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UBC Theses and Dissertations

Regulation of glycogen synthase kinase-3 (GSK-3) and β-catenin by CD40 in B lymphocytes Biagioni, Bradly Joseph 2004

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R E G U L A T I O N O F G L Y C O G E N S Y N T H A S E K I N A S E - 3 (GSK-3) A N D | 3 - C A T E N I N B Y C D 4 0 IN B L Y M P H O C Y T E S B y B R A D L Y J O S E P H B I A G I O N I B . S c , The University of Calgary, 2001 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (In Microbiology and Immunology) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A November 2004 © Bradly Joseph Biagioni 2004 Abstract Antibodies are specialized molecules produced by B-lymphocytes to combat infection by binding to and targeting infectious agents for destruction. To produce antibodies, B-cells require signals from multiple receptors, including the B-cel l antigen receptor (BCR) and CD40. (3-catenin is a transcriptional co-activator that regulates important pro-proliferative genes. Phosphorylation of (3-catenin by glycogen synthase kinase-3 ( G S K -3) in resting B cells targets (3-catenin for proteasome-mediated degredation, keeping levels of (3-catenin low. B C R engagement results in (3-catenin upregulation by phosphorylating G S K - 3 on negative regulatory sites and inhibiting G S K - 3 activity. (3-catenin then localizes to the nucleus, and activates transcription. I show that CD40 also regulates G S K - 3 , but does so through a different intracellular signaling pathway than the B C R . I show that C D 4 0 induces the phosphorylation of G S K - 3 on negative regulatory sites via a pathway that is independent of phosphatidylinositol-3 kinase (PI3K) and Akt , but requires the activity of M E K - 1 . Also , I used a reporter gene assay to show that B C R and C D 4 0 stimulation act together to induce a greater level of |3-catenin-dependent transcription than B C R stimulation alone. Two alternate hypotheses are possible to explain this effect. Either C D 4 0 enhances the upregulation and nuclear localization of (3-catenin caused by the B C R , or CD40 signaling upregulates (3-catenin binding partners. The binding partners of (3-catenin are transcription factors of the T C F / L E F family. Recent reports showed that in response to CD40 signaling L E F - 1 m R N A increases. This suggests that the synergistic increase in (3-catenin mediated transcription could be due to C D 4 0 induced upregulation of (3-catenin binding partners. I used quantitative real-time polymerase chain reaction to show that one possible mechanism for the CD40-mediated ii increase of |3-catenin-mediated transcription is CD40 induced upregulation of TCF-1 m R N A . The GSK-3/|3-catenin /TCF-1 pathway may be a key point of integration between B C R and C D 4 0 signaling which regulates B cell differentiation. In addition, GSK -3 and |3-catenin have been implicated in the development of B-cel l malignancies. Greater understanding of the interactions of these key regulatory pathways in the activation of B-cells is essential to our understanding of how humoral immune responses clears infection, and could provide insight into the development of B-cell leukemias. Table of Contents Abstract i i Table of Contents iv List of Figures vi List of Abbreviations vii Acknowledgements ix Dedication x Chapter 1 - Introduction 1 L I B lymphocytes 1 1.1.1 Role of B lymphocytes in immunity 1 1.1.2 B cell development 1 1.2 B cell activation 6 1.2.1 Structure and function of the B cell antigen receptor 6 1.2.2 B cell activation by T cell-dependent antigens 7 1.2.3 C D 4 0 signaling in B cells 7 1.2.4 Mechanisms of B C R and CD40 synergy in B cell activation 10 1.3 Intracellular signaling events regulated by the B C R 11 1.3.1 The initiation of B C R signaling 11 1.3.2 Downstream events in B cell signaling: Akt , and G S K - 3 12 1.4 (3-catenin and T C F / L E F family transcription factors 14 1.5 Summary of the thesis 21 1.5.1 Hypothesis and Objectives 21 1.5.2 Summary of results 22 Chapter 2 - Methods 23 Chapter 3 - Results 30 3.1 C D 4 0 induces G S K - 3 and A K T phosphorylation via two distinct signaling pathways 30 3.1.1 C D 4 0 stimulation induces the phosphorylation of G S K - 3 and A k t . . . . 30 3.1.2 Phosphorylation of G S K - 3 is not dependent on A k t 31 3.1.3 G S K - 3 phosphorylation by C D 4 0 requires M E K 1 / E R K activity 36 3.2 C D 4 0 signaling leads to an increase in the transcription of (3-catenin binding partners 42 3.2.1 Expression of (3-catenin and T C F / L E F family members in B cell lines and splenic B cells 42 3.2.2 C D 4 0 can influence |3-catenin-mediated transcription 46 3.2.3 C D 4 0 signaling increases TCF-1 m R N A in WEHI-231 cells 50 Chapter 4 - Discussion 55 4.1 Summary 55 4.2 Pathway by which CD40 regulates G S K - 3 58 4.3 Downstream of CD40-mediated regulation of G S K - 3 63 4.4 T C F / L E F family members in B cells and their regulation by C D 4 0 . . . . 66 4.5 Conclusions, perspectives and future directions 69 References 74 Appendix A - Detailed protocol for Q R T - P C R 93 Appendix B - SDF-1 stimulation of WEHI-231 cells results in G S K - 3 and A k t phoshorylation 100 Lis t of Figures Figure 1.1: B cell development 3 Figure 1.2: BCR-mediated inactivation of G S K - 3 and stabilization of |3-catenin 15 Figure 3.1: C D 4 0 stimulation induces the phosphorylation of G S K - 3 and A K T in W E H I -231 cells 32 Figure 3.2: C D 4 0 stimulation induces the phosphorylation of G S K - 3 and A K T in Bai 17 and CH31 cells 34 Figure 3.3: CD40-induced phosphorylation of G S K - 3 is not dependent on PI3K 37 Figure 3.4: CD40-induced phosphorylation of G S K - 3 depends on M E K 1 activity 39 Figure 3.5: Expression of P-catenin and |3-catenin binding partners in B cell lines and splenic B cells 43 Figure 3.6: Both B C R stimulation and CD40 stimulation increase (3-catenin-dependent transcription 48 Figure 3.7: C D 4 0 stimulation increases TCF-1 m R N A levels in WEHI-231 cells. ..51 Figure 4.1: Summary of the findings of this thesis 56 Figure 4.2: Pathways activated by the B C R and C D 4 0 61 Figure B . l : SDF-1 stimulation leads to the phoshorylation of G S K - 3 and Ak t 101 Lis t of abbreviations A b Antibody A P C Adenomatous polyposis coli B C R B cell antigen receptor B S A P B cell specific activator protein C D 4 0 L C D 4 0 ligand C L P Common lymphoid progenitor E B F Early B cell factor E G F Epidermal growth factor E R K Extracellularly regulated kinase F A C S Fluorescence activated cell sorting F C S Fetal calf serum G A P D H Glyceraldehyde 3-dehydrogenase G C Germinal center G S K - 3 Glycogen synthase kinase-3 H R P Horseradish peroxidase H S C Hematopoetic stem cell Ig Immunoglobulin IL-7R Interleuken-7 receptor I L K Integrin linked kinase J N K c-Jun N-terminal kinase L E F - 1 Lymphoid enhancing factor-1 M A P K Mitogen activated protein kinase M A P K A P Kinase-2 Mitogen activated protein kinase activated protein kinase-2 N F - K B Nuclear factor-kappa B N F - A T Nuclear factor of activated T cells N G F Nerve growth factor N K cell Natural killer cell PBS Phosphate-buffered saline P C R Polymerase chain reaction PI3K Phosphatidylinositol 3-kinase P K C Protein kinase C Q R T - P C R Quantitative real-time polymerase chain reaction R A G Recombination-activating gene R T - P C R Reverse transcriptase polymerase chain reaction SDF-1 Stromal cell derived factor-1 T B S T Tris-buffered saline containing 0.01% Tween 20 T C F T cell factor T C R T cell antigen receptor T N F a Tumor necrosis factor alpha T N F R Tumor necrosis factor receptor T R A F Tumor necrosis factor receptor associated factor v i i i Acknowledgments I would like to thank my supervisor Mike Gold for giving me the opportunity to pursue my own ideas, and his ability to put a positive spin on any situation. I would like to thank the following past and present members of the Gold lab, Rosaline Lee, May Dang-Lawson, Kevin L i n , and Caylib Durand for their constant advice and support. Two other lab members especially standout and deserve special thanks: Sarah McLeod , thanks for keeping my sanity intact with such quotables as, "Don' t worry, nothing ever works," and Kathy Tse for crucial assistance with the PI3K inhibitor experiments and unquenchable enthusiasm even in the face of blank blots, undecipherable results and other minor catastrophes. In addition, I would like to thank the members of Team Tomorrow for constantly brewin' and given'er and reminding me of what is really important. Lastly, I would like to thank my family for everything that they have given me, without them, none of this would be possible. IX For Sarah Dawn, who adds joy to my life. Chapter 1 - Introduction 1.1 B lymphocytes 1.1.1 Role ofB lymphocytes in immunity B lymphocytes produce antibodies that combat infections by pathogens (1). Antibodies bind to specific molecular patterns on antigens called epitopes (1,2). Antibodies have two main functions. First, they bind to and inactivate extracellular antigens, such as bacteria, viruses and toxins, and second, they activate components of the innate immune system, including complement, N K cells, and macrophages which help destroy and eliminate the invading pathogens (1, 2). Defects in the ability to produce antibodies are found in patients with a variety of immunodeficiency diseases, and these patients are susceptible to recurrent bacterial infections (1,3). Circulating B cells do not constitutively produce antibodies, but need to be activated by antigen, which causes them to proliferate and then differentiate into antibody secreting plasma cells (1,4). In order to differentiate into plasma cells, B lymphocytes require signals from multiple cell surface receptors including the B cell antigen receptor (BCR) and CD40 (1, 4-6). These receptors initiate signaling pathways that regulate the expression of genes and activity of proteins that control B cell proliferation and their subsequent differentiation into antibody-producing plasma cells (7, 8). 1.1.2 B cell development The development of B lymphocytes from haematopoetic stem cells is a tightly regulated and complex process that originates in the fetal liver and continues in the bone marrow of adult mice and humans. B cell differentiation is delineated into stages, starting from the haematopoetic stem cell (HSC), followed by the common lymphoid progenitor (CLP) , an on through pro- and pre-B cell stages in which rearrangement of the immunoglobulin (Ig) genes occurs to generate a functional B C R . The major steps and checkpoints in B cell development are depicted in Figure 1.1. The first stage in B lymphopoesis is the differentiation of a H S C to a C L P , which can generate all three lymphoid lineages, N K , T and B cells (9). The transcription factors P U . l and Ikaros are important for this first differentiation step, and these transcription factors drive the expression of the IL-7 receptor (IL-7R), which is important in generating survival signals for early B and T cell progenitors (10, 11). After commitment to the lymphoid lineage, the expression of B cell-specific transcription factors drives the expression of other B cell-specific genes that commit the C L P to differentiate into a B lymphocyte, while suppressing its ability to differentiate into a T cell or N K cell (9, 10). The expression of B cell specific transcription factors drives the cell to a more committed B cell fate, the first stage of which is known as the pro-B cell, which can be identified by its expression of B220, an isoform of CD45 found on all B lineage cells (9, 11). It is at this stage that the rearrangement of the immunoglobulin (Ig) genes begins, starting with the recombination of the D and J segments of the heavy chain locus (9). The transcription factors E 2 A and early-B-cell factor (EBF) are key regulators of the transition from C L P to pro-B cell (10, 11). Ectopic expression of E 2 A , and E B F drives the expression of an entire B cell differentiation program, activating the expression of Pax-5, surrogate light chains, and the recombination activating genes, R A G - 1 and R A G - 2 (11). One of the key genes activated by E 2 A and E B F is pax-5 (BSAP) , which promotes the expression of genes important for further differentiation of B cells, such as C D 19, N-myc, and L E F - 1 , among others, while repressing non-B lineage genes such as Notch (10-13). In the absence of pax-5 expression, recombination of the D- J segments of the Ig 2 Figure 1.1: B cell development The first step in generation of a mature B cell is the differentiation from a hematopoetic stem cell (HSC) to a common lymphoid progenitor (CLP) . The genes PU.l and Ikaros are involved in this step, driving the expression of IL-7R, which is necessary for the survival of C L P s . The B cell development program is then initiated by the expression of E2A and E B F , transcription factors able to drive the expression of pax-5 and other genes important for B cell lineage commitment. The expression of E2A and E B F ultimately drive the cell to the pro-B cell stage, which are the first B220+ cells in B cell development. The expression of B220 marks the pro-B cell stage, where the Ig heavy chain (p) locus is rearranged. Successful rearrangement of the p locus and its expression on the cell surface along with the surrogate light chain is an important checkpoint in the transition between the pro- and pre-B cell stages. After entering the pre-B cell stage, signaling through the pre-BCR causes a proliferative burst and allows the rearrangement of the light chain locus. If this rearrangement is successful, the expression of the light chain on the cell surface along with the heavy chain marks the transition from the pre-B cell stage to the immature B cell stage. Then negative selection deletes self-reactive B cells, generating a mature B cell population. 3 pre-B cell receptor: -LI chain -VpreB - Ig-ot 4 heavy chain locus still occurs. However, no further recombination events occur, leading to a profound block in B lymphopoiesis at the pro-B cell stage (10, 11). If a successful D H - J H rearrangement occurs, then the developing B cell continues to rearrange the heavy chain locus, recombining the V H segment to the already joined DJ segments (12). A n in-frame V H - D J H rearrangement allows the expression of the heavy chain (u,) on the cell surface along with the surrogate light chain, which is made up of the X.5 and VpreB polypeptides, as well as the signaling molecules Igct and Ig|3 (11). This receptor complex is known as the pre-B cell receptor (pre-BCR) and marks entry into the pre-B cell stage (9, 11, 12). The pre-BCR has a similar structure to the B C R that is expressed on immature B cells (see section 1.2.1). Signaling through the pre-BCR constitutes an important checkpoint in B cell development, as signaling mediated by the pre-BCR is required for further development (14). Furthermore, the importance of pre-BCR signaling in pro- to pre- B cell transition is highlighted by studies in which disrupting genes that encode important B C R signaling molecules, such as syk, results in a block in B cell development at the pro- to pre-B cell checkpoint (14). P re -BCR signals result in allelic exclusion of the heavy chain locus and initiate a proliferative burst of 3-5 cycles, followed by the rearrangement of the light chain genes, starting with the kappa locus and then, if necessary, the lambda locus (9, 12). Successful rearrangement of the light chain, and its expression along with the heavy chain and Ig-a and Ig-|3 molecules on the cell surface is the final step in the generation of an immature B cell. The expression of the B C R allows a round of negative selection by which autoreactive B cells are either deleted through apoptosis, become anergic, or are induced to edit their antigen receptors through further rounds of Ig gene rearrangement (12). B cells surviving negative selection migrate to the secondary lymphoid organs, such as the spleen, where they 5 encounter antigen and participate in immune response (12). The differentiation of mature B cells into antibody producing plasma cells in the secondary lymphoid organs can be considered the final stage in their development, and this differentiation step is regulated by multiple receptors on the cell surface, including the B C R and CD40 (1, 4-6). 1.2 B cell activation 1.2.1 Structure and function of the B cell antigen receptor The B C R is a multi-protein complex composed of membrane bound immunoglobulin (mlg) subunits, as well as the Ig-ct and Ig-|3 subunits (1, 12, 15). The cytoplasmic tails of the mlg subunits are short, and have no intrinsic intracellular signaling activity (12, 15). The cytoplasmic domains of Ig-ci and Ig-p\ however, contain immunoreceptor tyrosine-based activation motifs ( ITAMs) , that, when phosphorylated on the tyrosine residues, recruit and activate Src family protein tyrosine kinases (PTKs) (1, 12). In particular, recruitment and activation of the Syk tyrosine kinase by the B C R , plays a key role in activating downstream signaling pathways, including those regulated by PLC-y , Phosphatidylinositol 3-kinase (PI3K), and Ras (1, 12). Signaling events are initiated when antigens bind to the mlg subunits and cluster several receptors together (1, 12, 15). This promotes the proliferation and differentiation of B cells (1, 12, 15). Eventually, the antibodies produced by the differentiated B cell wi l l bind to the same epitope that activated B C R signaling via the mlg (1,4, 12, 15). Some highly repetitive antigens, such as bacterial flagellin, cluster large numbers of B C R complexes, and are able to drive B cell activation in the absence of other signals (1, 16, 17). However, most antigens that activate B C R signaling are unable to drive the B cell into an 6 activated, antibody producing state, without additional T cell derived signals (1,7, 16, 17). Such antigens are called T cell-dependent antigens (1, 16, 17). 7.2.2 B cell activation by Tcell-dependent antigens In order to be activated by T cell-dependent antigens, B cells require a second signal from another cell surface receptor, CD40, which is a member of the tumor necrosis factor receptor (TNF-R) family of receptors (6, 7, 17). C D 4 0 is activated by C D 4 0 ligand (CD40L, CD154), which is found on the surface of activated T lymphocytes (6, 7, 17). If B cells do not receive this second signal from CD40, they undergo apoptosis or become anergic, which is a state of unresponsiveness to antigen (4, 6, 7, 18). This two-signal system ensures that B cells do not become activated by self-antigens, as C D 4 0 L is only highly expressed on T cells after they have also been activated by antigen (6, 7, 17). Signals originating from the B C R and CD40 have a synergistic effect on the activation of B lymphocytes (7, 19, 20). That is, CD40 stimulation along with B C R engagement results in a dramatic increase in the production of IL-6, TNF-ct, and I g M by B cells which is greater than stimulation of the B C R alone (20, 21). In addition, apoptosis induced by the B C R in the absence of C D 4 0 signaling can be prevented by the addition of C D 4 0 L , so C D 4 0 is important for the survival, as well as the activation of B lymphocytes (4, 7, 20, 22). Signals from the B C R and C D 4 0 mediate their effects on B cell fate by activating signaling pathways that regulate the expression of genes important for B cell survival and activation (4, 7, 20, 23). 1.2.3 CD40 signaling in B cells C D 4 0 is a ~50-kDa transmembrane receptor of the T N F - R superfamily (7, 8, 16). Loss-of-function mutations in the gene encoding the C D 4 0 ligand (CD40L; CD154) lead to defects in 7 humoral immunity. Specifically, genetic abnormalities in CD154 result in X-l inked hyper-IgM syndrome in humans, which is characterized by the inability to undergo Ig class switching to other isotypes such as IgG (7, 8, 24). Additionally, mice in which the genes encoding either CD40 or CD154 have been disrupted fail to form germinal centers, have a defect in isotype switching, have and defects in the generation of long-lived memory B cells (7, 25-27). These defects, however, have no effect on the response of B cells to T-independent antigens (7). Conversely, inappropriate expression of C D 4 0 L on B cells may lead to the induction of autoimmune disease. For example, mice overexpressing C D 4 0 L specifically on B cells develop an inflammatory bowel disease similar to colitis (28). The functions of C D 4 0 in B cells have been reviewed recently (6, 7, 29). C D 4 0 signaling is initiated when CD154 on the surface of activated T cells clusters CD40 molecules into trimers on the surface of the B cell (7). Since C D 4 0 contains no intrinsic kinase domain it uses members of the T N F - R associated factor ( T R A F ) family of adaptor proteins to recruit and activate intracellular kinases and activate downstream signaling events (6, 7, 29). The first T R A F identified as a CD40-associated protein was T R A F 3 (30, 31). C D 4 0 has also been shown to bind T R A F 1 , 2, and 6 directly, but the interaction of T R A F 5 with C D 4 0 is controversial (7). T R A F 2 and 3 are recruited to the consensus sequence (P /S /A/T)X(Q/E)E (where X is any amino acid) in the cytoplasmic tail of CD40 , whereas T R A F 6 binds to a different juxtamembrane binding motif P-X-E-X-X-(aromatic residue/acidic residue) (7, 32). The binding of different T R A F molecules to CD40 mediates the activation of differential downstream signaling events, each with specific cellular outcomes (7, 32). For example, CD40-mediated activation of the p38 mitogen activated protein kinase ( M A P K ) pathway depends primarily on T R A F 6 (33) and controls affinity maturation and the survival of plasma cells (5). 8 In addition to the recruitment of T R A F s , C D 4 0 also recruits the E3 ubiquitin ligase adaptor protein Cbl an event that is required for the activation of the serine/threonine kinase Ak t (34). Downstream of the adaptor proteins recruited by CD40, are multiple signaling cascades that lead to the activation of transcription factors, such as N F - K B , N F - A T , and AP-1 (7, 32). One of the most important pathways regulated by CD40 in B cell leads to the activation of transcription factor N F - K B , since N F - K B is a key regulator of the survival of B lymphocytes (1, 4, 35, 36). In unstimulated cells N F - K B is retained in the cytoplasm by inhibitor of N F - K B (IKB) proteins that block its nuclear import and ability to activate transcription (37). After receiving appropriate stimuli, I K B kinases ( IKK) are activated and phosphorylate I K B . This targets I K B for degradation by the proteasome, allowing N F - K B to translocate into the nucleus and activate transcription (37). Although recruitment of T R A F 2 and T R A F 6 to C D 4 0 is required for optimal N F - K B activation (33), the exact mechanism of CD40-mediated N F - K B activation is not completely understood (7). In other cell types, N F - K B activation by T R A F s is mediated by N I K , a M A P 3 K that phosphorylates and activates the I K K complex (32, 38). C D 4 0 engagement may lead to the activation of I K K by another mechanism, since a recent study showed that N I K -deficient mice can increase the DNA-binding ability of N F - K B normally for short time courses of C D 4 0 stimulation, but are unable to sustain this normally after 4 hours (39). C D 4 0 also regulates the expression of the pro-survival serine/threonine kinase Pim-1, and the c-myc transcription factor, via the activation of N F - K B (7,40-42). In addition to activation of N F - K B , CD40 also activates the p38, E R K , and J N K M A P K pathways (43, 44). The effect of the activation of these M A P K s is to phosphorylate and activate transcription factors (45, 46). For example, J N K and p38 are able to phosphorylate S M A D 3 , a transcription factor, and this phosphorylation results in its nuclear translocation (45). Although many downstream signaling targets of C D 4 0 have been 9 identified, C D 4 0 signaling and its effects on B cell biology are not yet completely understood. Furthermore, it is still unclear how signals from C D 4 0 are integrated with signals from other cellular receptors that are important for B cell survival and activation, such the I L - 4 receptor and the B C R . What is clear is that the co-regulation of genes by the B C R and C D 4 0 is one way by which the two signals are integrated. 1.2.4 Mechanisms of BCR and CD40 synergy in B cell activation In response to antigen, the B C R produces both activation and survival signals, such as the nuclear localization of N F - K B , as well as apoptotic signals, such as an increase in intracellular calcium ( 4 , 4 7 ) . C D 4 0 signaling synergizes with the B C R induced activation and survival signals and opposes B C R induced death signals. For example, the activation and nuclear localization of the N F - K B transcription factor is an important survival signal in B cells that is regulated by both C D 4 0 and the B C R ( 1 , 4 , 6 , 7 , 17) . N F - K B in turn regulates the transcription of several other genes important for B cell survival, such as bcl-xl, c-myc and the pim-1 kinase (4 , 7 , 4 1 , 4 7 - 4 9 ) . In response to B C R signals, c-myc and pim-1 expression is transiently increased, but after a few hours drops below basal levels ( 4 1 , 4 8 , 5 0 ) . The eventual drop in c-myc and pim-1 levels when no C D 4 0 signal is received along with B C R engagement leads to apoptosis ( 4 1 , 4 8 , 5 0 ) . A combination of C D 4 0 and B C R signaling prolongs the expression of c-myc and pim-1, allowing the B cell to avoid B C R - i n d u c e d apoptosis ( 4 1 , 4 8 ) . B C R signaling also induces the expression and activation of both pro- and anti-apoptotic members of the bcl-2 family (4 , 5 1 ) . Some of the pro-apoptotic members of this family, such as Box and Bim are activated in response to calcium mobilization by BCR-med ia t ed signaling events (4) . However, B C R engagement also increases the expression of anti-apoptotic members 1 0 of this family, through the activation of N F - K B (4, 51). The regulation of bcl-2 family genes is another important site of C D 4 0 and B C R signal integration, as C D 4 0 can also increase the expression of anti-apoptotic bcl family members, such as bcl-xl, and the expression of these genes can protect B cells from BCR-induced apoptosis (49, 52, 53). There are undoubtedly more sites of B C R and C D 4 0 signal integration that regulate the survival and activation of B cells. 1.3 Intracellular signaling events regulated by the BCR The clustering of B C R receptor complexes on the surface of a B cell activates multiple signaling pathways (1,4, 12). Reviewed here are the pathways that regulate glycogen synthase kinase-3 (GSK-3) and its downstream target (3-catenin, the focus of this thesis. 1.3.1 The initiation of BCR signaling After receptor clustering by antigen, Src family tyrosine kinases that are associated with the B C R in a phosphorylation-independent manner phosphorylate the I T A M motifs in the cytoplasmic tails of the Igct and Ig(3 subunits of the B C R (1,4,12) . These initial phosphorylation events allow the SH2 domain-mediated recruitment of additional Src family tyrosine kinases as well as the Syk tyrosine kinase (1,4, 12). The activation of these P T K s leads to the activation of downstream signal transduction cascades that are regulated by PLC-y , phosphatidylinositol dependent kinase-3 (PI3K), and GTPases such as Ras, Rac, and Rap (1,4, 12). Adaptor proteins play a key role in coupling BCR-associated tyrosine kinases to the activation of PLC-y , PI3K and GTPases (1,4, 12). A key adaptor protein in B C R signaling is B L N K (also known as SLP-65 or B A S H ) (1,4, 12). B L N K contains several functional domains, 11 such as SH2 domains, and the recruitment of B L N K to the B C R complex (54), and its subsequent phosphorylation by F T K s allows for the assembly of the B cell signalosome that includes P L C - y and the Btk tyrosine kinase (1,4, 12). A n analogous pathway exists for the recruitment and activation of another important signaling molecule, PI3K, by the adaptor proteins G a b l , Gab2 and B C A P (1, 55). The recruitment and activation of PLC-y2 and PI3K allows for the production of important second messengers. Activated PLC-y2 generates the second messenger diacylglyerol ( D A G ) , which leads to the activation of protein kinase C ( P K C ) and the R a p l GTPase (1, 56). PLCy2 activation also leads to the production of the second messenger inositol (1,4,5)-trisphosphate (IP 3), which induces the release of C a 2 + from intracellular stores (4, 12). The recruitment of PI3K to the plasma membrane in response to B C R engagement allows it to phosphorylate phosphatidylinositol 4,5 bisphosphate (PIP 2), resulting in the generation of phosphatidylinositol 3,4,5 trisphosphate (PIP 3) (1, 4, 14, 57-59). PIP 3 can be converted to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2), by SHIP (SH-domain containing inositol phosphatase) an inositol phosphatase (60). Both PIP 3 and PI(3,4)P 2 function as second messengers, recruiting enzymes that contain pleckstrin homology (PH) domains, such as A k t ( P K B ) , phosphoinositide dependent-kinase 1 (PDK1) and Btk to the plasma membrane, where they are activated (58, 61). 1.3.2 Downstream events in B cell signaling: Akt, and GSK-3 A k t is a serine/threonine kinase that is activated upon B C R engagement after recruitment to the plasma membrane via its P H domain and subsequent phosphorylation by P D K 1 and P D K 2 (1, 57, 62). A k t can then promote cell survival by phosphorylating proteins involved in 12 regulation of apoptosis and metabolism (62-65). Important targets of A k t include Bad, which blocks the anti-apoptotic effect of bcl-2. A k t also negatively regulates the Forkhead transcription factors which regulate the expression of the p27 k i p l cell cycle inhibitor as well as the pro-apoptotic Bcl -2 family member B i m which plays a key role in B cell apoptosis (65, 66-68). Other important targets of Ak t are the two isoforms of G S K - 3 , G S K - 3 c i and GSK-3|3 (69, 70). Phosphorylation of G S K - 3 by Ak t is the major pathway by which G S K - 3 is inactivated in response to insulin (71-73). G S K - 3 is a constitutively active serine/threonine kinase that has two isoforms G S K - 3 a and GSK-3|3 (74-76). G S K - 3 has multiple functions including the regulation of glycogen synthase in response to insulin signaling, the regulation of cell cycle progression, the regulation of transcription and translation and the establishment of cellular polarity (71, 76-83). Generally the actions of G S K - 3 on these diverse cellular functions are inhibitory. For example, G S K - 3 negatively regulates translation by phosphorylating the eukaryotic initiation factor eIF2B (78, 84). The phosphorylation of eIF2B results in its inactivation, and a block in the initiation of translation (78, 85, 86). G S K - 3 can also inhibit the progression of the cell cycle by phosphorylation of cyclin D l , which results in its cytoplasmic retention and degradation (80, 87, 88). G S K - 3 also negatively regulates, via phosphorylation, a broad array of transcription factors including c -Myc , N F - A T , N F - K B , A P - 1 , and (3-catenin (86). Lastly, the inactivation of G S K - 3 by the PAR/cdc42 complex plays a role in the polarization of and formation of extensions by migrating cells (82, 83, 89). Phosphorylation of GSK-3ct and (3 on serine residues in their N -terminal regions inhibits their kinase activity (86, 90). The inactivation of G S K - 3 by receptor-mediated phosphorylation, then allows progression of these diverse cellular processes that are negatively regulated by G S K - 3 . 13 Along with Akt , other kinases, such as protein kinase C ( P K C ) enzymes, protein kinase A ( P K A ) , p90Rsk, and integrin linked kinase ( ILK) can phosphorylate the negative regulatory sites on G S K - 3 and inhibit G S K - 3 kinase activity (73, 91-94). Since B C R engagement causes Ak t activation, as well as the phosphorylation-dependent inactivation of G S K - 3 , Christian et al., hypothesized that the B C R regulates G S K - 3 via A k t (57, 95). However, this turned out not to be the case. This group found that the B C R regulates G S K - 3 primarily via the activation of P K C (95). Furthermore, they showed that the inactivation of G S K - 3 by P K C in response to B C R ligation leads to the accumulation and transcriptional activation of P-catenin Figure 1.2 and (95). 1.4 6-catenin and TCF/LEF family transcription factors One important target of G S K - 3 is the transcriptional co-activator P-catenin (86, 90). G S K - 3 was first identified as a regulator of P-catenin through study of the Wnt signaling pathway in Drosophila and Xenopus (86). G S K - 3 and P-catenin interact in a multi-protein complex comprised of axin, A P C , (3-catenin, and G S K - 3 (76, 86). Phosphorylation of P-catenin by G S K - 3 is facilitated by this complex, which targets P-catenin for ubiquitination and rapid destruction by the proteasome (86,96-98). In response to Wnt hormones interacting with Frizzled-family receptors, G S K - 3 activity is inhibited by the Disheveled (dsh) gene product (86, 98). The mechanism by which Disheveled inhibits G S K - 3 activity, allowing the accumulation of P-catenin, is not well characterized, but may be by competitively excluding G S K - 3 from the P-catenin degradation complex (99). Alternatively Wnt may inhibit G S K - 3 by the activation of P K C , which phosphorylates G S K - 3 (100), or via the recruitment of Akt , another kinase able to inactivate G S K - 3 , to the P-catenin destruction complex (70). In response to signaling events, such as Wnt/Wingless interactions, or the BCR-mediated activation of P K C , G S K - 3 is 14 Figure 1.2: The BCR regulates GSK-3 and B-catenin by a PLC/PKC dependent pathway Engagement of the B C R leads to the phosphorylation of G S K - 3 on negative regulatory sites, primarily via a PKC-dependent pathway, although A k t may play a minor role. The resulting inhibition of G S K - 3 leads to the accumulation of B-catenin. Adapted from (95). 15 PKC Major GSK-3 'Minor T p-catenin inactivated, and levels of free |3-catenin in the cell rise (86, 95, 96). The accumulation of |3-catenin allows it to translocate to the nucleus, interact with high-mobility group ( H M G ) transcription factors of the L E F / T C F family, and thereby activate transcription (86, 101-105). There are four members of the L E F / T C F family of transcription factors L E F - 1 , T C F - 1 , T C F - 3 and T C F - 4 (106-108). L E F - 1 and TCF-1 were the first members identified, and were characterized as T-cell specific transcription factors (106, 107). In 1998, two more mammalian T C F / L E F family members, TCF-3 and T C F - 4 were identified (108). A l l L E F / T C F family members have several different splice variants and transcriptional start sites (109-112). For example, L E F - 1 has two splice variants, and has two alternative promoters, while TCF-1 has at least eight protein isoforms (109-113). Members of the L E F / T C F family bind as monomers to the D N A consensus sequence 5 ' C T T G W W 3 ' (where W is an A or T) in the promoters of target genes (113, 114). L E F - 1 and other members of the L E F / T C F family are known as architectural proteins as they introduce large bends in D N A upon binding to their consensus sequences (115, 116). Knockout mice have been generated for all members of the L E F / T C F family of transcription factors (117-121). Knockout mice for T C F - 4 and T C F - 3 have defects in early embryonic axis induction and intestinal development, respectively (118, 119). L E F - 1 , originally cloned as a transcription factor involved in the induction of T cell antigen receptor (TCR) expression in developing T cells (106) was the first member of the family to be knocked out in mice. L E F - 1 knockout mice were generated by the insertion of a Neo r-cassette into the exons of L E F - 1 that encode for the H M G domain (121). Although these mice have defects in several L E F - 1 expressing organs and die shortly after birth, they show no obvious defects in their lymphoid cell populations, a surprising result as L E F - 1 is expressed in pre B-cells and all T cells 17 (114, 121). These L E F ' " mice were later shown to have a deficiency in the numbers of pro-B cells which are actively proliferating, as well as a greater number of pro-B cells undergoing apoptosis, although the remaining pro-B cells differentiate normally (122). A different knockout L E F - 1 gene of was generated by a lacZ insertion into the L E F - 1 locus, resulting in a fusion protein that is dominant-negative for all L E F / T C F isoforms (120). These mice have a profound embryonically lethal neurological phenotype in that they lack a hippocampus (120). A n analysis of the pattern of L E F - 1 and TCF-1 expression during development showed some partial overlap (123) . This, along with the difference between the two phenotypes of the different LEF-1"'" alleles suggested that in the case of the Neor-cassette insertion, T C F - 1 is able to substitute for L E F - 1 in development. TCF-1 knockout mice differ from the LEF-1 knockouts in that they are viable and have defects only in the development of T lymphocytes, which are blocked in the transition from the double negative to CD4+/CD8+ double positive stage (117). However, similar to the defect in B cell development in the L E F - 1 knockout mice, the T lymphocytes of T C F - 1 knockouts were deficient in numbers of precursor cells, but the small numbers of precursor T cells that survive and proliferate develop normally, leading to full immunocompetence (117). Crossing of the L E F ' and TCF-1"'" mice allowed the assessment of functional redundancy between the TCF-1 and L E F - 1 gene products (124). These double knockout mice have a. phenotype that is very similar to that of Wnt3a-/- mice, they lack limbs and have defects in placenta formation, whereas either of the single knockouts showed no similarity to any known Wnt knockout phenotype (124) . The redundant functions of TCF-1 and L E F - 1 in T cell development were again demonstrated in organ culture assays using the double and single L E F ' / T C F - l " ' " knockout mice (125) . In these assays, cells derived from the double knockout mice, are unable to rearrange or 18 express the T C R a locus, leading to a more severe block in T cell development than is observed either of the single L E F ' " or TCF-1"'" single knockout mice (125). Although members of the L E F / T C F family of transcription factors bind D N A in the absence of other proteins, they require interactions with P-catenin as well as accessory proteins such as pygopus and legless (BCL-9) in order to induce the transcription of target genes (101, 105, 126, 127). The target genes of the (3-catenin-LEF/TCF transcription complex, which include c-Myc, cyclin Dl, and survivin, a pro-survival protein, are important for normal development, cell cycle control, and the regulation of apoptosis (98, 101, 128-135). Normal activation of (3-catenin-mediated transcription is required for the formation of anterior-posterior axis Xenopus and mice, the development of segment polarity in Drosophila, and gut formation in C. elegans (98, 127, 136-140). In addition, the progression of many types of cancer, especially colon cancers, have been linked to the abnormal accumulation of P-catenin (128, 129, 132, 141-148). Abnormal activation of P-catenin leads to cancer through the transcription of cellular oncogenes (149). For example, transgenic mice expressing cyclin Dl, a downstream target of P-catenin (131), develop mammary hyperplasia and most die of breast cancer (150, 151). c-myc, another target of P-catenin (152), is associated with aggressive, poorly differentiated tumors, and plays a role in the loss of terminal differentiation (153). c-myc is also important for the regulation of apoptosis in many cell types, including B cells (47,48, 50, 153, 154). Furthermore, P-catenin likely plays a role in the metastatic potential of certain types of cancers, as its dysregulation leads to the loss of cellular adhesion and the expression of matrix metalloproteinases (129, 155-158). For example, in multiple myeloma, stimulation with Wnt proteins leads to an increase in P-catenin levels and P-catenin-mediated transcription, as well as rearrangement of the actin cytoskeleton and dramatic changes in cellular morphology (146). 19 Thus, through the transcription of cellular proto-oncogenes, (3-catenin regulates cellular survival, differentiation, and proliferation, and abnormal transcription of its target genes may result in carcinogenesis (149). Thus, (3-catenin, is an important regulator of cellular proliferation and differentiation (98, 149). It is normally found at low levels in the cell, due to phosphorylation by G S K - 3 , a constitutively active kinase that phosphorylates |3-catenin, targeting it for degradation by the proteasome (76, 135). Multiple signaling pathways, including those involving Wnt hormones and A k t negatively regulate G S K - 3 (76, 144, 159). Activation of these pathways inhibits G S K - 3 kinase activity and allows the accumulation of (3-catenin (86). (3-catenin can then translocate to the nucleus and activate transcription in cooperation with L E F / T C F transcription factors (101, 103, 149). Christian et al. showed that in response to B C R engagement, (3-catenin-dependent transcription increases in B cells (95). Neither the target genes nor the role of (3-catenin in B cell activation is known, but in early B cell development, (3-catenin, in concert with L E F - 1 , plays a role in the survival and proliferation of pro-B cells (122). |3-catenin also regulates the expression of the recombination-activating gene-2 ( R A G ) in developing B cells (160). Furthermore, a number of recent papers implicate (3-catenin and other members of the Wnt signaling pathway in the development of B and T cell lymphomas (146, 161, 162). (3-catenin may also be involved in the development of lymphoproliferative diseases caused by Epstein-Barr virus ( E B V ) infection. Infection with E B V causes various B cell malignancies, through the expression of latent membrane protein 2 A ( L M P 2 A ) , which mimics a constitutively active B C R (163-165). Infection of B cells with E B V or ectopic expression of L M P 2 A in B cells results in the accumulation and transcriptional activation of (3-catenin by a PI3K-independent mechanism (166, 167). In addition 20 to its association with the development of B cell malignancies, (3-catenin has been implicated in the normal development, activation and survival of T cells (162, 168-173). The finding that the B C R regulates the stability and transcriptional activity of (3-catenin (95) and the activation of (3-catenin by L M P 2 A , as well as the activation of |3-catenin during T and B cell development (122, 169, 171), and T cell activation (169) suggest a role for (3-catenin in the activation of B cells. 7.5 Summary of the thesis 1.5.1 Hypothesis and Objectives We have previously shown that stimulation of the B C R leads to phosphorylation and inactivation of G S K - 3 (57, 95) as well as the accumulation and transcriptional activation of |3-catenin (95). However, B C R stimulation alone is unable to drive the activation of B cells in response to T cell-dependent antigens (1, 4, 60). The result of prolonged B C R stimulation of ex vivo B cells and immature B cell lines, such as the WEHI-231 cell line, is receptor-mediated apoptosis (1, 4, 60). Signals from co-stimulatory molecules, such as CD40 , can rescue B C R -stimulated B cells from apoptosis, and help to drive B cells to differentiate into antibody-producing plasma cells (6, 7, 17). Since G S K - 3 negatively regulates the survival and differentiation of other cell types (75, 174), I propose that C D 4 0 stimulation results in the phosphorylation of G S K - 3 on its negative regulatory sites. I also hypothesize that CD40 regulates G S K - 3 by a PI3K/Akt-dependent mechanism, since C D 4 0 activates the PI3K/Akt pathway (34, 175-177) and A k t phosphorylates G S K - 3 in other cell types (94, 178,179). Furthermore, based on recent evidence that C D 4 0 signaling can regulate the expression of LEF-1 in murine splenic B cells (180, 181), I propose that C D 4 0 signals may regulate the expression of 21 other (3-catenin binding partners and increase the amount of |3-catenin-mediated transcription that is initiated by B C R signaling. Based on the hypothesis that C D 4 0 regulates G S K - 3 and is able to influence (3-catenin-mediated transcription, this thesis has several main objectives: 1. Determine i f C D 4 0 stimulation leads to the phosphorylation of G S K - 3 , and if so, determine the mechanism by which this occurs. 2. Determine which members of the L E F / T C F family of transcription factors are present in various B cell lines and splenic B cells, and determine whether they are regulated by C D 4 0 signaling. 3. Investigate the role of CD40 in regulating (3-catenin-mediated transcription. 1.5.2 Summary of results In this thesis I show that CD40 stimulation leads to the phosphorylation of G S K - 3 a and (3 on negative regulatory sites, Ser21 and Ser 9, respectively. I also demonstrate that in response to C D 4 0 stimulation the phosphorylation of G S K - 3 is not dependent on A k t but instead requires the activity of M E K - 1 , a kinase that activates the E R K M A P K (45, 46). In addition to delineating the signal transduction pathway by which CD40 promotes the phosphorylation of G S K - 3 , 1 investigated the role of CD40 in regulating |3-catenin-mediated transcription. I provide evidence that C D 4 0 may increase P-catenin-mediated transcription by itself, and can augment B C R -mediated P-catenin-dependent transcription. I also show that one possible mechanism for this effect is an increase in expression of T C F - 1 , a L E F / T C F family member. 22 Chapter 2 - Mater ia ls and Methods Antibodies and oligonucleotides Goat antibodies (Ab) against mouse I g M (u, chain-specific) were purchased from Jackson ImmunoReasearch Laboratories (West Grove, P A ) . The 1C10 anti-murine C D 4 0 mAb (182, 183) was purified from hybridoma supernatants using a protein-A Sepharose column. Abs specific for Akt , Ak t phosphorylated on Ser473 (anti-P-Ser473 Akt) , the phosphorylated forms of E R K (P-ERK1/2) and GSK-a /GSK-3(3 phosphorylated on Ser21 and Ser9, respectively (anti-P - G S K - 3 a / G S K - 3 p ) were purchased from Cel l Signaling Technologies (Beverly, M A ) . The monoclonal antibodies (mAbs) to |3-catenin and GSK-3(3 were obtained from B D Transduction Laboratories (Lexington, K Y ) and the A b specific for E R K 1 / 2 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, C A ) . Primers for reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (QRT-PCR) were designed as follows: Gene Forward Primer Sequence Reverse Primer Sequence |3-catenin: 5 ' - A C G C A C C A T G C A G A A T A C A A - 3 ' 5 ' -CTT A A A G A T G G C C A G C A A G C - 3 ' (3-actin 5 ' - G A T G T C A C G C A C G A T T T C C - 3 ' 5 ' - G T C C C T G T A T G C C T C T G G T C - 3 ' L E F - 1 5 ' - T C A C A G G C A T G G T T T C T A A G G C - 3 ' 5 ' -TCT C A G G A T G C A G C A T C C T A G G - 3 ' T C F - 1 : 5 ' - A G A C T T T T C C C G G A C A A A C T - 3 ' 5 ' - T G A G C A G A T T G A A G G C A G A - 3 ' T C F - 3 : 5 ' - T G C A G T G A G T G C G A A A T C C - 3 ' 5 ' - T G G C C C T C A T C T C C T T C A T A - 3 ' T C F - 4 : 5 ' - A G G A G A T G A C C T A G G C G C T A A - 3 ' 5 ' - C T T G A C A T C G G C T A A A T C C C T - 3 ' G A P D H : 5 ' - A T G T G T C C G T C G T G G A T C T G A - 3 ' 5 ' - T G C C T G C T T C A C C A C C T T C T T - 3 ' 23 Cell lines and murine splenic B cells The WEHI-231 IgM+ immature murine B cell line and the A 2 0 and 2PK3 IgG+ murine B cell lines were obtained from American Type Culture Collection (Manassas, V A ) . The K40-B l (183) and 300-19 pro-B cell line, as well as the 70Z/3 pre-B cell line, the CH31 IgM+ immature B cell line, and the Bai 17 IgM-i- murine B cell line were gifts from Dr. A . DeFranco (University of California, San Francisco, C A ) . A l l cell lines were maintained in RPMI-1640 supplemented with 10% heat inactivated fetal calf serum (FCS), 50 p M 2-mercapthoethanol, 1 m M pyruvate, 2 m M glutamine, 15 U/ml penicillin, and 50 pg/ml streptomycin (complete medium). Small resting B cells were isolated from spleens of C 5 7 B L / 6 mice by Percoll density centrifugation after A b - and complement-mediated lysis of T-cells (57). B cell stimulation and preparation of cell lysates To reduce basal signaling by serum components, WEHI-231 cells were grown in complete medium with the F C S reduced to 0.5% for 12-18 h before stimulation. The cells were washed once with modified HEPES-buffered saline (57) and then resuspended in this buffer at 1 x 10 7 cells/ml and warmed to 37°C for 10-30 min. Where indicated, cells were incubated with the kinase inhibitors U0126, wortmannin or Ly294002 for 20 min prior to stimulation. Cells were then stimulated with anti-IgM and/or 1C10. For isolation of R N A , WEHI-231 cells were stimulated with 1C10 for 0-8 hours in complete medium without prior serum starvation. The reactions were terminated by adding ice cold PBS containing I m M N a 3 V 0 4 and then centrifuging the cells for 3 min at 800 x g in a cold microfuge. WEHI-231 cells were solubilized in Triton-X 100 lysis buffer (20 m M T r i s - H C l , pH 8.0, 1% Triton X-100, 137 m M N a C l , 2 m M E D T A , 25% glycerol, 0.5 m M D T T , 1 m M phenylmethylsulfonyl fluoride, 1 pg/ml aprotinin, 10 24 pg/ml leupeptin, 1 m M N a 3 V 0 4 , 25 m M P-glyerophosphate). After 10 min on ice, insoluble material was removed by centrifugation and the protein concentration determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Immunoblotting Total cell extracts (30 pg protein, unless otherwise indicated) were separated on SDS-P A G E gels and then transferred to nitrocellulose membranes. A l l membranes were blocked for 0.5-1 hour with 5% (w/v) skim milk powder in Tris-buffered saline containing 0.01% Tween 20 (TBST). The membranes were then incubated overnight at 4°C with primary A b . A l l Abs were diluted in T B S T / 5 % bovine serum albumin (BSA) with the exception of the mAbs to P -ERK1/2 and E R K 1 / 2 , which were diluted in T B S T / 5 % milk, and the A b to phosphorylated A k t which was diluted in T B S T / 1 % B S A . The membranes were washed with T B S T and incubated with the appropriate HRP-conjugated secondary A b (Bio-Rad, Hercules, C A ) for 1 h at room temperature. Immunoreactive bands were visualized using E C L (Amersham Pharmacia Biotech, Baie d'Urfe, Quebec, Canada). To reprobe the membranes, bound Abs were eluted by incubating the membrane in 10 m M Tr i s -HCl (pH 2.0), 150 m M N a C l for 30 min. The membranes were then reblocked and probed as described above. Luciferase reporter gene assays The TOPtk and FOPtk plasmids were obtained from Dr. M . Waterman (University of California, Irvine, C A ) . Transient transfection of WEHI-231 cells was performed using the D M R I E - C lipid reagent (Invitrogen, Burlington, Ontario, Canada). Briefly, l i p i d : D N A complexes were formed by adding the D M R I E - C lipid reagent (12 pi) and D N A (4 pg) to 1 ml 25 of O P T I - M E M medium and incubating for 45 min at 2 l ° C for 45 min. WEHI-231 cells (4 x 106) that had been resuspended in 0.2 ml complete medium lacking antibiotics were added to D N A d i p i d complexes and grown for an additional 20 hours at 37°C. The batch-transfected cells were then resuspended in fresh complete medium and divided into multiple wells of a 24-well dish. The cells were then cultured with medium only, 10 \xg/ml goat anti-mouse I g M , 5 u,g/ml 1C10, or a combination of both Abs for 3 h at 37°C. After washing with P B S , cells were lysed in Reporter Lysis Buffer (Promega, Madison, WI). Luciferase activity was determined using the Promega luciferase assay system (Promega, Madison, WI). Readings were made using a MircoLumat Plus luminometer ( E G & G Berthold, Bad Wildbad, Germany) set for a 10 sec acquisition window. Relative luciferase units were obtained by normalizing the luciferase activity to the amount of protein as determined using the bicinchoninic acid assay. Preparation of total cellular RNA and RT-PCR Total cellular R N A was isolated from WEHI-231 cells using TRIzo l (Invitrogen, Carlsbad, C A ) according to the manufacturer's instructions. R N A quality was checked by agarose gel electrophoresis and the concentration determined by spectrophotometry after every manipulation. Contaminating genomic D N A was removed by DNase treatment using the D N A -free kit from Ambion Inc. (Austin, T X ) . Briefly, 10 \ig of total R N A was incubated with 5 Units DNase for 30 min at 37°C. After DNase treatment, c D N A was prepared from the extracted R N A using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, C A ) , according to the manufacturer's specifications. P C R reactions were carried out using puRe Taq Ready-To-Go P C R beads (Amersham Pharmacia Biosciences, Piscataway, NJ) using the following conditions: 1 min at 95°C, followed by 35 cycles of 45 sec at 95°C, 45 sec at 56°C, and 45 sec at 72°C, 26 followed by 5 min at 72°C. P C R products were visualized by ethidium bromide staining after electrophoresis on 2% agarose gels. Real-time quantitative PCR R N A was isolated and purified as described above. c D N A was generated by reverse transcription using an oligo d(T) 1 6 primer and the TaqMan Reverse transcription reagents (P/N N808-0234, Applied Biosystems Inc., Foster City, C A ) . Briefly, 0.5 pg of purified R N A in a final volume of 10 p\ was incubated with I X TaqMan R T buffer, 5.5 m M M g C l 2 , 500 p M dNTP, 2.5 p M oligo d(T) ] 6 , 4 Units RNase inhibitor and 12.5 Units MultiScribe reverse transciptase for 10 min at 25°C, followed by a 30 min incubation at 48°C and a 5 min incubation at 95°C to inactivate the enzyme. Real-time P C R (QRT-PCR) was performed using the S Y B R Green P C R master mix (P/N 4309155, Applied Biosystems Inc.) following the manufacturer's protocol as follows. Briefly, the c D N A generated from unstimulated cells was titrated to a concentration range of 0.3125-20 ng and was incubated with I X S Y B R Green P C R master mix and 300 n M of the TCF-1 or G A P D H primers for the generation of standard sample curves. For unknown samples to be quantified, the c D N A generated from 10 ng R N A was used, with the same concentrations of primers and S Y B R Green as used for the generation of the standard curves. P C R reactions (25 pi final volume) each were added to the 96-well A B I Optical Reaction Plate (P/N 4306737). The thermal cycling conditions were as follows: 10 min at 95°C for Ampl iTaq Gold activation followed by 43 cycles of 15 sec at 95°C and 1 min at 60°C. Double-stranded P C R amplicons fluorescently labeled with S Y B R Green were detected using the A B I sequence detection system. 27 To calculate the relative levels of starting template in the experimental samples a relative standard curve approach was used. Standard curves of C t vs log(ng c D N A ) were generated for both the target gene of interest (TCF-1) and an endogenous control gene ( G A P D H ) using the titrations of c D N A generated from unstimulated samples as above. Representitive standard curves are shown. **8 *> <] n , _o -0.25 0.25 0.7S , . y --3.2128* + 25.511 Log(ng) cDNA R°-0.8955 TCF-1 std curve 23-o.rs 0.25 0.25 0.75 log(ng) cONA GAP0 std curve 1-3.213SX* 20.636 R! = 0.9968 For each experimental sample, the amount of target (TCF-1) and endogenous reference ( G A P D H ) were determined from the appropriate standard curve using the following equations: A ) For T C F - 1 : B) For G A P D H : IA) C t = m[log (ngXCF.,)] + b IB) C t = m[log (ngG A P D H)] + b 2A) log (ngXCF.,) = (C, - b)/m 2B) log (ngG A P D H) = ( C t - b)/m 3A )ng T C F . 1 = 10(C<-b>/m 3 B ) n g G A P D H = 10(C<-b)/m Where m and b represent the slope and intercept of the appropriate standard curve respectively B y dividing the result of equation 3A by the result of equation 3B, a normalized "amount" of m R N A is obtained, with arbitrary units: 3A/3B = ng T C F.,/ ng G A PDH 28 n g T C F - / n g c A P D H = X = "arbitrary value of TCF -1 cDNA" The "arbitrary value of TCF-1 m R N A , " X , is calculated for all experimental samples, including the unstimulated control. X for each stimulated sample is divided by X for the unstimulated control to obtain a fold increase in expression as follows: X s t i m u l a t e d / X u n s t i m u l a t e d = fold increase in TCF -1 mRNA A more detailed protocol for Q R T - P C R is attached as Appendix 1. 2 9 Chapter 3 - Results 3.1 CD40 induces GSK-3 and Akt phosphorylation via two distinct signaling pathways 3.1.1 CD40 stimulation induces the phosphorylation of GSK-3 and Akt C D 4 0 activates multiple signal transduction pathways that regulate B cell proliferation and differentiation (6, 7, 184). Since B C R signaling leads to the phosphorylation of A k t and G S K - 3 (57, 95), we investigated whether CD40 also activated A k t and G S K - 3 in B cells. G S K - 3 is a constitutively active kinase that normally opposes cell growth and proliferation by phosphorylating and negatively regulating proteins such as (3-catenin, c -Myc , Cycl in D l , and N F - A T (76, 185). Receptor-induced phosphorylation of G S K - 3 a and GSK-3(3 on Ser21 and Ser9, respectively, inhibits their kinase activities (71, 76) and can be assessed using phospho-specific antibodies. Since C D 4 0 induces B cell activation and proliferation, we tested the hypothesis that C D 4 0 induces the phosphorylation and inactivation of G S K - 3 . G S K - 3 can be phosphorylated by a variety of kinases including Akt , I L K , p90Rsk, and P K C (71, 79, 88, 174, 178). Since C D 4 0 has been shown to induce Ak t phosphorylation and activation (34, 175-177), we tested whether C D 4 0 regulates G S K - 3 , and i f so, whether it does so via Akt . To determine if C D 4 0 induces the phosphorylation of A k t and G S K - 3 , we used the WEHI-231 cell line. WEHI-231 cells represent an immature B cell line that is similar to normal splenic B cells which undergo apoptosis after prolonged B C R stimulation. The apoptosis induced by B C R stimulation of these cells can also be rescued by stimulation with C D 4 0 L or anti-CD40 antibodies, and they are often used as a model of normal B cells undergoing B C R -mediated clonal deletion. WEHI-231 cells were stimulated with the 1C10 anti-CD40 antibody and analyzed by immunoblotting with the anti-P-Ser473-Akt, and P-GSK-3a /P-GSK-3 |3 antibodies (Figure 3.1 A , B) . The phosphorylation of G S K - 3 in response to C D 4 0 stimulation 30 was apparent after 5 minutes of stimulation, and begins to decline after 60 min (Figure 3.1A). The phosphorylation of A k t in response to C D 4 0 stimulation is not sustained as long as G S K - 3 phosphorylation. Figure 3. IB shows that CD40 stimulation induces the phosphorylation of Ak t on Ser 473 after 5 minutes, but the phosphorylation of A k t only continues up to 30 min. Thus, Figure 3.1 shows that stimulation of WEHI-231 cells leads to the phosphorylation of Ak t and G S K - 3 . To confirm that the effect of CD40 stimulation on the phosphorylation of G S K - 3 is not unique to the WEHI-231 cell line, I stimulated the Bai 17 and CH31 cell lines with the 1C10 anti-C D 4 0 antibody and analyzed the phosphorylation of G S K - 3 and A k t using the same anti-P-GSK-3a/(3 and anti-P-Ser473-Akt (Figure 3.2). The Bai 17 cell line represents a mature IgM+ B cell which is resistant to BCR-mediated apoptosis, whereas the CH31 cell line has similar properties to the WEHI-231 cell line (186, 187). A s shown in Figure 3.2A, C D 4 0 stimulation, leads to an increase in the phosphorylation of G S K - 3 a and G S K - 3 p \ in both Bai 17 cells and CH31 cells. However, in contrast to WEHI-231 cells, C D 4 0 stimulation led to a very weak and transient increase in the phosphorylation of A k t on Ser473 in Bai 17 cells and did not cause any detectable phosphorylation of A k t in CH31 cells (Figures 3.2B). The finding that G S K - 3 phosphorylation occurs in cell lines that have very little or undetectable amounts of A k t phosphorylation in response to C D 4 0 stimulation suggests that CD40-mediated G S K - 3 phosphorylation does not depend on Akt . 3.1.2 Phosphorylation of GSK-3 is not dependent on Akt A k t is able to phosphorylate G S K - 3 in response to other signals, such as insulin (62, 70). The ability of C D 4 0 to induce G S K - 3 phosphorylation in the absence of A k t phosphorylation in 31 Figure 3.1: CD40 stimulation induces the phosphorylation of GSK-3 and AKT in WEHI-231 cells A and B , Upper panels: WEHI-231 cells were stimulated with 20 u.g/ml of the 1C10 anti-CD40 mAb for the indicated times. Cel l extracts (30 [ig protein) were analyzed for G S K - 3 phosphorylation using the anti-P-GSK-3a/GSK-3|3 A b (A) and for A k t phosphorylation using the anti-P-Ser473 A k t A b (B). To ensure equal loading, the membranes were stripped and reprobed with Abs against GSK-3(3 or A k t (A and B , lower panels). Similar results were obtained in at least 3 independent experiments. 32 A* anti-CD40 (min) anti-P-GSK-3a/p blot anti-GSK-3p reprobe 0 5 15 30 60 >66 «+- P-GSK-3a ^ P - G S K - 3 p GSK-3 P B. anti-CD40 (min) anti-P-Ser473 Akt blot anti-Akt reprobe 0 5 15 30 60 66 P-Ser473-Akt 66 Akt 33 Figure 3.2: CD40 stimulation induces the phosphorylation of GSK-3 and AKT in Ball 7 and CH31 cells A , Upper panels: B a l l 7 and CH31 cells were stimulated with 20 pg/ml of the 1C10 anti-CD40 mAb for the indicated times. Cel l extracts (30 pg protein) were analyzed for G S K - 3 phosphorylation using the ant i-P-GSK-3a/GSK-3|3 A b . B , Upper panels, the same cell extracts were analyzed for A k t phosphorylation using the anti-P-Ser473 A k t A b , and were subject to long exposure times to detect the phosphorylation of Atk in Bai 17 cells. To ensure equal loading, the membranes were stripped and reprobed with Abs against GSK-3(3 or A k t (A and B , Lower panels). In these experiments the lysates from the two cell lines were probed concurrently using the same antibody solutions. Similar results were obtained in 3 independent experiments. 34 A. a n t i - C D 4 0 ( m i n ) ant i -P-GSK-3a/ |3 blot anti-GSK-3{3 reprobe B. a n t i - C D 4 0 ( m i n ) anti-P-Ser473 Akt blot anti-Akt reprobe BAL17 CH31 0 5 15 30 0 5 15 30 66 h 4 5 "45 P-GSK-3a P-GSK-3P GSK-3 p BAL17 CH31 0 5 15 30 0 5 15 30 •66 P-Ser473-Akt Akt 35 CH31 cells suggested that C D 4 0 does not regulate G S K - 3 via Akt . To test this hypothesis, I utilized the PI3K inhibitors LY294002 and wortmannin, effective inhibitors of the PI3K/Akt signaling pathway. Pre-treatment of WEHI-231 cells with the PI3K inhibitor LY294002 or wortmannin completely abrogated CD40-induced A k t phosphorylation (Figure 3.3A), but had little or no effect CD40-induced phosphorylation of GSK-3ct or GSK-3|3 (Figure 3.3B). Thus, the phosphorylation of Akt , but not G S K - 3 , in response to C D 4 0 stimulation is dependent on PI3K activity. 3.1.3 GSK-3 phosphorylation by CD40 requires MEK1/ERK activity A s well as activating PI3K, J N K and p38 M A P kinase; C D 4 0 also activates the M E K 1 / E R K pathway (16, 43, 44, 184). Recently, Davies et al., showed using HeLa cells ectopically expressing CD40, that CD40 activates p90Rsk (also known as M A P K A P Kinase-1) via the M E K 1 / E R K pathway (188). In PC12 cells, chemoattractant-stimulated neutrophils, and embryonic Xenopus cells, inactivation of G S K - 3 in response to various stimuli occurs via a M E K / E R K / R s k pathway (71, 74, 90, 174, 178, 189, 190). Thus, I hypothesized that CD40 may regulate the phosphorylation of G S K - 3 via this pathway. To determine i f CD40-induced phosphorylation of G S K - 3 was dependent on M E K 1 / E R K activity, I used the M E K 1 inhibitor U0126, and analyzed the phosphorylation of G S K - 3 a and (3 by Western blotting with the anti-phospho-GSK-3a/p antibody (Figure 3.4). Pre-treatment of WEHI-231 cells with 50 p M of the M E K 1 inhibitor or U0126 led to the complete abrogation of G S K - 3 phosphorylation after 15 minutes of stimulation with the 1C10 antibody (Figure 3.4A). In contrast, pre-treatment of WEHI-231 cells with these inhibitors had no effect on BCR-induced phosphorylation of G S K - 3 (Figure 3.4A). The effectiveness of the 36 Figure 3.3: CD40-induced phosphorylation of GSK-3 is not dependent on PI3K. A and B : WEHI-231 cells were pre-treated with 30 n M wortmannin (W), 25 pM LY294002 (Ly) or an equivalent volume of D M S O for 20 min at 37°C and then stimulated with 20 u,g/ml of the 1C10 anti-CD40 m A b for the indicated times. Upper panels, Cel l extracts were analyzed for G S K - 3 and A k t phosphorylation by immunoblotting with the anti-P-GSK-3a/|3 and anti-P-Ser473 A k t antibodies. Lower panels: To ensure equal loading the blots were then reprobed the anti-GSK-3|3 and anti-Akt Abs. Similar results were obtained at least 3 independent experiments. 37 Inhibitor anti-CD40 (min) P-Ser473-Akt Akt-DMSO Ly W 0 15 15 15 -66 . anti-P-Ser473 Akt blot -66 anti-Akt ....... reprobe Inhibitor anti-CD40 (min) P-GSK-3a-* -P-GSK-3p-*-GSK-3p - • DMSO 0 15 Ly W 15 15 anti-P-GSK-3o:/p .45 b l o t anti-GSK-3p •45 reprobe 38 Figure 3.4: CD40-induced phosphorylation of GSK-3 depends on MEK1 activity. A and B : WEHI-231 cells were pre-treated for 20 min at 37°C with U0126 (U) at 50 u M , or with an equivalent volume of D M S O . The cells were then stimulated with 20 n g /ml of the 1C10 anti-CD40 mAb, or 40 [Ag /ml of anti-IgM for the indicated times. Upper panel: Cel l extracts were analyzed for G S K - 3 and E R K phosphorylation by immunoblotting with anti-P-GSK-3a/(3 Abs or with an t i -P -ERKl /2 Abs. Lower panels: The blots were then reprobed the anti-GSK-3(3 and a n t i - E R K l / 2 Abs, as a loading control. Similar results were obtained in 3 independent experiments. 39 A . Inhibitor anti-IgM (min) anti-CD40 (min) U DMSO U 15 15 0 0 0 0 0 0 15 15 P-GSK-3<x.#J P-GSK-3|3-*-GSK-3(3-^ anti-P-GSK-3a/p AC blot 45 anti-GSK-3p 4 5 reprobe B . Inhibitor U DMSO U anti-IgM (min) 15 15 0 0 0 anti-CD40 (min) 0 0 0 15 15 P-ERK-1-*J P-ERK-2-^ L > -ERK-1-ERK-2" 1 •45anti-P-ERK-1/2 blot -45 anti-ERK-1/2 reprobe 40 inhibitor's ability to block M E K 1 activity was determined by immunoblotting using an antibody specific for the phosphorylated forms of E R K 1 and 2, which are direct downstream targets of M E K 1 (Figure 3.4B). A s shown in Figure 3.4B, CD40 stimulation of WEHI-231 cells leads to the phosphorylation of E R K 2 , but phosphorylation of E R K 1 is undetectable. In contrast, B C R stimulation leads to the phosphorylation of both E R K isoforms (Figure 3.4B). U0126 very effectively blocked the phosphorylation of E R K 2 by CD40, and of E R K 1 and 2 by the B C R (Figure 3.4B). The data in Figure 3.4 show that engagement of either C D 4 0 or the B C R results in the phosphorylation of G S K - 3 , but that only C D 4 0 uses a MEK-1-dependent pathway to phosphorylate G S K - 3 . These results show that CD40 stimulation of B cells leads to the phosphorylation of G S K - 3 and A k t (Figure 3.1 and 3.2). The effect of C D 4 0 stimulation on the phosphorylation on these important protein kinases occurs via two independent pathways. CD40-induced phosphorylation of A k t is dependent on PI3K activity (Figure 3.3A) whereas the phosphorylation of G S K - 3 is independent of PI3K and is instead dependent on the activity of M E K 1 (Figure 3.4A). Thus, C D 4 0 induces G S K - 3 phosphorylation via a MEK-dependent pathway while the B C R indues G S K - 3 phosphorylation via a MEK-independent pathway that involves P K C (95). Since G S K - 3 can be phosphorylated by the p90Rsk kinase (74, 174), a downstream target of E R K , a reasonable hypothesis is that CD40 induces G S K - 3 phosphorylation via a M E K / E R K / p 9 0 R s k pathway. C D 4 0 has been shown to induce the activation of p90Rsk in HeLa cells ectopically expressing C D 4 0 (188). 41 3.2 CD40 leads to an increase in the transcription of B-catenin binding partners 3.2.1 Expression of B-catenin and TCF/LEF family members in B cell lines and splenic B cells G S K - 3 regulates the stability and activity of many cellular proteins, including the multifunctional protein P-catenin (74, 174). p-catenin is able to bind cadherins in adherens junctions (158), as well as drive transcription in the nucleus (74, 174). P-catenin-mediated transcription is dependent on the formation of complexes between P-catenin and members of the T C F / L E F family of HMG-box-containing transcription factors (105, 113). We recently showed that stimulation of B cells through the B C R causes the accumulation of P-catenin in the nuclei of B cells and stimulates P-catenin-dependent transcription from a promoter that contains T C F / L E F binding sites (95). However, it was not well defined which of the various T C F / L E F transcription factors were present in B cells. Since P-catenin has no intrinsic D N A binding properties, and requires interactions with members of T C F / L E F family of transcription factors in order to activate transcription (103, 105, 113), it is important to know which T C F / L E F family members are present in B cells and could therefore mediate P-catenin-mediated transcription in response to receptor engagement. Therefore, I performed R T - P C R with primers specific for P-catenin, T C F -1, L E F - 1 , T C F - 3 , and T C F - 4 to investigate the expression pattern of the T C F / L E F family of transcription factors in cell lines representing various stages of B cell development, and in splenic B-cells. The early stages of B cell development are represented by the the pro-B cell lines 300-19, and K 4 0 B 1 , and the pre-B cell line 70Z/3 (Figure 3.5, Upper panels). A l l of these cell lines express high levels of P-catenin, and at least two T C F / L E F family members. For example, 70Z/3 cells express LEF-1 and T C F - 1 . None of these cell lines express T C F - 3 . 42 Figure 3.5: Expression of B-catenin and /3-catenin binding partners in B cell line and splenic B cells Total RNA was isolated from the indicated B cell lines or from murine splenic B cells and analyzed by RT-PCR with primers specific for (3-catenin, LEF-1, TCF-1, TCF-3, or TCF-4. (3-actin-specific primers were used to amplify actin as a loading control and to ensure that no genomic DNA contamination was present. Genomic DNA contamination would be indicated by the generation of PCR products from RNA samples that had not been incubated with reverse transcriptase (Actin: No R-T). 43 CO CP __. 0 0 n ro > o •a m o o N \ U J 7? O CD U J O o P-catenin The WEHI-231, C H 3 1 , and Bai 17 cell lines represent IgM+ B cells. WEHI-231 and CH31 cells are immature IgM+ B cells that are susceptible to BCR-induced apoptosis (186, 191), whereas Bai 17 represents a mature IgM+ B cell that does not undergo apoptosis in response to B C R signaling(187). Both WEHI-231 and CH31 express p-catenin and T C F - 1 but do not express L E F - 1 , T C F - 3 , or T C F - 4 . (Figure 5, Centre panels). Bai 17 cells have a slightly different profile than WEHI-231 and CH31 cells in that they express L E F - 1 along with |3-catenin and TCF-1 (Figure 3.5). IgG+ memory B cells, the last stage of B-cell development, are represented by the A 2 0 and 2PK3 cell lines (Figure 3.5, Lower panels). I found that A 2 0 cells express P-catenin, LEF-1 and T C F - 3 , while 2PK3 cells express p-catenin, L E F - 1 , T C F - 1 and T C F - 4 . Lastly, small resting B-cells were isolated from C 5 7 B L / 6 mice and total R N A was isolated. This R N A was also analyzed by R T - P C R using the P-catenin, L E F - 1 , T C F - 1 , -3 and - 4 specific primers. I found that splenic B-cells express P-catenin, L E F - 1 , T C F - 1 and T C F - 4 (Figure 3.5). Thus, cell lines representing the various stages of B-cell development show different patterns of expression for the members of the T C F / L E F family of transcription factors. A l l of the B cell lines examined, excluding the 300-19 pro-B cell line and the A 2 0 mature B cell line express T C F - 1 . The expression of LEF-1 and T C F - 4 seems to depend on the developmental stage of the B cell. L E F - 1 and T C F - 4 are present in the cell lines representing earliest stages of B cell development, (300-19,70Z/3, and K40B-1) and in the cell lines representing the final stages of B cell development, mature IgM+ (Bai 17) and IgG+ B cells (A20 and 2PK3) but are not expressed in the immature IgM+ cell lines, WEHI-231 and CH31 . 45 The expression pattern of splenic B-cells most resembles that of the mature B cell lines B A L 1 7 and 2PK3 . Since the population of cells isolated is greater than 90% IgM+ B cells, it could be that the difference between the expression pattern of the IgM+ cell lines (WEHI-231 and CH31) is due to differences between cell lines and normal B cells, or that immature B cells are different than mature B cells. However, I cannot completely rule out the possibility that some contaminating cells were present in the population of spleen cells used to generate the total R N A used for R T - P C R analysis. 3.2.2 CD40 can influence 6-catenin-mediated transcription We have previously shown that stimulation of the B C R increases P-catenin-dependent transcription (95) in WEHI-231 cells. Since CD40 stimulation of WEHI-231 cells prevents apoptosis that is caused by B C R stimulation of these cells (6, 7, 49, 52, 53, 192, 193), and (3-catenin activates the transcription of pro-proliferative genes, such as c-myc (152) and cyclin Dl (131), we hypothesized that C D 4 0 might increase P-catenin-dependent transcription. Furthermore, C D 4 0 induces the phosphorylation of G S K - 3 (Figure 3.1, 3.2) a key regulator of (3-catenin in B cells and other cell types (76, 174, 194, 195). In addition, two recent reports (180, 181) showed that C D 4 0 stimulation of splenic B cells increases the transcription of the T C F / L E F family member L E F - 1 . Thus, we examined the effect of C D 4 0 stimulation on P-catenin-mediated transcription in the WEHI-231 murine B cell line. We used a luciferase reporter gene assay employing the TOPtk and FOPtk reporter plasmids to assess P-catenin-mediated transcription in WEHI-231 cells. The TOPtk plasmid contains multiple L E F / T C F binding sites as well as a minimal thymidine kinase promoter upstream of the luciferase gene. The FOPtk plasmid is identical except that it contains mutated 46 L E F / T C F binding sites. Binding of p-catenin-LEF/TCF complexes to the L E F / T C F binding sites in the TOPtk plasmid stimulates transcription of the luciferase reporter gene. Since the FOPtk plasmid contains mutated L E F / T C F binding sites, (3-catenin-TCF/LEF complexes cannot bind to the FOPtk plasmid D N A , thus, no transcription of the luciferase gene occurs. Figure 3.6 shows that, as previously reported (95), clustering the B C R with anti-IgM Abs caused a 2-3 fold increase in luciferase activity in cells transfected with the TOPtk plasmid but did not cause an increase in luciferase activity in cells transfected with the FOPtk plasmid. Stimulation of the transfected WEHI-231 cells with the 1C10 anti-CD40 antibody alone, or with a combination of 1C10 and anti-IgM, results in a 6-8 fold increase in transcription from the TOPtk plasmid, along with a slight increase in trascription from the FOPtk plasmid (Figure 3.6). The increase in luciferase transcription that occurs after C D 4 0 stimulation alone or in combination with B C R stimulation, is greater than that which occurs when the B C R alone is stimulated using anti-IgM antibodies (Figure 3.6). The effect of C D 4 0 signaling on P-catenin-mediated transcription requires further investigation, since the data presented in Figure 3.6 regarding the effect of CD40 stimulation alone on P-catenin-mediated transcription is based on only one experiment. However, the co-stimulation experiments show conclusively that C D 4 0 stimulation along with B C R stimulation leads to an increase in luciferase activity that is significantly (p<0.05, student's t test) greater than B C R stimulation alone (Figure 3.6). The mechanism by which CD40 is able to regulate P-catenin-mediated transcription is not clear. However, since P-catenin is negatively regulated by G S K - 3 (174) and C D 4 0 stimulation leads to the phosphorylation, of G S K - 3 (Figure 3.1A) it is likely that CD40 , is able to regulate P-catenin levels in the cell via its inactivation of G S K - 3 . 47 Figure 3.6: Both BCR stimulation and CD40 stimulation increase B-catenin-dependent transcription. WEHI-231 cells were transiently transfected with either the TOPtk or FOPtk reporter plasmids and then cultured for 20 h at 37°C. Duplicate samples were then cultured for 3 h with 10 u,g/ml anti-IgM Abs (IgM), 5 u.g/ml of the 1C10 anti-CD40 mAb (CD40), or a combination of anti-IgM Abs and the 1C10 anti-CD40 mAb (Both). Unstimulated samples (Unstim) were cultured for 3 hours with no addition of stimulating Abs. Luciferase assays were performed on cell extracts. Luciferase units were normalized to the protein concentration for each sample. The values for the unstimulated cells that had been transfected with either the TOPtk or FOPtk plasmids were arbitrarily set to one, and the values for the anti-IgM, anti-CD40 or combination anti-IgM and anti-CD40 stimulation are reported as "Relative fold increase" relative to the corresponding unstimulated sample. Each data point represents the mean ± S E M for n number of experiments as indicated. 48 8 StopTK • fopTK 0) 6 w CO a> o 5 c 2 4 CD > i s 3 a> fT 1 H 0 n=4 n=4 n=1 • R i l l H H I l B l i l l » n=3 Unstim anti-IgM anti-CD40 B o t h 49 3.2.3 CD40 stimulation increases TCF-1 mRNA in WEHI-231 cells The data presented in Figure 3.6 shows that C D 4 0 stimulation, either alone, or in combination with B C R stimulation, leads to an increase in (3-catenin-mediated transcription that is greater than that caused by B C R stimulation alone. There are two possible explanations for this effect. The first is that C D 4 0 stimulation leads to an increase in the level of nuclear (3-catenin that is greater than that which occurs after B C R stimulation. This hypothesis assumes that the amount of T C F / L E F family members is not a limiting factor in |3-catenin-mediated transcription in WEHI-231 cells. Another possible hypothesis is that the availability of T C F / L E F factors limits the amount of (3-catenin-mediated transcription in response to B C R stimulation, and that CD40 stimulation is able to increase the level of T C F / L E F factors available to form complexes with nuclear |3-catenin. The second hypothesis is supported by two recent papers (180, 181) which use D N A microarrays and Northern blotting to show that CD40 signaling causes an increase in LEF-1 m R N A in splenic B cells. To examine the effect of CD40 stimulation on the levels of L E F / T C F , expression, both semi-quantitative R T - P C R and quantitative real-time P C R ( Q R T - P C R ) were performed on total cellular R N A isolated from WEHI-231 cells after stimulation with anti-CD40 Abs. Semi-quantitative R T - P C R revealed that culturing WEHI-231 cells with anti-CD40 Abs for 4 or 8 h increased the level of TCF-1 m R N A (Figure 3.7A). A s a loading control, R T - P C R was also performed using |3-actin specific primers, and no significant difference was observed in the intensity of the |3-actin bands, indicating that similar amounts of c D N A were present in the P C R reaction mix. Negative controls in which no reverse transcriptase or no R N A were used during the production of c D N A were also analyzed, and these controls showed no bands for (3-actin, indicating that no contaminating genomic D N A was present in the R N A used for analysis (data 50 Figure 3.7: CD40 stimulation increases TCF-1 mRNA levels in WEHI-231 cells A . WEHI-231 cells stimulated with 5 pg/ml 1C10 anti-CD40 m A b for the indicated times before performing R T - P C R on total cellular R N A , using primers specific for TCF-1 or |3-actin. A representative experiment is shown. B . Quantitation of the increase in T C F - 1 m R N A by Q R T - P C R . After stimulation of WEHI-231 cells with 5 pg/ml of the 1C10 anti-CD40 mAb for the indicated times, total R N A was isolated for analysis by Q R T - P C R . The data for 2 or 3 independent analyses for each sample were averaged. The results are expressed after normalization against G A P D H . Each value represents the mean ± S E M for n number of independent experiments as indicated. The statitistical significance of the fold change compared to the unstimulated control sample was assessed using Student's one-tailed t-test: **,p< 0.005; *,p< 0.05 51 A . Anti-CD4Q stimulation (hours) TCF-1 0 0.5 1 2 4 8 Actin B . 0 1 4 Anti-CD40 stimulation (hours) 52 not shown). I also carried out R T - P C R reactions with primers specific for the other members of the L E F / T C F family of transcription factors, including L E F - 1 , T C F - 3 and T C F - 4 . There was no change in the expression of these transcription factors in response to C D 4 0 stimulation (data not shown). In order to quantify the transcriptional upregulation of TCF-1 caused by C D 4 0 stimulation in WEHI-231 cells, Q R T - P C R was performed. First, c D N A was generated from total R N A isolated from anti-CD40-stimulated WEHI-231 cells. This c D N A was used for the amplification of TCF-1 and G A P D H during the P C R reaction. In order to compare the levels of TCF-1 between the stimulated and unstimulated samples, c D N A from the unstimulated sample was titrated and used to generate a standard curve for each of the two genes. These standard curves are then used to calculate the amount of G A P D H and TCF-1 in each experimental sample The amount of TCF-1 was normalized to the level of G A P D H in each sample, and expressed as fold change relative to the unstimulated control. A statistically significant (p<0.005, student's t test) 2-fold increase in the level of TCF-1 m R N A was observed after 1 h of C D 4 0 stimulation (Figure 3.7B). After 4 h and 8 h, the increase in the level of TCF-1 m R N A by C D 4 0 stimulation was 2.5- to 3-fold (p<0.05, student's t test, Figure 3.7B). The results presented in Figure 3.7 show that C D 4 0 can influence the expression of T C F -1, a member of the T C F / L E F family of transcription factors. The effect of C D 4 0 signals on the expression of TCF-1 may be one way by which C D 4 0 is able to influence the level of P-catenin-mediated transcription in WEHI-231 cells (Figure 3.6). Since the experiment showing an increase in P-catenin-mediated transcription was only carried out one time, it is still unclear if C D 4 0 itself is able to increase p-catenin-mediated transcription. However, the ability of CD40 53 to regulate G S K - 3 suggests that C D 4 0 may regulate not only the nuclear binding partners of (3-catenin, but (3-catenin itself. P-catenin has been implicated in the progression of various types of cancers, especially colon cancers (103, 132, 143, 145, 148, 196). P-catenin causes the abnormal growth of cancer cells by increasing the transcription of pro-proliferative genes such as c-myc and cyclin Dl (75, 103, 129-131, 142, 143, 145, 148, 152, 196-199). c -Myc has previously been shown to play an important role in the ability of C D 4 0 to block BCR-induced apoptosis (42, 50, 67, 200). Thus the ability of C D 4 0 to promote P-catenin-dependent transcription may allow C D 4 0 to regulate the expression of anti-apoptotic or pro-proliferative genes in WEHI-231 cells. 54 Chapter 4 - Discussion 4.1 Summary C D 4 0 is an important co-stimulatory molecule that promotes the activation of B lymphocytes. G S K - 3 is a constitutively active kinase that negatively regulates many genes and proteins important for cellular survival such as c-Myc (81), cyclin D l (87), and |3-catenin (76, 174). The phosphorylation of G S K - 3 causes the inactivation of its kinase activity, leading to the upregulation and/or activation of these proteins, usually resulting in increased cellular survival and proliferation (76, 174). In this thesis I show for the first time that C D 4 0 stimulation of B cells leads to the phosphorylation of G S K - 3 (Figure 3.1, 3.2). I also show that C D 4 0 mediates this phosphorylation event by a pathway that is independent of P I3K/Akt (Figure 3.3), but which requires the activity of M E K - 1 / - 2 , an upstream activator of the E R K pathway (Figure 3.4). I also investigated an event downstream of G S K - 3 , the regulation of (3-catenin-dependent transcription. When this thesis was started, it was not known which of the T C F / L E F family of transcription factors were present in B lymphocytes. Since interactions between P-catenin and the T C F / L E F family of transcription factors are required for P-catenin to activate transcription (174), I used R T - P C R to investigate which members of this family were present in B cells (Figure 3.5). I provide evidence that C D 4 0 is able to regulate P-catenin-dependent transcription in WEHI-231 cells (Figure 3.6) and that one possible mechanism for this effect is an increase in the transcription of TCF-1 (Figure 3.7). A summary of these CD40-induced events is presented in Figure 4.1. 55 Figure 4.1: Summary of the findings of this thesis C D 4 0 engagement leads to the phosphorylation of both A k t and G S K - 3 by different pathways. The phosphorylation of A k t is dependent on PI3K activity, whereas the phosphorylation of G S K - 3 requires M E K - 1 activity. In addition, C D 4 0 positively affects (3-catenin-dependent transcription, either directly, via its regulation of G S K - 3 , or indirectly, via regulation of the expression of T C F - 1 , or both. See text for details. CD40 P I 3 K MEK-1 Akt E R K - - > p 9 0 ' ^ ? GSK-3 P-catenin t TCF-1 Transcription 57 4.2 Pathway by which CD40 regulates GSK-3 C D 4 0 induces the phosphorylation of G S K - 3 via a PI3K-independent pathway that did not involve A k t (Figure 3.3). I found that C D 4 0 was able to induce strong phosphorylation of G S K - 3 , even under conditions in which Ak t was not activated or in which PI3K activity was inhibited (Figure 3.2, 3.3). PI3K-independent inactivation of G S K - 3 has been observed in monocytes (201) and in tumor necrosis factor-a (TNFa)-stimulated muscle cells (202). I found that CD40-induced G S K - 3 phosphorylation in B cells was dependent on the activity of M E K - 1 and was reduced by the M E K - 1 / - 2 inhibitor U0126 (Figure 3.4). Although C D 4 0 stimulation of B cells leads to the phosphorylation of G S K - 3 , it remains to be formally shown that CD40-induced phosphorylation of G S K - 3 inhibits its kinase activity. U0126 exerts its inhibitory effect by blocking the activation of M E K - 1 (203). The specificity of U0126 has been shown in vitro, and in whole-cell based assays, U0126 inhibits the activity of M E K - 1 by about 50% at a concentration of 10 u M (203). The treatment of cells with U0126 no effect on the activity of a panel of other protein kinases including the closely related M K K - 3 , -4, -6 and - 7 , thus, U0126 is a specific inhibitor of the E R K pathway downstream of M E K - 1 (203). U0126 was able to block the phosphorylation of E R K in response to B C R signaling as well as the phosphorylation of E R K in response to C D 4 0 signaling (Figure 3.4). However, another structurally unrelated inhibitor of M E K - 1 , PD98059 was able to block the phosphorylation of E R K in response to C D 4 0 stimulation, but was unable to block the activation of E R K by the B C R (data not shown). This could be because PD098059 at 50 pM does not inhibit the in vivo activation of the E R K pathway when stimulated with potent activators of M A P K K , such as the B C R , and its low solubility in aqueous solutions precludes its use at higher 58 concentrations (204). Thus the different effects of these two inhibitors on E R K activation in response to C D 4 0 stimulation versus B C R stimulation could be due to the much stronger phosphorylation of E R K in response to B C R signaling (Figure 3.4). However, the use of U0126 shows that M E K - 1 is a principle activator of the E R K pathway in both B C R signaling and CD40 signaling (Figure 3.4). A key unanswered question raised by the data in Figure 3.4 is how the CD40-mediated activation of M E K - 1 / E R K leads to the phosphorylation of G S K - 3 . The inactivation of G S K - 3 downstream of E R K has been documented in the response of PC12 cells to nerve growth factor (NGF) and epidermal growth factor (EGF), as well as in developing Xenopus embryos (74, 90, 190, 206, 207). A good candidate for a kinase downstream of E R K that is able to phosphorylate G S K - 3 is p90Rsk. p90Rsk is able to phosphorylate G S K - 3 in EGF-stimulated 3T3 cells and in chemoattractant-stimulated neutrophils (178, 189). The regulation of p90Rsk activation is complex, and not completely understood. However, phosphorylation of p90Rsk by E R K is absolutely required for p90Rsk activation (208-210). In HeLa cells expressing transfected CD40, C D 4 0 stimulation leads to the MEK-1/ERK-dependent activation of p90Rsk (188). Since C D 4 0 mediated G S K - 3 phosphorylation in B cells is M E K - 1 / E R K dependent (Figure 3.4), as is the CD40-mediated activation of p90Rsk in other cell types (188), p90Rsk may phosphorylate G S K - 3 in response to C D 4 0 signals in B cells. Western blotting could be used to assess whether C D 4 0 induces the phosphorylation, and presumably the activation of p90Rsk in response to C D 4 0 signaling. A s it has no intrinsic kinase activity, C D 4 0 activates downstream signaling pathways via the recruitment of T R A F proteins. T R A F 2, 3, 5 and 6 have been shown to associate with CD40 (32). T R A F 6 is the major transducer of signals from the IL-6 receptor and the Toll- l ike receptor 59 (TLR) family (211). Its role in C D 4 0 signaling is to activate the p38 M A P K (33) and E R K pathways (212, 213). Genetic ablation of the T R A F 6 - C D 4 0 interaction leads to defects in the generation of long-lived memory B cells and differentiation into antibody-producing plasma cells (5). Using C D 4 0 constructs with truncated cytoplasmic tails I attempted to map which region of the C D 4 0 important for G S K - 3 phosphorylation. These cells were previously derived in our lab and used to map the regions important for the activation of p38 M A P K , M A P K A P Kinase-2, and J N K via their interactions with T R A F 2,3, and 5 (44). However, these cells were unable to activate E R K (data not shown, (44)) and thus never consistently showed G S K - 3 phosphorylation in response to stimulation. In addition, C D 4 0 stimulation of the parental WEHI-231 cells transfected with these constructs does not lead to the phoshorylation of E R K (C. Sutherland and M . R . Gold, unpublished observations). It could be that these WEHI-231 cells do not express an important factor upstream of E R K , such as T R A F 6 . Re-deriving the cell lines could help provide evidence that G S K - 3 lies downstream of TRAF6-mediated M E K / E R K activation in CD40-stimulated B cells. It is interesting to note that although the phosphorylation of G S K - 3 in response to CD40 stimulation is blocked by U0126, the phosphorylation of G S K - 3 in response to B C R signaling is unaffected, even though E R K phosphorylation is completely blocked (Figure 3.4). The regulation of G S K - 3 in response to B C R stimulation is mediated primarily via P K C , but is also partially dependent on the PI3K activity (57, 95). The inactivation of G S K - 3 by the B C R resembles that of the canonical Wnt signaling, in which both P K C and A k t play a role (70, 91, 100, 214). Figure 4.2 summarizes the pathways that lead to G S K - 3 phosphorylation in response to B C R and C D 4 0 stimulation. 60 Figure 4.2: Pathways activated by the BCR and CD40 Both CD40 and BCR engagement activate both the PI3K/Akt pathway and the M E K -1/Erk pathway. However, the activation of these pathways by the different receptors have different outcomes. See text for details. 61 CD40 PI3K X T R A F 6 BCR Akt MEK-1 MEK-1 PI3K Akt P L C ERK t ERK t PKC t GSK-3 p90Rsk GSK-3 62 Both the B C R and C D 4 0 can activate the PI3K7Akt pathway (Figure 3.1, 3.2, (57, 95)), as well as the M E K / E R K pathway (Figure 3.4, (43)). The activation of the M E K / E R K pathway by CD40, however, leads to the phosphorylation of G S K - 3 , whereas the activation of this same pathway by the B C R does not (Figure 3.4). The regulation of G S K - 3 in other cell types is dependent on its ability to interact in several different multi-protein complexes. For example, canonical Wnt signals inactivate the "pool" of G S K - 3 that interacts with A P C and A x i n and leads to the stabilization of (3-catenin (76, 174). Stimulation of P C 12 or R A T - 1 cells with insulin, however, leads to the inactivation of G S K - 3 via a P I 3 K / A K T pathway, and has no effect on the stability of (3-catenin (69, 174, 215, 216). In these cases, the different "pools" of G S K - 3 are insulated from each other by their interactions with different protein complexes. A similar system could exist in B cells to insulate the "pool" of G S K - 3 that is inactivated by Ak t and P K C signals from the B C R from the "pool" of G S K - 3 that is inactivated by M E K 1 / E R K signals from CD40. A n interesting experiment to determine i f there are indeed two differentially regulated pools of G S K - 3 in B cells would be to separate cell lysates on sizing columns. Then the various fractions could be assayed by blotting for phospho-GSK-3. If B C R - and CD40-mediated signals inactivate G S K - 3 that is in different fractions, then it is likely that differentially regulated "pools" of G S K - 3 exist in B cells. 4.3 Downstream of CD40-mediated regulation of GSK-3 The existence of two separate pathways in B cells able to regulate G S K - 3 implies the existence of separate "pools" of G S K - 3 , and could reflect different roles for G S K - 3 in signal transduction by the B C R and C D 4 0 in B cells. B C R signaling leads to the stabilization and 63 activation of (3-catenin (Figure 1.2, (95)), whereas CD40-induced inactivation of G S K - 3 in B cells could have different functions. G S K - 3 regulates the stability or activity of a number of other proteins that have been implicated in C D 4 0 signaling, in particular c -Myc and N F - A T . G S K - 3 has been implicated in the regulation of c -Myc protein stability in other cell types (76, 81, 174, 217). It carries out this effect by phosphorylating c-Myc on Thr-58, which targets c-Myc for destruction by ubiquination and subsequent destruction by the proteasome (81, 184). The stability of c -Myc is also regulated by E R K , which phosphorylates c -Myc on Ser-62, the consequence of which is to stabilize the protein (184, 217). CD40 regulates both E R K and G S K -3 by the same pathway, which is dependent on the activity of M E K - 1 (Figure 3.4). The ability of C D 4 0 to activate both E R K and inactivate G S K - 3 via a MEK-dependent signaling pathway would allow C D 4 0 to promote the stability of c -Myc in B cells. The use of G S K - 3 inhibitors, which mimic the receptor-induced inactivation of G S K - 3 , coupled with the M E K - 1 inhibitor U0126 could test this model. In other systems, G S K - 3 negatively regulates a number of important transcription factors such as p-catenin, N F - K B , N F - A T , and C R E B (76, 174). The regulation of any of these transcription factors by C D 4 0 could be mediated by G S K - 3 . Since G S K - 3 inactivation by the B C R leads to activation of P-catenin (95), I investigated if C D 4 0 signaling activates a similar pathway downstream of G S K - 3 . I provide evidence that CD40 can positively regulate P-catenin-mediated transcription (Figure 3.6), and that at least one way by which this may occur could be via the increase in transcription of an important binding partner of P-catenin, TCF-1 (Figure 3.7). P-catenin is a multifunctional protein, which is able to bind to adherins junctions at the cell membrane, the G S K - 3 / A P C / A x i n multi-protein complex in the cytoplasm and to members of the T C F / L E F 64 family of transcription factors in the nucleus (71, 76, 174). The interaction of P-catenin with these various complexes in the cell regulates the stability and functionality of P-catenin. |3-catenin, when stabilized by the inactivation of G S K - 3 , translocates to the nucleus where it binds to the T C F / L E F transcription factors (71, 76, 174). In order to activate transcription, these interactions with D N A binding proteins must occur, as P-catenin has no intrinsic DNA-binding properties (105, 218). Previously, we showed that inactivation of G S K - 3 by the B C R led to the accumulation and stabilization of P-catenin (95). This accumulation of P-catenin leads to an increase in P-catenin-dependent transcription using a reporter gene assay (Figure 3.6) and (95). Figure 3.6 shows that CD40 stimulation by itself is able to increase transcription from the TOPtk plasmid, and this increase in transcription is greater than that observed for B C R stimulation alone (Figure 3.6), although more replicates of this experiment are needed. In combination with B C R stimulation, CD40 is also able to positively affect P-catenin-dependent transcription. Again, the level of transcription induced by C D 4 0 stimulation in combination with B C R stimulation is greater than that induced by B C R stimulation alone (Figure 3.6). The increase in transcription from the FOPtk plasmid observed in response to C D 4 0 stimulation indicates that C D 4 0 induces some non-specific transcription of the luciferase gene (Figure 3.6). This non-specific increase in transcription could be due to the presence of the minimal thymidine kinase promoter in the TOPtk and FOPtk plasmids. In any case the non-specific effect of CD40 on the FOPtk plasmid is by itself not enough to account for the entire difference between the level of transcription observed for C D 4 0 stimulation alone or in combination with B C R stimulation when compared to B C R stimulation alone (Figure 3.6). What is not clear from Figure 3.6 is the mechanism by which C D 4 0 increases B C R -induced P-catenin-dependent transcription. The simplest explanation is that the effect could 65 simply be additive. That is, that CD40 stimulation, through its inhibition of G S K - 3 (Figure 3.1, 2.2), also promotes the accumulation of (3-catenin, and is thereby able to drive transcription of luciferase from the TOPtk plasmid. This hypothesis is supported by the single luciferase experiment in which C D 4 0 was used as the stimulant (Figure 3.6). However, previous data in our lab (S. Christian, P. Sims and M . Gold, unpublished observations) indicated that C D 4 0 stimulation alone might not drive the transcription of luciferase from the TOPtk plasmid, in direct conflict with the data presented here. I attempted to investigate the effect of C D 4 0 stimulation on the accumulation of (3-catenin by Western blotting nuclear and cytosolic cell extracts. However, I was unable to obtain clean nuclear and cytosolic fractions. Further, I attempted to repeat the luciferase experiments in which C D 4 0 alone was used as a stimulant. Transient transfection of B cells only occurs at a very low efficiency, and after attempting to transfect cells using lipid reagents and two types of electroporators, we decided to focus on other experiments. Generation of stable reporter cell lines using retrovirus-mediated gene transfer could help to further explore the role of C D 4 0 in induction of (3-catenin-dependent transcription. Experiments to determine i f the CD40-induced phosphorylation of G S K - 3 leads to the accumulation of P-catenin and activation of P-catenin-dependent transcription are underway in our lab. 4.4 TCF/LEF family members in B cells and their regulation by CD40 Figure 3.6 clearly shows that CD40 is able to augment p-catenin dependent transcription that is initiated by the B C R . Another hypothesis to explain the ability of C D 4 0 to augment B C R -mediated P-catenin dependent transcription is that CD40 increases the expression of a limiting factor that is required for P-catenin to stimulate transcription. Good candidates for these limiting 66 factors are the T C F / L E F family of transcription factors, as they are necessary for (3-eatenin to activate transcription of its target genes (103, 105, 218, 219). A s the expression of these factors in B cells was not well defined when this thesis began, I used R T - P C R to investigate which of these factors were present in B cells at different developmental stages (Figure 3.5). In the earliest developmental stage, the pro-B cell stage, represented by the K40B1 and 300-19 B cell lines, I found the presence of T C F - 4 , but K40B1 cells seem to express more T C F - 4 than 300-19 cells. Reya et al, showed that Wnt signals, which activate |3-catenin-mediated transcription, are important for the proliferation and survival of pro-B cells (122). I found no expression of L E F - 1 in these the K40B1 pro-B cell line, and 300-19 cells only express very low levels of LEF-1 (Figure 3.5). A s L E F - 1 knockout mice die shortly after birth (121), Reya et al, examined primarily fetal liver pro-B cells, but also show that there is a much lower expression of LEF-1 in adult bone marrow when compared to fetal liver (122). The difference in the results could be due to the use of cell lines versus primary cells and could also reflect my use of adult-derived cells that have much lower LEF-1 expression. The next stage of development, pre-B cells are represented by the 70Z/3 cell line, and show expression of L E F - 1 and TCF-1 (Figure 3.5). This cell line was used for cloning the L E F -1 gene, and was shown by Northern blotting to express L E F - 1 (106). Other pre-B cell lines also express L E F - 1 (106), but to my knowledge, this is the first report of T C F - 1 expression in pre-B cells. The WEHI-231 and CH31 cell lines represent immature I g M + B cells. These cells express only TCF-1 (Figure 3.5). The B a l l 7 cell line represents I g M + B cells that are resistant to antigen receptor-mediated apoptosis. Along with T C F - 1 , Bal-17 cells also expressed LEF-1 (Figure 3.5). T C F - 1 , along with L E F - 1 , is also expressed in the IgG + memory B cell lines A 2 0 67 and 2PK3 (Figure 3.5). These cell lines also express T C F - 3 and T C F - 4 , respectively (Figure 3.5). The expression of TCF-1 in B cells is controversial. Qiang et al, found that only L E F - 1 , but not T C F - 1 , -3, or -4 , were expressed in the Daudi, Namalwa and ST486 human B cell lines (146). However, L u et al., show that normal peripheral B cells express all four members of the T C F / L E F family (161), and I found the expression of T C F - 1 , T C F - 4 , and L E F - 1 in B cells isolated from mouse spleen (Figure 3.5). Overall, the data in Figure 3.5 suggests that the expression of these transcription factors is developmentally regulated in B cells. For example, LEF-1 is present in the early stages of B cell development, the pro- and pre- B cell stages. L E F - 1 then is absent in immature B cells when they are most susceptible to antigen-receptor mediated apoptosis. It is again expressed in the final stages of B cell development, memory B cells (Figure 3.5). F A C S sorting of developing B cells, followed by R T - P C R analysis could be used to show the developmental regulation of these transcription factors in mice. After showing that WEHI-231 cells express only TCF-1 (Figure 3.5) and that C D 4 0 increases (3-catenin-dependent transcription induced by the B C R (Figure 3.6), I asked whether C D 4 0 stimulation increases the expression of members of the T C F / L E F family which mediate the effects of p-catenin on transcription. I found that C D 4 0 engagement increased the expression of T C F - 1 in WEHI-231 cells (Figure 3.7). Other groups have reported that C D 4 0 signaling increases the expression of LEF-1 in splenic B cells (180, 181). A n increase in the level of m R N A for a particular gene does not always guarantee a similar increase in the level of corresponding functional protein in the cell. Thus, I attempted to use Western blotting to determine i f the level of TCF-1 protein increases in response to C D 4 0 signaling. However, I was 68 unable to obtain any consistent results, likely due to the poor quality of the commercial antibodies available and low levels of expression of these proteins in B cells. The level of P-catenin-dependent transcription observed for cells stimulated with CD40 alone or in combination with the B C R is greater than that observed for the B C R stimulation alone (Figure 3.6). Assuming that the CD40-induced increase in T C F - 1 m R N A corresponds to a similar increase in the level of TCF-1 protein, the data in Figures 3.6 and 3.7 suggest that in the case of B C R signaling alone the limiting factor in P-catenin-dependent transcription could be the availability of members of the T C F / L E F family of transcription factors. Thus, the regulation of P-catenin by the BCR-mediated inactivation of G S K - 3 , along with the CD40-mediated upregulation of T C F / L E F family members (Figure 3.7, and (180, 181)) could be one way by which signals from the B C R and C D 4 0 are integrated into a transcriptional response. 4.5 Conclusions, persectives and future directions B cells that detect antigen via their antigen receptor require T cell mediated "second signals" to avoid apoptosis (7, 29). The activation of C D 4 0 by its cognate ligand, CD154, which is highly expressed on activated T cells, constitutes one of these important second signals (7, 29). This two-signal system is important for eliminating self-reactive B cells, as well as for activating B cells that have bound foreign antigens (1, 4, 7, 29). A breakdown of this two-signal system, which can be caused by the aberrant expression of the C D 4 0 ligand, CD154, on B cells leads to a lupus-like autoimmune disease in mouse models (220), and may contribute to the development of Hodgkin's lymphoma (221). A s they are important components of the two-signal system, the signaling pathways activated by the B C R and CD40 have a synergistic effect on the activation and differentiation of B cells into plasma cells and memory B cells (19, 20, 222, 223). These 69 final differentiation steps, which require B cell-T cell interactions, occur in secondary lymphoid organs within specialized microdomains called germinal centers (16), which are also absent in mice unable to activate T R A F 6 via C D 4 0 (5). It is in germinal centers that B cells receive the T-cell help required to avoid receptor mediated-apoptosis, which is induced when B cells receive only signals through the B C R (1, 4, 7, 29). The signals initiated by the B C R and CD40 activation of these two receptors must be integrated in order to have these effects. A good example of integration between B C R and C D 4 0 signaling pathways is the regulation of c -Myc by both the B C R and CD40. c-myc is a cellular oncogene implicated in the development of many types of aggressive human cancers including Burkett's lymphoma, a malignant cancer of B cells (153, 224). When the B C R is activated without co-stimulation of CD40, the levels c -Myc protein rise transiently (36, 48, 67, 225). This transient increase and subsequent decrease in the levels of c-Myc to below basal levels plays a role in the activation-induced cell death that occurs when B cells receive only signals through the B C R (36,48, 67, 225). CD40 , which is able to rescue a B cell from BCR-induced apoptosis, is able to cause a sustained increase in the levels of c-Myc in the cell (36, 48, 67, 225). Thus, the level of c-Myc in the B cell remains high and the cell does not undergo apoptosis. The exogenous expression of c-Myc is sufficient to prevent cell death, showing that c -Myc is a critical determinant of life and death decisions, at least in WEHI-231 cells (42). It could be that the lack of germinal centers, long-lived memory cells and plasma cells in mice unable to recruit T R A F 6 in response to CD40 stimulation (5), and therefore activate E R K (212, 213) could be in part due to an inability to upregulate c -Myc via a T R A F 6 / M E K - 1 / E R K / G S K - 3 pathway, leading to excessive apoptosis of antigen-stimulated B cells in the secondary lymphoid organs. 70 A key step in the differentiation of B cells into antibody-producing plasma cells is the clonal expansion of antigen-specific B cell subsets (1,4). During this proliferation in germinal centers, somatic hypermutation of Ig V regions occurs and selection for B cells that bind antigen with high affinity occurs (1, 2 ,4 , 226). Only those cells that bind antigen displayed on the surface of follicular dendritic cells receive survival signals (1, 2 ,4 , 226). The rapid proliferation and somatic hypermutation in G C B cells can sometimes be accompanied by chromosomal rearrangements or other mutations and thus many B cell lymphomas exhibit a G C B cell phenotype (1, 2, 4, 226). G S K - 3 and P-catenin are key regulators of proliferation in many other cell types and have been implicated in the progression of several types of cancer, especially colon cancer (76, 128, 129, 132, 141-143, 155, 196, 227, 228). Mounting evidence implicates disregulation of GSK-3/(3-catenin signaling in various B cell lymphomas and in multiple myeloma (146, 229-232). Recently a N F - K B family member, B C L - 3 , implicated in the development of B cell chronic lymphocytic leukemia, was shown to be a target of G S K - 3 and of E R K 1 (233). Phosphorylation of B C L - 3 by G S K - 3 leads to its degradation (233). Thus CD40 mediated regulation of E R K and G S K - 3 could lead to the activation of B C L - 3 and transcription of pro-proliferative genes. P-catenin, another target of G S K - 3 , can drive the transcription of pro-proliferative genes such as c-myc (130, 152) and cyclin Dl (131). Thus, the proliferation of cells is a direct downstream effect of increased P-catenin stability. Increases in |3-catenin-mediated transcription induced by C D 4 0 either alone or in conjunction with the B C R (Figure 3.6) could promote the proliferation or survival of B cells. The development of proper humoral immune responses, the production of high affinity antibodies, and the development of long-lived memory B cells requires both B C R - and CD40-mediated pro-proliferative signals (1, 4, 7, 17). Support for a pro-proliferative effect of P-catenin on 71 lymphocytes is well documented. For example, T cell antigen receptor signaling increases (3-catenin levels in T cells (169), and abnormal expression of Wnt proteins have been observed in multiple myeloma cells (146, 229). In addition, the deletion of L E F - 1 blocks the development of B cells due to a profound block in the proliferation of B cell precursors in the fetal liver (122). The synergistic induction of P-catenin-dependent transcription by the B C R and CD40 could be an important site of integration for these two signals. A good experiment that could be done to examine if (3-catenin promotes B-cell proliferation would be to place a non-degradable mutant of (3-catenin under the control of an inducible promoter, such as a T E T on/off system. Then the effect of (3-catenin overexpression on B cell proliferation could be studied. Although interactions between T C F / L E F family members and P-catenin are well understood, recent papers show that in Drosophila, at least two other gene products, pygopus and legless, are required for P-catenin-dependent transcription to occur (126, 127, 234). It is thought that these proteins recruit transcriptional machinery to DNA-bound TCF/LEF-P-catenin complexes (126, 127, 234). The human homologue of legless is B C L 9 , a proto-oncogene first identified due to its frequent rearrangement in Burkitt's lymphoma (230, 231), and it is required for the activation of P-catenin-dependent transcription in mammalian systems (232). Nothing is yet known about the regulation of these genes in normal B cell development and activation, but they could be important in the regulation of P-catenin-dependent transcription by the B C R and CD40. In conclusion, in this thesis I have identified a novel pathway by which CD40 induces the phosphorylation of G S K - 3 in B cells. In addition, I have shown that C D 4 0 signaling can affect P-catenin-mediated transcription and that this effect may be due to the ability of C D 4 0 to increase the transcription of T C F - 1 , a binding partner of P-catenin. Several testable questions 72 remain unanswered. First, what are the kinases that link M E K - 1 to G S K - 3 ? A recent paper (188) showed that CD40 activates the M E K / E R K pathway, leading to the activation of p90Rsk in HeLa cells exogenously expressing CD40. p90Rsk is able to phosphorylate G S K - 3 in other cell types (71, 174, 190, 235), but further evidence is required to prove this in B cells. Second, what are the downstream consequences of G S K - 3 phosphorylation in response to C D 4 0 signals, specifically, does G S K - 3 regulate P-catenin and/or c-Myc in C D 4 0 stimulated B cells? The use of pharmacological inhibitors of G S K - 3 and M E K - 1 could help elucidate the role of G S K - 3 in B cells, specifically to determine if G S K - 3 has an effect on B C R and CD40-mediated B cell activation. Alternatively, a non-phosphorylatable GSK-3(3 could be expressed in B cells by infecting B M cells with retroviruses and then using these cells to reconstitute irradiated mice. This system was used to study the role of G S K - 3 in T lymphocytes (236). The same system could be used to study the role of G S K - 3 inhibition in B cells. Lastly, what is the mechanism by which C D 4 0 affects P-catenin-mediated transcription? Specifically, is it only by the CD40-mediated upregulation of TCF-1 that accounts for the relatively large increase in transcriptional activity, when compared to the B C R , or is CD40 able to regulate P-catenin itself? Further study of the mechanisms that regulate this important signaling pathway in normal B cells could lead to the development of new treatments for various B cell malignancies, as well as providing insights into the normal regulation of B cell-mediated immune responses. 73 References 1. Gold , M . R. 2002. To make antibodies or not: signaling by the B-cel l antigen receptor. Trends Pharmacol Sci 23:316. 2. Defrance, T., M . Casamayor-Palleja, and P. H . Krammer. 2002. The life and death of a B cell. Adv Cancer Res 86:195. 3. Gauld, S. B . , J. M . Dal Porto, and J. C. Cambier. 2002. B cell antigen receptor signaling: roles in cell development and disease. Science 296:1641. 4. Niiro , H . , and E . A . Clark. 2002. Regulation of B-cell fate by antigen-receptor signals. 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Always use RNase-free tips, and for the pipetting of template into the 96-well R T -P C R plates use the Ambion plO tips (they are more accurate). 1. Isolate R N A from cells Use TRIzol Spec R N A for concentration, run 1% agarose gel for R N A integrity 2. DNase treatment Use D N A free kit (Ambion) Treat 10 pg R N A in 30 pi H 2 0 Spec R N A for concentration Check for genomic D N A contamination by running 30 cycles of regular P C R with a primer that you know amplifies from genomic D N A 3. Reverse Transcribe RNA using TaqMan (ABI) reagents For each rxn make R T master mix*: Component: pL : lOx Buffer 1.0 25 m M M g C l 2 2.2 2.5 m M dNTP mix 2.0 Primers** 0.5 RNase Inhibitor (20 U / L ) 0.2 Reverse Transcriptase(50 U / p L ) 0.25 U S E 6.15 pi of RT-master mix for each reaction Dilute R N A to use .5pg of R N A in 3.85 pi for each rxn Each rxn should be lOpl total volume *Ensure that you have enough RT master mix for n+2 pts for your experiment with duplicate c D N A of the negative control (to make a standard curve in the P C R reaction). If this is the first time you are doing Q R T - P C R , you wi l l need to make even more c D N A , to ensure that you have enough to optimize your primers. For example, if you have an experiment with 4 time points then make enough master mix for 6 reactions 93 Time course (min) number of rxns needed 0 1 5 15 2 1 1 1 This wi l l ensure that you (1) don't run out of RT-master mix (because you have enough for 1 extra tube and (2) you have enough c D N A for the standard curve that you wil l need to make for analysis of the P C R reaction **Primers for reverse transcription should be optimized (can use oligo-d(T) n , random hexamers or gene-specific primers) The thermal cycler in rm. 32 has a program ( Q R T * C D N A ) that contains the correct temperature cycle as follows: 1. 1 0 m i n @ 2 5 ° C 2. 3 0 m i n @ 4 8 ° C 3. 5 m i n @ 9 5 ° C 4. °o m i n @ 4 °C 4. Primers Primers should be designed using ABI-primer express software (The disc is in a binder above Brad's desk - marked R T - P C R info). Keep several things in mind: a) Primers should be designed for a 50-150 bp amplicon b) G C content should be in 20-80% range c) Melting temperature (T m ) should be 58-60°C d) The five nucleotides at the 3' end should have no more than 2 C ' s or G ' s Design 2-3 primers for each gene, since not all primers wil l work for the amplification of Your Favorite Gene ( Y F G ) . I usually run the primer sequence through a B L A S T search to ensure that it is specific for Y F G . Ordering 2-3 different primers for the same gene at a time wil l reduce lag time if you are in a hurry. When the primers are delivered reconstitute them to 100 p M . a) A d d lOOpl H 2 0 to the lyophilized primers b) Measure absorbance at 260 nm of a 1:100 dilution c) Calculate the concentration using the extinction coefficient as follows: A 2 6 0 = sum of extinction coefficients x cuvette path length x Cone. (pM)/100 d) Calculate and add the correct amount of H 2 0 to each primer to make the final The sum of extinction coefficient is the contribution of each nucleotide to the absorbance at 260. For each nucleotide they are: concentration 100 p M A - 15,200 C - 7050 G - 12,010 T - 8400 94 So for the sequence A T C C G , the sum of the extinction coefficients is: 46100 5. PCR Did you do the tutorial? If not do it now. This means you. Really, it's important. For each well , use a 25[ih reaction volume as follows: 8 u.1 of diluted template 12.5 ul S Y B R green 4.5 [i\ of diluted primers I find that using 10 ng of template/well is a good amount for the amplification reaction. I assume that the RT-reaction is very efficient and converts all of the m R N A into c D N A . Thus all calculations of template concentration are based on: [ cDNA stock] = 50 ng/ul This is because we used 0.5 u,g R N A for the RT reaction. Therefore the template should be diluted 1:40 to use 10 ng c D N A / 8 \x\ total volume for each well . Before you start use a 96-well template to map out your plate. This wi l l allow you to easily figure out the volumes of each dilution that you wi l l need. For the following discussion we wi l l use the above example of an experiment with a 0, 1,5, and 15 minute time course where you are quantifying the relative level of Your Favorite Gene ( Y F G ) , using the level of G A P D H as a reference (non-changing) gene. A. Primer optimization The first thing that must be done is the concentration of primers must be optimized. Most primers work well in a 50 to 900 n M range. That is, each well requires 50-900 n M of the forward and 50-900 n M of the reverse primer. To optimize the primer concentration run a P C R reaction with 50 n M primers, 300 n M primers and 900 n M primers for Y F G and examine if there is any difference in the amount of amplification between the three concentrations. If there is not, then use the lowest primer concentration that still gives maximum amplification. Also , use this optimization experiment to check that there is no non-specific amplification in the well . Do this 2 ways: 1. Compare the dissociation curve for the No Template Controls (NTC) and the unknown wells. The N T C should have a peak at a lower temperature than that of the unknowns. If the unknowns have more than one peak, then there is more than one P C R product in the well and you have non-specific amplification, and you should use a different primer set. 2. Run the P C R products on an agarose gel, if there is more than one band on the gel, there is non-specific amplification and you should use a different primer set 95 B. Relative Quantification Relative quantification is the use of a normalizing gene (one that does not change) to quantify the fold change in the expression level of YFG. The best (fastest, easiest to optimize) way to do relative quantification is to use standard curves, which are made with serial dilutions of your negative control cDNA (For example, the 0 time point of the experiment described under "reverse transcription"). Remember we assume that the stock [cDNA] = 50 ng/pl. Serial dilutions for standard curves: Dilution factor [cDNA] (ng/pl) 8pl = ng cDNA 1 50 20 2.5 20 40 1.25 10 80 .625 5 160 .3125 2.5 320 .15625 1.25 640 .078125 .625 1280 .0390265 .3125 Make sure that you have enough of each dilution for the number of wells that require template. See attached example template and count the number of wells for each. So, for our example for the quantification of YFG, with GAPDH as a normalizing reference gene, we refer to the attached 96-well template and see that we need enough of the 20 ng dilution for 6 wells: 3 for the YFG standard and 3 for the GAPDH standard, a total of 53 pi (6 wells x 8pl/well x 1.1 = 53pi). After making the dilutions for the standard curve, dilute the other time points 1:40 for use in the unknown wells, again remember to make excess dilution to ensure that you have enough template for all the wells. Dilute the primers to the correct concentration that you determined empirically (i.e. either 50-900 nM). Ensure that you have enough of each primer for all the wells that require it (I usually do this by multiplying all the volumes by 1.1). Back to our example of YFG and GAPDH: Stock primer concentrations = lOOpM Use primers at 300 nM/well # of wells = 36/each gene Primer name Vol. stock (pi) Vol. H2Q (ul) Total vol. YFG forward 3 174 180 YFG reverse 3. GAPDH forward 3 174 180 GAPDH reverse 3 Then mix the diluted primers with 2x SYBR green solution. Again multiply the volumes by 1.1, to ensure you have enough mix. Use 17 pi of this mixture for each well: 96 Gene Y F G G A P D H #wells 36 36 xl.l 40 40 vol. S Y B R (uL) 500 500 V o l . Primers 180 180 Pipette the S Y B R + Primers mixture into the appropriate wells (17 u,l). Then add diluted template (8ul). Note: the Q R T - P C R machine is sensitive enough to detect very small differences in the amount of template thus, i f you are sloppy about pipetting your samples, you wil l introduce large amounts of error into you data. This is bad. Ergo, make sure you prime your pipette tips (pipette up and down a few times) and use the plO whenever possible. After pipetting cover the 96-well optical plate with an adhesive cover, and spin the liquid to the bottom of the plate using the plate adaptors for the Beckman centrifuge. Go to the Schulte lab in Bio-Sci . Set up a plate document (.sds document) on the laptop. The tutorial shows you how to do this. Remember to change the reaction volume to 25ui and to have the program generate a dissociation curve. Wait about 2 hours. Go get your mountain of data for analysis. Bring a blank C D to save it on. C . Data Analysis Before exporting your data to Excel , you should check several things: 1. Baseline. Under the amplification tab of results in the ABI-pr i sm program, there is a setting called baseline. I think that this is a measurement of the autoflourescence of the sample. The default for this option is for the baseline to be taken from cycles 6-15. Make sure that the baseline measurement ends 1-2 cycles before amplification begins. 2. Threshold. Threshold is the arbitrary point that you choose to compare Y F G to the normalizing gene. Make sure the threshold line passes through the amplification plot where it is still linear. It is best if you can choose a threshold which passes through the linear sections of all the curves, but which the N T C does not cross. 3. Check the dissociation curve to ensure that there is no non-specifc amplification in the wells. 4. Export the data to Excel , and analyze for fold increase or decrease relative of Y F G compared to the normalizing gene. The basic idea for data analysis of these results is that you use the standard curves to convert the C T value that is measured by the P C R machine into "ng". The "ng" of c D N A for Y F G is divided by the "ng" for G A P D H , to normalize the data. Then the normalized "ng" of Y F G are divided by the 0 time point to get a "relative fold increase" in m R N A compared to unstimulated 97 control. The excel spreadsheet called " Q R T - P C R analysis" in the shared folder on the G4 is set up such that you should only have to cut and paste your data into the correct place. If you want to know any more about any of this, the binder on Brad's desk labeled Q R T - P C R information is where to look.. . 98 1 2 3 4 5 6 7 8 9 10 11 12 A Y F G N T C Y F G N T C Y F G N T C Y F G Std 20ng Y F G Std 20ng Y F G Std 20ng Y F G Std lOng Y F G Std lOng Y F G Std lOng Y F G Std 5ng Y F G Std 5ng Y F G Std 5ng B Y F G Std 2.5ng Y F G Std 2.5ng Y F G Std 2.5ng Y F G Std 1.25ng Y F G Std 1.25ng Y F G Std 1.25ng Y F G Std . 0.625 n? Y F G Std 0.625 ng Y F G Std 0.625 ng Y F G Std 0.3125 ng Y F G Std 0.3125 ng Y F G Std 0.3125 ng C G A P D H N T C G A P D H N T C G A P D H N T C G A P D H Std 20ng G A P D H Std 20ng G A P D H Std 20ng G A P D H Std lOng G A P D H Std lOng G A P D H Std lOng G A P D H Std 5ng G A P D H Std 5ng G A P D H Std 5ng D G A P D H Std 2.5ng G A P D H Std 2.5ng G A P D H Std 2.5ng G A P D H Std 1.25ng G A P D H Std 1.25ng G A P D H Std 1.25ng G A P D H Std 0.625 G A P D H Std 0.625 ng G A P D H Std 0.625 r\ cr G A P D H Std 0.3125 t-i cr G A P D H Std 0.3125 rt cr G A P D H Std 0.3125 "f E Y F G 0 min Y F G 0 min Y F G 0 min Y F G 1 min Y F G 1 min Y F G 1 min Y F G 5 min Y F G 5 min Y F G 5 min Y F G 15 min Y F G 15 min Y F G 15 min F G A P D H 0 min G A P D H 0 min G A P D H 0 min G A P D H 1 min G A P D H 1 min G A P D H 1 min G A P D H 5 min G A P D H 5 min G A P D H 5 min G A P D H 15 min G A P D H 15 min G A P D H 15 min G H Appendix B - SDF-1 stimulation of WEHI-231 cells results in GSK-3 and Akt phoshorylation SDF-1 induces migration and morphological changes in WEHI-231 cells, and the activation of the P13K pathway has been implicated in lymphocyte polarization and migration induced by SDF-1 (237-239). Wnt stimulation of multiple myeloma cells leads to morphological changes and activation of the Rho GTPase (146), and G S K - 3 inactivation is important for polarization and lamellapodia formation in other cell types (82, 83, 89). I found that SDF-1 stimulation of WEHI-231 cells results in the transient phosphorylation of both A k t and G S K - 3 (Figure B . l ) . Thus, the SDF-1-mediated phosphorylation of A k t and G S K - 3 could play a role in the polarization of B lymphocytes in response to SDF-1 . 100 Figure B . l : SDF-1 stimulation leads to the phoshorylation of GSK-3 and Akt A and B , Upper panels: WEHI-231 cells were stimulated with 100 ng/ml of the SDF-1 for the indicated times. Cel l extracts (30 u,g protein) were analyzed for G S K - 3 phosphorylation using the anti-P-GSK-3cx/GSK-3|3 A b (A) and for A k t phosphorylation using the anti-P-Ser473 A K T A b (B). To ensure equal loading, the membranes were stripped and reprobed with Abs against GSK-3|3 or A k t (A and B , lower panels). Similar results were obtained in 2 independent experiments. 101 SDF-1 (min) ant i-P-GSK-3a/p blot anti-GSK-3|3 reprobe P-GSK-3a ^ P - G S K - 3 ( 3 r45 GSK-3|3 B. SDF-1 (min) anti-P-Ser473 Akt blot anti-Akt reprobe 0 5 15 30 45 - 6 6 P-Ser473-Akt 66 Akt 102 

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