LACTOSYLCERAMIDE STIMULATES RAS-GTP LOADING, KINASES (MEK, RAF), P44 MITOGEN-ACTIVATED PROTEIN-KINASE, AND C-FOS EXPRESSION IN HUMAN AORTICSMOOTH-MUSCLE CELLS

Previously, our laboratory has shown that lactosylceramide (LacCer) ca n serve as a mitogenic agent in the proliferation of aortic smooth mus cle cells ''a hallmark in the pathogenesis of atherosclerosis'' (Chatt erjee, S. (1991) Biochem. Biophys. Res. Commun. 181, 554-561). Here we report a novel aspect of LacCer-mediated signal transduction. We demo nstrate that LacCer (10 mu M) can stimulate the phosphorylation of mit ogen-activated protein (MAP) kinase p44(MAPK) to phosphorylated p44(MA PK) in aortic smooth muscle cells from rabbit or human origin. Western immunoblot assays and direct measurement of activity in immunoprecipi tated MAP kinase revealed that within 5 min of incubation of cells wit h LacCer there was a 3.5-fold increase in the activity of p44(MAPK). T his continued up to 10 min of incubation; thereafter, the MAP kinase a ctivity decreased in these cells. Phosphoamino acid analysis revealed that the tyrosine and threonine moieties of p44(MAPK) was phosphorylat ed by LacCer. Incubation of cells with ceramide and glucosylceramide d id not significantly stimulate p44(MAPK) activity. Preincubation with tyrphostin (20 mu M; a potent and specific inhibitor of tyrosine kinas e) markedly inhibited the LacCer mediated stimulation in p44(MAPK) act ivity. Next we investigated the upstream and downstream parameters in MAP kinase signaling pathways. We found that lactosylceramide stimulat ed (7-fold) the loading of GTP on Ras. Concomitantly, Lac-Cer stimulat ed the phosphorylation of MAP kinase kinases (MEK) and Raf within 2.5 min. Lactosylceramide specifically induced c-fos mRNA expression (3-fo ld) in these cells as compared to control. In summary, one of the bioc hemical mechanisms in LacCer mediated induction in the proliferation o f aortic smooth muscle cells may involve Ras-GTP loading activation of the kinase cascade (MEK, Raf, p44(MAPK)), and c-fos expression.