Potential role for ceramide in mitogen-activated protein kinase activation and proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein

Proliferation of vascular smooth muscle cells (SMC) is a hallmark in the pathogenesis of atherosclerotic lesions. Mildly oxidized low density lipoproteins (UV-oxLDL), which are mitogenic to cultured AG-08133A SMC, activate the sphingomyelin (SM)-ceramide pathway. We report here the following. (i) UV-oxLDL elicited a biphasic and sustained activation of MBP kinase activity, phosphorylation and nuclear translocation of p44/42 mitogen-activated protein kinase (MAPK), and [H-3]thymidine incorporation, which were inhibited by PD-098059, a MAPK kinase inhibitor. (ii) The use of preconditioned media (from SMC pre-activated by UV-oxLDL) transferred to native SMC and blocking antibodies against growth factors suggest that UV-oxLDL-induced activation of MAPK and [H-3]thymidine incorporation seem to be independent of any autocrine secretion of growth factors. (iii) UV-oxLDL-induced activation of a neutral sphingomyelinase, SM hydrolysis, ceramide production, and [H-3]thymidine incorporation were inhibited by two serine-protease inhibitors (serpins), suggesting that a serpin-sensitive proteolytic pathway is involved in the activation of the SM-ceramide signaling pathway. (iv) UV-oxLDL-induced MAPK activation and [H-3]thymidine incorporation were mimicked by ceramide generated in the plasma membrane by bacterial sphingomyelinase treatment or by addition of the permeant C-2-ceramide. Serpins did not inhibit the MAPK activation and [H-3]thymidine incorporation induced by C-2-ceramide, indicating that activation of the MAPK and [H-3]thymidine incorporation is subsequent to the stimulation of the SM-ceramide pathway. Taken together, these data suggest that mitogenic concentrations of UV-oxLDL are able to stimulate the SM-ceramide pathway through a protease-dependent mechanism and activate p44/42 MAPK, leading to proliferation of vascular SMC.