9 2 . These reports prompted this study to determine if PTX-sensitive G proteins are responsible for the inhibition of B M D M apoptosis by oxLDL. If that were the case, PTX would have induced apoptosis in cells incubated with oxLDL. The results presented here showed that PTX had the opposite effect, in that it protected B M D M from apoptosis induced by growth factor withdrawal, evidently by stimulating the same intracellular signaling pathways involved in the anti-apoptotic effect of oxLDL (Figure 4.10). In previous studies we showed that apoptosis of B M D M induced by M-CSF withdrawal involves stimulation of ASMase and ceramide accumulation 7 2 ' 1 0 ° . Activation of ASMase seems to be essential for apoptosis in macrophages because 80 BMDM obtained from ASMase knockout mice were resistant to apoptosis upon M-CSF withdrawal (Please see next section, Figure 5.1). In addition, treatment of macrophages with C1P, which is a potent inhibitor of ASMase, prevented ceramide accumulation and blocked macrophage death 1 0 1 , whereas incubation of the macrophages with the cell-permeable C2-ceramide induced apoptosis. One major finding of the present studies is that PTX inhibits ASMase activation and the subsequent accumulation of pro-apoptotic ceramide. The second important observation is that PTX stimulates the PI3K/PKB pathway, which is a major mechanism by which growth factors promote cell survival. These effects of PTX are also consistent with previous observations by Testai et al 2 8 5 who have recently demonstrated that inhibition of PI3K leads to ASMase activation in oligodendrocytes. Our results with mastoparan and B-oligomer suggest that Gi-proteins are involved in regulating ASMase activity and apoptosis in the macrophages. However, this is unlikely to be associated with an effect on the levels of intracellular cAMP since addition of 8-bromo-cAMP or stimulation of adenylyl cyclase with forskolin did not rescue macrophages from apoptosis. Some reports have suggested that ADP-ribosylation is not required for PTX to exert its function 5 6 ' m ' 3 9 4 , but others showed that this action is essential for PTX to induce intracellular signaling 3 7 2 ' 3 9 ° . In this study, we show that in BMDM, ADP ribosylation of G; proteins is required for PTX to provide macrophage survival, since the B oligomer of PTX was unable to mediate the same effects as intact PTX. We also show that PTX activates the PKB target N F K B , supporting our previous observation that PI3K is an important regulator of N F K B activation in B M D M 7 2 > 1 0 0 ' 1 0 1 . Moreover, we found 81 that PTX upregulated Bcl-X L, which is a downstream target of N F K B . This parallels the effects of oxidized LDL, sphingosine 1-phosphate, or C1P in BMDM, all of which cause upregulation of Bcl-X L via activation of PI3K/PKB and inhibition of ASMase 5 1 > 1 0 0 ' 1 0 1 . Inhibition of N F K B activation by the selective inhibitors CAPE or SC-514 abolished the anti-apoptotic effect of PTX suggesting that N F K B is required for PTX-mediated survival of macrophages. The above results provide the first evidence for a novel biological effect of PTX in the control of cell survival through inhibition of ASMase and stimulation of the P I 3 K / P K B / N F K B pathway in macrophages. It has been reported that PTX can trigger a tyrosine kinase signaling cascade in myelomonocytic cells 395.There is evidence showing that tyrosine-kinase receptors and G proteins may converge on a common effector(s) necessary for the regulation of macrophage survival ' . This may be one of the mechanisms by which PTX signals to inhibit B M D M apoptosis. Another mechanism by which PTX might promote macrophage survival is through activation of TLR4 receptors. In fact, PTX has been found to co-immunoprecipitate with CD 14 3 9 8 , which mediates the binding of lipopolysaccharide (LPS) to initiate signaling through TLR4. Also, it has been demonstrated that G; proteins are coupled to the TLR4 signaling pathway in RAW264.7 cells , and that TLR4 is a receptor for PTX in nervous system autoimmune disease In the latter study, it was demonstrated that leukocyte recruitment induced by PTX is TLR4 dependent. PTX also induces dendritic cell maturation in a TLR4-dependent manner 5 6 , and TLR4 stimulation activates N F K B through the PI3K/PKB pathway 3 9 9 . Our results are consistent with these observations in suggesting a cross-talk between TLR4 and Gi-protein signaling in the promotion of cell survival by PTX. 82 Recently a paper was published showing that ceramide activates TLR4 signaling, suggesting that this mechanism might allow pathogens to elicit TLR4 responses by perturbing sphingolipid receptors for virulence ligands 4 0 0 . LPS, a ligand for TLR4, is structurally similar to ceramide and stimulates some ceramide targets 4 0 1 . The fact that ceramide, and perhaps other related sphingolipids, can act as ligands for TLR4 adds a new dimension to the regulation of signal transduction processes by sphingolipids through toll-like receptors. However, results in Figure 4.7 suggest that at least under the conditions we used, TLR4 is not required for apoptosis or survival of B M D M in the absence or presence of MCSF. 83 BMDM a > FDCP-1 -BMDM -FDGP-1 +M-CSF 0.4 0.6 0.8 PTX (ug/ml) -MCSF -MCSF+PTX 58.4% +IL3 -IL3 -IL3+PTX 75.9% 79.2% Sub-dipolid DNA Figure 4.1 Pertussis toxin selectively protects macrophages apoptosis. (A) B M D M were seeded at 5xl04cells/well and FDCP-1 at 2xl04cells/well in 96-well plates. Cells were cultured in the absence of growth factors, but with PTX at indicated concentrations for 24 hours before adding MTS. Cell survival in the presence of M-CSF or IL-3 respectively was the reference for 100% survival. Data represent means ± SD of triplicate samples. Similar results were obtained in two replicate experiments. (B) B M D M or FDCP-1 cells were incubated in the presence or absence of respective growth factors with or without 0.5ug/ml of PTX for 24 hours. Cells were then stained for sub-diploid DNA with propidium iodide (PI) and analyzed by flow cytometry. 84 A 0:4 : r , , , 1 Q 0.1 0.2 0.3 0.4 0.5 PTX(ug/ml) B 160% -I Figure 4.2 Pertussis toxin can inhibit ASMase activation and exogenous ceramide blocks the anti-apoptotic effect of pertussis toxin. (A) B M D M were cultured in the absence of M-CSF and with the indicated concentration of PTX for 24 hours. Lysates were assayed for ASMase activity. Results are representative of three experiments. (B) B M D M were cultured for 24 hours in the absence of M-CSF with or without 0.5ug/ml of PTX together with the indicated concentration of C2-ceramide or dihydro-C2-ceramide before the addition of MTS. Cell survival in the presence of M-CSF (control) was the reference for 100% survival. Data represent means ± SD of three experiments each in triplicate. *p<0.005 versus PTX alone. 85 140% -Mastoparan 17 - Mastoparan 10 15 Pnhibitor](uM) 90 80 -I DAnnex in V Ul: 70 -60 -g; so -w : 40 o: 20 10 0 I PI 2 Time (hr) II Caspases 3/7 LD Control Mastoparan aPARP 0.5% 16.2% M M17 Uncleaved Cleaved Caspase Activation; 86 Figure 4.3 Mastoparan induces apoptosis in macrophages. (A) B M D M were plated at 5xl04cells/well and incubated overnight. Mastoparan or mastoparan 17 was added in the presence of M-CSF and incubated for 24 hours before adding MTS. Cell survival with only M-CSF was the reference for 100% survival. Data represent means ± SD of three independent experiments performed in triplicate. *p<0.005 versus mastoparan 17. (B) BMDM were treated with 20uM mastoparan in the presence of M-CSF for the indicated time. Cells were then stained for sub-diploid DNA and for phosphatidylserine exposure. Results are representative of two experiments. (C) B M D M were incubated with or without 20uM of mastoparan,for 4 hours, stained for activation of caspases 3/7, 8, or 9 and analyzed by flow cytometry. Results shown are representative of two experiments. (D) B M D M were cultured with 20uM of mastoparan or mastoparan 17 for 4 hours. Lysates were immunoblotted for PARP. Results are representative of two experiments. 87 A 3 - + Mastoparan --Mastoparan 100 200 Time (min) 300 400 B - M a s t o p a r a n - M a s t o p a r a n 17 Figure 4.4 Mastoparan activates ASMase and increases ceramide levels in macrophages. (A) B M D M were incubated in medium containing M-CSF with or without 20uM of mastoparan, lysed by three cycles of freeze/thawing and assayed for ASMase. Results are means ± SD of two experiments, except the value at 5 minutes, which is the mean of three experiments. *p<0.01 versus zero minutes. (B) B M D M pre-labeled with [3H]palmitate were treated with 20uM mastoparan or mastoparan 17 in medium containing M-CSF and ceramide levels were then determined. Data are means ± SD of two experiments done in duplicate. Results are the mean ± SD of two experiments. 88 A 6 5 :•> 0 -I : i , , , , 1. 0 50 100 150 200 250 300 Time (min) C 16 Time (hr) 89 Figure 4.5 Pertussis toxin confers partial resistance to mastoparan-induced cell death. (A) B M D M were pre-incubated with lug/ml PTX in the presence of M-CSF for 24 hours before the addition of 20uM of mastoparan. ASMase activity was then determined. Results are relative to time zero of mastoparan addition and are means ± SD of two experiments. (B) Cells were treated as in (A), and ceramide levels were determined. Results are relative to time zero of mastoparan addition and are means ± SD of three experiments done in duplicate. *p<0.05, **p<0.01 versus PTX treated at the respective times. (C) Cells were treated as in (A) and analyzed for sub-diploid DNA by PI staining and flow cytometry. Results are representative of two experiments. 90 -MCS'F -MCSF+PTX -MCSF+B-ol igo -MCSF X -MCSF+PTX; • Cell viability • DNA fragmentation X I -MCSF+B-oligo 91 Figure 4.6 Enzymatic activity of PTX is required for inhibition of apoptosis. (A) BMDM were treated with 0.5|ig/ml of PTX holotoxin or B-oligomer in the absence of M-CSF for 24 hours. Results for ASMase activity (open bars) and ceramide levels (solid bars) are expressed relative to cells incubated with M-CSF and are means ± SD of two (ceramide) or three (ASMase) experiments. **p<0.01 versus PTX treated. (B) For viability measurements, B M D M were seeded in 96-well plates and then incubated for 24 hours in the absence of M-CSF with 0.5ug/ml of PTX holotoxin or B-oligomer before adding MTS. Cell survival in the presence of M-CSF was the reference for 100% survival. Data represent means ± SD. Results are representative of two similar experiments performed in triplicate. For measuring cell death by DNA fragmentation, cells were treated as in (A), stained with PI and analyzed by flow cytometry. Data represent means ± SD of three experiments. *p<0.05 versus PTX. 92 93 Figure 4.7 Pertussis toxin may signal through the TLR4 receptor to block apoptosis. (A) For viability measurements, B M D M were incubated with 0.5(a.g/ml of PTX in the absence of M-CSF for 24 hours before the addition of MTS. Cell survival in the presence of M-CSF was the reference for 100% survival. Data represent means ± SD of two experiments performed in triplicate. (B and C) B M D M from TLR4+/+ and TLR4-/- mice were incubated with 0.5(ig/ml of PTX in the absence of M-CSF for 24 hours and then stained for phosphatidylserine exposure (B) or analyzed for sub-diploid DNA by flow cytometry (C). Results are representative of two similar experiments. (D) Macrophages were treated with 0.5ug/ml of PTX in the absence of M-CSF for 24 hours. Cells incubated with M-CSF were used as controls (CTRL). ASMase activity (D) or ceramide levels (E) were then determined. Results are expressed relative to control cells and are means ± SD of two (D) or three (E) experiments each in duplicate. 95 cAMP cAMP cAMP FK1 uM F K I O u M 50uM 100uM 200uM B Time (min) 0 15 30 45 120+MCSF MCSF- PTX PTX+UO PTX+PD D Time (min) 0 30 60 120 240 P-PKB | -• ~ ~ - ~ — | Actin MWHGjft ,nii Mn-ir —**• I -MCSF PTX PTX+LY 96 Figure 4.8 PKB is the major pathway required for the anti-apoptotic effect of PTX. (A) B M D M were placed in M-CSF-deficient medium containing 0.5 ug/ml of PTX with or without the indicated concentration of 8-bromo-cAMP (cAMP) or forskolin (FK) for 24 hours before addition of MTS. Cell survival in the presence of M-CSF was the reference for 100% survival. Data represent means ± SD of triplicates. Similar results were obtained in each of two replicate experiments. (B) B M D M were deprived of M -CSF for 4 hours before stimulation with 0.5ug/ml PTX for the indicated time. Cell lysates were blotted for phospho-ERKl/2 and actin as loading control. Results shown are representative of two experiments. (C) B M D M in M-CSF-deficient medium were treated with 0.5ug/ml of PTX with or without 2uM U0126 or lOuM PD98095 for 24 hours before addition of MTS. Cell survival in the presence of M-CSF was the reference for 100% survival. Data represent means ± SD of three experiments performed in triplicate. (D B M D M were deprived of M-CSF for 4 hours before addition of 0.5p.g/ml of PTX for the indicated time. Cell lysates were blotted for phospho-PKB and actin as loading control. Results shown are representative of two experiments. (E) B M D M in M-CSF-deficient medium were treated with 0.5ng/ml of PTX with or without 5uM LY294002 or lOOnM wortmannin for 24 hours before addition of MTS. Cell survival in the presence of M-CSF was the reference for 100% survival. Data represent means ± SD of three experiments performed in triplicate. *p<0.001 versus PTX alone. 97 B Time(min) 0 2 P - I K B p85 PTX(ug/ml) M C S F a B c l - X L a Actin 5 10 20 0.05 0.1 0.5 1 Probe PTX - + -MCSF PTX PTX+SC PTX+CAPE 98 Figure 4.9 Pertussis toxin signals through N F K B to mediate macrophage survival. (A) BMDM were deprived of M-CSF for 4 hours and then treated with 0.5ug/ml of PTX for the indicated time. Cell lysates were blotted for phospho-IicB and p85, as loading control. Similar results were obtained in each of three experiments. (B) Nuclear N F K B DNA binding activity was determined by EMSA after stimulation of B M D M for 6 hours with or without 0.5ug/ml PTX in the absence of M-CSF. Similar results were obtained in two experiments. (C) Macrophages were cultured in the presence or absence of M-CSF with the indicated concentration of PTX for 30 hours. Lysates were immunoblotted for BC1-XL, or for actin as loading control. Results shown are representative of two experiments. (D) B M D M were treated with 0.5ug/ml of PTX with or without SC514 or CAPE for 24 hours before addition of MTS. Cell survival in the presence of M-CSF was the reference for 100% survival. Data represent means ± SD of three experiments performed in triplicate. *p<0.001 versus PTX alone. 99 PTX oxLDL Figure 4.10 A working model of PTX induced macrophage survival. Like oxLDL, PTX prevents macrophage apoptosis following M-CSF withdrawal by at least two primary mechanisms. One is by activating the PI3K/PKB pathway. PKB-mediated phosphorylation of IKB leads to the release and activation of N F K B . This maintains Bcl-X L expression, which leads to the inhibition of the caspase cascade and subsequent apoptosis. The second one is by inhibiting ASMase, thereby preventing ceramide generation, a process mediated through Gy0 proteins. PTX also acts through TLR4 on unidentified targets to regulate apoptosis. 100 5 Regulation of ceramide generation during macrophage apoptosis 5.1 Introduction Ceramide is a key mediator of apoptosis triggered by various stimuli such as ionizing radiation, TNFa, chemotherapeutic agents and Fas ligand 229>402>403. Ceramide is thought to induce apoptosis either as a second messenger or by modulating membrane structure and dynamics. Some evidence suggests that ceramide may mediate raft clustering into macrodomains for transmembrane signaling, or alternatively, it may promote mitochondrial membrane permeability and channel formation for cytochrome c release 1 9 1 . Ceramide can be generated from the hydrolysis of sphingomyelin (SM) via the activity of sphingomyelinase (SMase). Acid sphingomyelinase (ASMase) is a lysosomal enzyme that belongs to a family that also includes neutral and alkaline SMases 1 9 6 . Deficient ASMase activity is the cause of human type A and B Niemann-Pick disease (NPD) in which SM degradation is impaired 3 0 1 . It has been shown that ASMase activity is essential for ceramide-mediated apoptosis. For example, cells from NPD patients or ASMase-/- mice were resistant to ionizing radiation with regard to ceramide generation and apoptosis 3 0 2 - m - 4 0 5 m Furthermore, ASMase-/- mice also showed defects in ceramide generation and apoptosis in lung endothelium 4 0 4 and throughout the central nervous system 4 0 6 . Interestingly, thymic cells from ASMase-/- mice remain sensitive to apoptosis induced by ionizing radiation 4 0 4 . Ceramide can also be produced from the de novo synthesis pathway regulated by enzymes such as serine palmitoyltransferase (SPT) and ceramide synthase (CS). SPT catalyzes the condensation of serine and palmitoyl-CoA while CS acylates sphinganine 101 to produce dihydroceramide, which is then desaturated to give ceramide. De novo synthesis of ceramide has been implicated in responses to TNFa 2 3 2 , heat shock 4 0 7 , exogenous ceramide 4 0 8 , and several chemotherapeutic agents 2 0 9 ' 2 1 0 . Recently, we showed that ASMase activation and ceramide generation were involved in apoptosis of bone marrow-derived macrophages (BMDM) induced by growth factor withdrawal 7 2 . To further investigate ceramide production in cells undergoing apoptosis, we used B M D M generated from ASMase-/- mice. In the present study we demonstrate that loss of ASMase confers partial resistance to apoptosis, with less ceramide being generated in response to growth factor withdrawal. In addition, the de novo pathway of ceramide synthesis is implicated in the accumulation of ceramide in BMDM undergoing apoptosis. 5.2 Results 5.2.1 ASMase is only partly responsible for ceramide generated in response to M-CSF withdrawal in BMDM We have previously shown that increased ceramide production following M-CSF withdrawal was due to the activity of ASMase 7 2 . Inhibition of ASMase activity by oxLDL or desipramine increased cell viability 7 2 . To further elucidate the role ASMase plays in the B M D M apoptosis, we used ASMase knockout mice generated in Dr. R. Kolesnick's laboratory 4 0 9 . As one would predict, in ASMase knockout cells, the increase in ceramide content as well as the cell death resulting from cytokine withdrawal were less than that in wild type cells (Figure 5.1). ASMase-/- BMDMs also showed less caspase 9 activation in response to M-CSF withdrawal (Figure 5.2B), and were partially protected from apoptosis as reflected by DNA fragmentation (Figure 5.2A). 102 5.2.2 Ceramide generation in ASMase-/- cells is unlikely to arise from degradation of sphingomyelin It is noteworthy that in ASMase -/- cells, ceramide generation was only partially blocked even though these cells were expected to be totally deficient in ASMase. To verify that ASMase activity was undetectable in the knockout cells, we performed in vitro assays of SMase activity in cell lysates. Results in Figure 5.3A confirm that ASMase activity in wild type cells increased in response to M-CSF withdrawal while this activity was absent in ASMase-/- cells. Ceramide generation in response to stimuli such as TNF has been attributed to NSMase activity 4 1 °, and therefore we also assayed NSMase in the lysates. However, as seen in Figure 5.3B, NSMase activity in ASMase-/-cells is low and actually decreases in response to cytokine withdrawal so it is unlikely to account for the ceramide generation by means of SM hydrolysis in ASMase knockout cells. 5.2.3 Accumulation of ceramide mass from de novo synthesis upon M-CSF withdrawal As we failed to find an increase in SMase activity in ASMase -/- macrophages after M-CSF deprivation, the obvious alternative explanation for increased ceramide radioactivity was that ceramide synthesis was increased. Ceramide production in response to death stimuli such as daunorubicin 2 1 0 , etoposide 2 0 9 , heat shock 4 0 7 or photodynamic therapy 2 1 5 has been reported to be due to accelerated de novo synthesis, not increased SM degradation. As demonstrated in Figure 5.4, ceramide mass increased rapidly after M-CSF withdrawal. Fumonisin B l (FBI), an inhibitor of ceramide synthase, blocked the accumulation of ceramide after M-CSF withdrawal. 103 Congruent results were obtained when [3H] palmitoyl-ceramide was monitored. Incubation of FBI in the absence of M-CSF almost completely abolished the increase in [3H] palmitoyl-ceramide in ASMase-/- B M D M (Figure 5.5). Incubation with SPT inhibitor, myriocin (Myr) also inhibited M-CSF withdrawal-induced ceramide increase in ASMase-/- BMDM. Both inhibitors also reduced the [3H] palmitoyl-ceramide accumulation in wild type B M D M although not to the level seen in control cells incubated with M-CSF. The residual ceramide in these cells likely reflects the action of ASMase. 5.2.4 De novo production of ceramide is not dependent on serine palmitoyltransferase (SPT) but ceramide synthase (CS) activities The findings that the production of ceramide upon cytokine withdrawal can be attenuated by inhibitors of SPT and CS led us to investigate the activities of SPT and CS. SPT is the rate limiting enzyme for de novo ceramide synthesis 2 0 8 and its activity has been shown to be required for ceramide increase during etoposide-induced apoptosis 2 0 9 . However, as shown in Figure 5.6, there was no change in SPT activity in B M D M at the end of 24- hour incubation in response to cytokine withdrawal and only a small increase in CS activity was observed. A possible explanation for this unexpected result is presented in the Discussion. 5.2.5 Ceramide-l-phosphate inhibits ceramide generation despite the absence of ASMase We previously showed that ceramide-l-phosphate (C1P) can inhibit ceramide generation observed in B M D M when M-CSF is absent 1 0 1 . Exogenous C1P inhibited 104 ASMase in BMDMs at concentrations that also prevented apoptosis 1 0 1 . . To determine whether C1P also inhibits de novo ceramide synthesis, we treated both ASMase+/+ and ASMase-/- B M D M with C1P. Ceramide accumulation was dramatically inhibited (Figure 5.7A). C1P also rescued cells from cytokine apoptosis after M-CSF withdrawal (Figure 5.7B). 5.3 Discussion In this study, we have shown that in the absence of ASMase, B M D M generated less ceramide and were partially resistant to apoptosis after M-CSF withdrawal. However, there was still ceramide generation even though we confirmed there was no ASMase and a decrease in NSMase activity in ASMase-/- cells. We concluded that the ceramide accumulation was likely due to de novo synthesis in these cells. Our observation that the resistance to apoptosis in ASMase-/- was not 100% (Figure 5.1) is probably because the role of ASMase in apoptosis is dependent upon the type of stress and may also be cell type specific. This is supported by the observation of Lozano et al that ASMase-/- murine embryonic fibroblasts (MEFs) were completely protected from radiation-induced apoptosis but only partially resistant to low serum induced cell death and offered no protection to staurosporine treatment 4 1 ' . Moreover, ASMase is essential for chemotherapy-induced apoptosis in oocytes 3 3 4 but not required for testicular ceramide production or for the ability of the germ cells to undergo apoptosis 412 Without significant SMase activity in ASMase-/- cells, it is unlikely the ceramide accumulation is from SM hydrolysis. Using inhibitors for SPT and CS, FB1 and Myr, we confirmed that the de novo synthesis pathway also contributed to ceramide generation 105 upon cytokine withdrawal (Figures 5.4B & 5.5). However, results to measure SPT and CS activities at the end of the 24 h incubation were inconclusive, as the changes seemed insufficient to explain the observed increase in ceramide. There are reports demonstrating that the changes in ceramide level can be biphasic in certain conditions 2 3 4> 4 1 3. Sumitomo et al 2 3 3 reported that etoposide induced early ceramide increase was due to the transient and rapid activation of de novo pathway while the ceramide level was sustained in the longer term by the activity of SMase. Thus it is possible that CS activity increased transiently in our model but we only measured a mild increase after 24 hours of cytokine withdrawal. This may also explain our observation that FBI was able to significantly block ceramide mass accumulation within 6 hour as in Figure 5.5B but only attenuate ceramide generation in the ASMase+/+ at 24 hour as in Figure 5.4. Similarly, SPT activity was observed to be activated within 15 minutes by etoposide treatment in Molt-4 cells 2 0 9 . Although we did not observe a change in SPT activity (Figure 5.6), its inhibitor, myriocin reduced incorporation of [3H] palmitate into ceramide (Figure 5.5). Hence, it is possible that SPT as well as CS were transiently activated in the first few hours. Under normal pathways of sphingolipid synthesis, ceramide is considered an intermediate rather than an end product. It serves as a precursor for assembly of more complex sphingolipids such as sphingomyelin and glucosylceramide. Ceramide may accumulate if its conversion to complex sphingolipids is blocked, for example by inhibition of sphingomyelin synthase (SMS) and glucosylceramide synthase (GCS). Interestingly, ceramide itself is reported to inhibit SMS 4 1 4' . Whereas SPT and CS reside on the endoplasmic reticulum 4 1 5 , SMS and GCS are located on the Golgi apparatus and/or plasma membrane 4 1 6> 4 1 7. Therefore another regulatory point for accumulation of 106 newly synthesized ceramide during apoptosis in B M D M may be at the level of ceramide transport. C1P was able to promote cell survival and block the ceramide generation despite the absence of ASMase (Figure 5.7). In addition to its effect on inhibiting ASMase activity in B M D M 1 0 1 , C1P also stimulates PI3K to phosphorylate PKB to promote survival 5 1 . It was demonstrated that besides inhibition of ASMase, PI3K can also activate GCS and SMS to reduce ceramide production 4 1 8 . It is reasonable to expect that in the absence of ASMase, C1P still activates PI3K/PKB and possibly GCS and SMS to reduce ceramide production. Ceramide synthesis and metabolism is a complex process. Besides the enzymes discussed here, there are still many regulatory enzymes that might be involved in modulating the concentration of this molecule. For example, cytokine withdrawal may activate ceramide kinase to produce C1P, which was shown by us and others to be pro-survival 5 1 ' 1 0 1 . A recent report demonstrated the involvement of dihydroceramide desaturase in cell cycle progression 4 1 9 . Another potential regulator is sphingosine kinase which generates the mitogenic metabolite sphingosine-1-phosphate that can inhibit ceramide production and block apoptosis in BMDM upon M-CSF withdrawal 1 0 ° . These additional effects could work in concert with ASMase to regulate ceramide levels and cell survival. For example, a recent observation showed that CS activation depended on ceramide generated by ASMase activity 2 3 4 . Therefore, although the individual contributions of either pathway of ceramide generation may vary with cell type, they appear to play complementary roles. 107 ASM+/+ A S M - / -Figure 5.1 A S M deficiency confers partial resistance to cytokine withdrawal-induced apoptosis and ceramide increase. (A) ASMase +/+ and ASMase-/- B M D M were incubated with [3H]palmitate and without M-CSF for 24 hours. Cells labeled in the presence of M-CSF served as the control. Ceramide was then isolated by TLC and counted. Radioactivity in ceramide relative to that in control cells was then calculated. Data are means ± SD of six independent experiments done in duplicate (dotted bars). BMDM from ASMase +/+ and ASMase-/- were seeded at 5xl04cells/well and in 96-well plates overnight to allow cells to adhere. Cells were cultured in the absence of growth factors for 24 hours before adding MTS. Cell survival in the presence of the respective cytokine was the reference for 100% survival. Data represent means ± SD of three experiments performed in triplicate (hatched line bars). *p<0.05, **p<0.01 vs. ASM+/+ cells. 108 ASM+/+ ASM-/-Sub-diploid DNA CTRL M-CSF-ASM+/+ 77.3% 1 1 1 , , u | i I ASM-/-Caspase 9 activation: CTRL M-CSF-Figure 5.2 ASM deficiency confers partial resistance to cytokine withdrawal induced DNA fragmentation and caspase 9 activation. (A) B M D M from ASMase +/+ and ASMase-/- were incubated in the presence or absence of M-CSF for 24 hours. Cells were then stained for sub-diploid DNA with propidium iodide (PI) and analyzed by flow cytometry as described in Materials and Methods. (B) B M D M from ASMase +/+ and ASMase-/- were incubated with or without M-CSF for 24 hours, stained for activation of caspase 9 and analyzed by flow cytometry. Results shown are representative of two independent experiments. 109 A. Figure 5.3 Ceramide generated in ASMase -/- BMDM is not due to SM hydrolysis. B M D M were cultured in the presence or absence of M-CSF for 24 hours. Lysates were assayed for ASMase (A) and NSMase (B) activity as described in Materials and Methods. Results were means ± SD of at least three independent experiments. *p<0.05 vs. control in ASM+/+ cells; **p<0.01 vs. ASM+/+ cells. 110 A. Time(hr) B. 4.5 -I o I— 0> 4 -N CD 3.5 -£ *-> 3 -o > 2.5 -2 -1.5 -TJ I 1 < 2 0.5 -o 0 C 6 6 9 9 24 24 • C1P* — Origin •CTRL •FB1 10 15 Time (hr) 20 25 Figure 5.4 Time course for the change in ceramide mass after M-CSF withdrawal. ASMase+/+ B M D M were cultured without M-CSF for 0 to 24 hours. At the indicated times, the cells were harvested, and ceramide mass was quantified using the diglyceride kinase assay and normalized to lipid phosphate. 32P-labeled lipids were separated by TLC and quantified with a phosphorimager. (A) shows a scan from one of three independent experiments. (B) shows means ± SD of three experiments done in duplicate, except 24 hour time point is of two experiments and FBI is of one experiment in duplicate. *p<0.05, **p<0.01 vs. time zero. I l l 3 o o o •ASM+/+ • ASM- / -N Myr FB1 Figure 5.5 Inhibitors of the de novo ceramide synthesis pathway are able to block ceramide production in ASMase-/- BMDM. ASMase +/+ and ASMase-/- BMDM were incubated with [3H]palmitate and without M-CSF for 24 hours in the absence or presence of 100 nM Myr or 5OuM FBI. Ceramide was then isolated by TLC and counted. Cells incubated in the presence of M-CSF served as control. Radioactivity in ceramide relative to that in control cells was then calculated. Data are means ± SD of three independent experiments done in duplicate. *p<0.05, **p<0.01 vs. control. 112 2 • SPT • C S ASM+/+ A S M - / -Figure 5.6 SPT is unlikely to be the enzyme responsible for the de novo synthesized ceramide during macrophage apoptosis. ASMase +/+ and ASMase-/- B M D M were cultured in the absence or presence of M-CSF for 24 hours. B M D M cultured in the presence of M-CSF are used as control. The microsomes were isolated and used to determine the in vitro SPT and CS activity as described in materials and methods. Data are expressed as fold change in the absence of MCSF relative to control and as means ± SD of five and three independent experiments for SPT and CS respectively.*p<0.05 vs. control. 113 A. • M C S F -• C 1 P ASM+/+ ASM-/ -160 • M C S F -• C 1 P ASM+/+ ASM- / -Figure 5.7 C1P can inhibit the ceramide generation and promote cell survival independent of ASMase. (A) For ceramide level, ASMase +/+ and ASMase-/- B M D M were labeled with [3H]palmitate when M-CSF was withdrawn in the absence or presence of 30 uM C1P for 24 hours. Cells labeled in the presence with M-CSF served as control cells. Ceramide was then isolated by TLC and counted. Data are means ± SD of three independent experiments done in duplicate.*p<0.05 vs. MCSF-. (B) B M D M from ASMase +/+ and ASMase-/- were seeded at 5xl04cells/well and in 96-well plates overnight to allow cells to adhere. Cells were cultured in the absence of growth factors with or without 30 uM C1P for 24 hours before adding MTS. Cell survival in the presence of the respective cytokine was the reference for 100% survival. Data represent means ± SD of four experiments performed in triplicate (closed bars). *p<0.01, vs. MCSF-. 114 Serine + Palmitoyl-CoA Serine Palmitoyltransferase (SPT) Growth factor withdrawal Ceramice-1 j-phosphate KPI3K; 3Ketosphingan ne Sphinganine ER Sphingomyelin Ceramide Synthase (CS! Dihydroceramide wi: Sphingomyelin Synthase (SMS; Dihydroceramide Desaturase Sphingomyelinase (SMase! ->• Ceramide''.— ' Glucosyl ceramide synthase (GCS) ->• Glucosylceramicles i Golgi/ Lysosome/ sPlasmairriembrarie Ceramide synthase Ceramidase SSpirigoishre:;^^ iSy'X'^ *• Sphingosine kinase : Spingosine-1 -phosphate Figure 5.8 A working model of ceramide generation pathways in response to cytokine withdrawal. Ceramide generation induced by cytokine withdrawal can be regulated by the activation of sphingomyelinase. It can also be regulated by increasing de novo synthesis of ceramide. It is also possible ceramide accumulation is due to decrease synthesis of complex sphingolipids by inhibition of SMS and/or GCS activities. All these enzymatic activities may be controlled by PI3K. 115 6 Summary At the outset of my studies, the aim was to investigate the events that regulated apoptosis of B M D M following cytokine starvation, as well as the survival of BMDM in the presence of oxidized LDL. In particular, the focus was on the family of proteins that are key to the regulation of the intrinsic pathway of apoptosis, the Bcl-2 family of proteins. An initial hypothesis pursued was that incubation with oxLDL may regulate the expression of multiple members of this family. In fact, we found that oxLDL induced Bax degradation as well as increased Mcl-1 expression in B M D M to promote cell survival. Both of these effects were mediated through the PI3K/PKB pathway. A recent model proposed that BH3-only proteins promote apoptosis by displacing pro-survival Bcl-2 family members resulting in the release of Bax and Bak. Since we did not observe any change in the interaction between Bim and Mcl-1, it is possible that Bim may act to displace other pro-survival family members to cause the release of Bax and Bak. Because Bcl-2 was not found to interact with Bax and Bak, BC1-XL may be a candidate to explore, especially since we showed that B c l - X L is involved in oxLDL mediated macrophage survival against cytokine withdrawal induced apoptosis 1 2 . Other BH3-only proteins, such as Bad or Noxa may also play a role in macrophage apoptosis. In U V induced apoptosis, Bad was shown to displace BC1-XL while Noxa was shown to bind to Mcl-1, allowing Bak to be free to induce apoptosis 1 7 0 . OxLDL may promote macrophage survival by regulating these proteins and their interactions. Regulation of Mcl-1 level by oxLDL is also worth exploring for therapeutic purpose in atherosclerosis. In cancer therapy, several reports have pointed out that the Mcl-1 level in cancer cell lines is the determinant of the efficacy of the BH3 mimetic 116 chemotherapeutic agent, ABT-737 1 8 8 > 4 2 0 . OxLDL may regulate Mcl-1 levels at the translational level by facilitating its translation. It may also increase the stability of Mcl-1 proteins. It was recently demonstrated that a BH3 like protein, M U L E , is able to bind to Mcl-1 and promote its degradation 4 2 1 . It would be interesting to see which mechanism oxLDL employs to regulate Mcl-1 level. Next, a collaborative study with Dr. Anton Gomez-Munoz followed up on an unexpected observation with PTX in BMDM. In the initial experiment, we used PTX to test if part of the survival effect of oxLDL involved G-protein coupled signalling. This hypothesis appeared to be incorrected, as incubation of PTX with B M D M in the absence of serum or oxLDL promoted cell survival. In this thesis, I demonstrated that this effect of PTX was mediated through the PI3K/PKB signalling cascade, Gj proteins and TLR4. Finally, our laboratory has been interested in a number of aspects of sphingomyelin and ceramide metabolism. We were able to obtain ASMase-deficient mice, from which we isolated BMDM. These macrophages had enhanced survival compared to wild-type BMDM, probably because they can not generate ceramide from sphingomyelin when starved of cytokine. However, the ASMase-deficient cells still generated some ceramide under these conditions, due to an increase in ceramide synthesis. Experiments in these cells allowed us to demonstrate that ASMase and de novo ceramide synthesis work in concert to regulate ceramide generation during macrophage apoptosis. This was important because apoptosis induced by some other stimuli was abrogated in cells from the ASMase-/- mice 4 1 1 . While we showed the enzymatic inhibitors for SPT and CS can reduce ceramide generation in response to cytokine withdrawal, in vitro enzymatic activities after 24 hour 117 withdrawal were not impressive. We propose this may be due to the transient activation of these enzymes. A shorter time course to probe for transient activation of these enzymes should be investigated. Enzymes downstream of the de novo pathway for synthesis of complex sphingolipids, such as GCS and/or SMS may also be involved in macrophage apoptosis (Figure 5.8). Their activities may be reduced during apoptosis and therefore contribute to the increased ceramide levels. Furthermore, like ASMase, oxLDL may also be able to regulate enzymes in the de novo pathway to inhibit ceramide generation to promote survival and this regulation may also be under the control of the PI3K/PKB pathway. It is interesting how various agnoists can contribute to macrophage survival through different mechanisms. OxLDL is able to do so by down-regulating the pro-apoptotic Bax and increasing the anti-apoptotic Mcl-1 expression. PTX is able to promote survival not only through GPCR but also partially through TLR4. C1P is able to promote survival through the inhibition of ceramide generation even in the absence of ASMase. Each of these individual events cannot completely explain the physiological response of the macrophage, but PI3K/PKB seems to play a central role in regulating all these events. In the future, being able to quantify the importance of each event and how PI3K/PKB can selectively control the outcome of macrophage apoptosis versus survival will provide a valuable tool for diseases intervention such as for atherosclerosis. 118 7 Bibliography 1. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W, Jr., Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. 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