Open Collections

Oxidized low density lipoprotein mediated macrophage survival : an essential role for PI3K/PKB pathway

University of British Columbia

Oxidized LDL (ox-LDL) has previously been shown to induce endothelial adhesion molecule, MCP-1 and M-CSF expression thereby leading to monocyte recruitment, adherence and macrophage differentiation and has recently been shown to induce macrophage proliferation. The purpose of this study was to examine an additional mechanism by which ox-LDL might increase macrophage populations - enhanced macrophage survival. I found that ox-LDL caused a dose-dependent inhibition of the apoptosis that occurs in cultured bone marrow-derived macrophages following M-CSF withdrawal. Incubation of macrophages with either native LDL or acetylated LDL had no effect on survival, thereby suggesting an oxidation specific event. The pro-survival effect of ox-LDL was not inhibited by neutralizing antibodies to GM-CSF, was maintained in mice homozygous for a mutation in the M-CSF gene, and was not due to other secreted cytokines or growth factors. This suggested that ox-LDL activated cytokine-independent intracellular survival signaling pathways. Ox-LDL caused activation of the MAP kinases ERK1/2 as well as protein kinase B (PKB), a target of phosphatidylinositol 3-kinase (PI3K). Furthermore, there was phosphorylation of two important PKB downstream targets lкB-α-Ser-32 and Bad- Ser-136, both of which act at the level of the pro-survival Bcl-2 protein, Bcl-Xι to inhibit apoptosis. The MEK inhibitors PD 98059 and U0126 blocked ERK1/2 activation, but did not diminish survival. Conversely, the PI3K inhibitors LY 294002 and wortmannin blocked PKB activation, and the ability of ox-LDL to promote macrophage survival. I also demonstrated that ox-LDL prevented the elaboration of ceramide following the withdrawal of M - CSF, and pre-treatment with soluble ceramides attenuated ox-LDL mediated macrophage survival and PKB activation. The source of ceramide elaboration was through the metabolism of sphingomyelin to ceramide rather than de novo synthesis as determined by inhibitor studies with fumonisin BI and desipramine; as well as by determination of levels of sphingomyelin. Ox-LDL was also able to prevent the decline in the levels of Bcl-XLthat occur following M - CSF withdrawal. Taken together, these results indicate that ox-LDL prevents ceramide elaboration, activates a PI3K/PKB pathway that reduces cytosolic sequestration of NF-кB , as well as phosphorylates Bad and thereby promotes macrophage survival in the absence of growth factors at the level of Bcl-Xι and caspase 3 activation.

Thesis/Dissertation

application/pdf

2009-11-03

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/14626

Experimental Medicine

University of British Columbia

UBCV

DSpace