REDOX-REGULATED SIGNALING BY LACTOSYLCERAMIDE IN THE PROLIFERATION OFHUMAN AORTIC SMOOTH-MUSCLE CELLS

Previously, our laboratory reported that lactosylcer-amide (LacCer) st imulated human aortic smooth muscle cell proliferation via specific ac tivation of p44 mitogen-activated protein kinase (MAPK) in the p21(ras )/Raf-1/MEK2 pathway and induced expression of the transcription facto r c-fos downstream to the p44 MAPK signaling cascade (Bhunia A, R., Ha n, H., Snowden, A., and Chatterjee S. (1906) J. Biol. Chem. 271, 10660 -10666). In the present study, we explored the role of free oxygen rad icals in LacCer-mediated induction of cell proliferation. Superoxide l evels were measured by the lucigenin chemiluminescence method, MAPK ac tivity was measured by immunocomplex kinase assays, and Western blot a nalysis and c-fos expression were measured by Northern blot assay, We found that LacCer (10 mu M) stimulates endogenous superoxide productio n tl-fold compared with control) in human aortic smooth muscle cells s pecifically by activating membrane-associated NADPH oxidase, but not N ADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants, N ace tyl-L-cysteine (NAC or pyrrolidine dithiocarbamate. NAC and DPI both a brogated individual steps in the signaling pathway leading to cell pro liferation. For example, the p21(ras.)GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduc ed glutathione (GSH), In contrast, depletion of GSH by L-buthionine (S ,R)-sulfoximine up-regulated the above described signaling cascade. In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxi de (a redox stress signaling molecule), which mediates cell proliferat ion via activation of the kinase cascade, Our findings may er;plain Sh e potential role of LacCer in the pathogenesis of atherosclerosis invo lving the proliferation of aortic smooth muscle cells.