Lactosylceramide stimulates ras-GTP loading, kinases (MEK, Raf), p44 mitogen-activated protein kinase, and c-fos expression in human aortic smooth muscle cells

Previously, our laboratory has shown that lactosylceramide (LacCer) can serve as a mitogenic agent in the proliferation of aortic smooth muscle cells 'a hallmark in the pathogenesis of atherosclerosis' (Chatterjee, S. (1991) Biochem. Biophys. Res. Commun. 181, 554-561). Here we report a novel aspect of LacCer-mediated signal transduction. We demonstrate that LacCer (10 μM) can stimulate the phosphorylation of mitogen-activated protein (MAP) kinase p44(MAPK) to phosphorylated p44(MAPK) in aortic smooth muscle cells from rabbit or human origin. Western immunoblot assays and direct measurement of activity in immunoprecipitated MAP kinase revealed that within 5 min of incubation of cells with LacCer there was a 3.5-fold increase in the activity of p44(MAPK). This continued up to 10 min of incubation; thereafter, the MAP kinase activity decreased in these cells. Phosphoamino acid analysis revealed that the tyrosine and threonine moieties of p44(MAPK) was phosphorylated by LacCer. Incubation of cells with ceramide and glucosylceramide did not significantly stimulate p44(MAPK) activity. Preincubation with tyrphostin (20 μM; a potent and specific inhibitor of tyrosine kinase) markedly inhibited the LacCer mediated stimulation in p44(MAPK) activity. Next we investigated the upstream and downstream parameters in MAP kinase signaling pathways. We found that lactosylceramide stimulated (7-fold) the loading of GTP on Ras. Concomitantly, LacCer stimulated the phosphorylation of MAP kinase kinases (MEK) and Raf within 2.5 min. Lactosylceramide specifically induced c-fos mRNA expression (3-fold) in these cells as compared to control. In summary, one of the biochemical mechanisms in LacCer mediated induction in the proliferation of aortic smooth muscle cells may involve Ras-GTP loading, activation of the kinase cascade (MEK, Raf, p44(MAPK)), and c-fos expression.