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

Abstract

Apoptosis is an important mechanism involved in regulating the number of macrophages present at sites of inflammation. Several lines of evidence indicate that blocking macrophage apoptosis can increase atherosclerosis. We previously reported that oxidized LDL (oxLDL) can inhibit apoptosis in cultured bone marrow-derived macrophages in part by activating the phosphoinositide 3 kinase (PI3K)/protein kinase B (PKB) pathway and subsequent expression of pro-survival protein Bcl-X L. Here we report that oxLDL also alters the levels of the pro-apoptotic protein, Bax. This effect of oxLDL on Bax regulation was at a post-transcriptional level, mediated by accelerated degradation via the ubiquitin-proteasome pathway. However, Bax knockout macrophages were not resistant to apoptosis following cytokine withdrawal, suggesting that the downregulation of Bax is only partially responsible for the pro-survival effects mediated by oxLDL in these cells. OxLDL is also able to increase the expression of the prosurvival relative, Mcl-1. The effect of oxLDL on Bax degradation and Mcl-1 expression was blocked by inhibitors of the PI3K/PKB pathway. To investigate the upstream receptor(s) activated by oxLDL to mediate macrophage survival, we used pertussis toxin (PTX) to test whether Gi protein coupled receptors are involved. Unexpectedly, we found that PTX by itself selectively blocks macrophage apoptosis in a dose-dependent manner. PTX acts in part by inhibiting acid sphingomyelinase activity which in turn prevents generation of ceramide during apoptosis. A Gi activator peptide, mastoparan, increased ceramide levels in macrophage and induced apoptosis, but pre-treatment with PTX partially overrode mastoparan-induced apoptosis. PTX failed to prevent ASMase activation or apoptosis in macrophages lacking toll-like receptor 4 (TLR4). Like oxLDL, the anti-apoptotic effect of PTX also activated the PI3K/PKB pathway which led to nuclear localization of the transcription factor NFκB and up-regulation of Bcl-X L. These results indicate that Gi proteins, TLR4, ASMase and the PI3K/PKB pathway are crucial components for regulation of macrophage apoptosis. We also looked at regulation of ceramide generation in response to apoptosis. Using ASMase-/- mice, we found that ceramide is still generated. Using inhibitors to enzymes involved in the de novo ceramide synthesis pathway, we concluded that both de novo synthesis and sphingomyelin hydrolysis can contribute to ceramide generation during macrophage apoptosis.

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