Oxidized low density lipoproteins stimulate galactosyltransferase activity, ras activation, p44 mitogen activated protein kinase and c-fos expression in aortic smooth muscle cells

0 Department of Pediatrics, Lipid Research Unit, The Johns Hopkins University, School of Medicine , Baltimore, MD 21287-3654 , USA 1 , Anil K.Bhunia , Ann Snowden and Hui Han Oxidized low density lipoproteins stimulate galactosyltransferase activity, ras activation, p 4 4 mitogen activated protein kinase and c-fos expression in aortic smooth muscle cells - Previously, our laboratory has shown that oxidized low density lipoproteins (Ox-LDL) can exert a concentrationdependent stimulation in the proliferation of aortic smooth muscle cells, "a hallmark in the pathogenesis of atherosclerosis" (ChatterjeeyS. (1992) Mol Cell Biochenu, 111, 143147). Here we report a novel aspect of Ox-LDL-mediated signal transduction. We demonstrate that in aortic smooth muscle cells, Ox-LDL stimulates the activity of a UDPgalactose:glucosylceramide pi*4 galactosyltransferase (GalT-2) and phosphorylation/activation of p44 mitogenactivated protein (MAP) kinase (p44 MAPK). The activity of GalT-2 increased about 2-fold within 2.5-5 min of incubation of cells with Ox-LDL (10 ug/ml). After 5 min of incubation of cells with Ox-LDL, but not LDL, there was a 2-fold increase in the activity of p44 MAPK. Phosphoamino acid analysis employing thin layer chromatography revealed that the tyrosine and threonine moieties of p44 MAPK was phosphorylated by Ox-LDL. D-l-Phenyl-2decanoylarnino-3-morpholino-l-propanol ( D - P D M P ; a potent inhibitor of GaIT-2) impaired the Ox-LDL mediated induction of p44 MAPK activity and the phosphorylation of tyrosine and threonine residues in p44 MAPK. This phenomenon was bypassed by the simultaneous addition of lactosylceramide. The upstream and downstream parameters in MAP kinase signaling pathways were investigated next We found that Ox-LDL stimulated (9-fold) the loading of GTP on Ras. Interestingly, Ox-LDL specifically induced c-fos mRNA expression (6.5-fold) in these cells, as compared to the control. Thus, one of the biochemical mechanisms in Ox-LDL mediated induction in the proliferation in aortic smooth muscle cells may involve GalT-2 activation, lactosylceramide production, Ras GTP loading, activation of the kinase cascade, and c-fos expression. MAPK/signaling/lactosylceramide/ Introduction The role of Ox-LDL in atherosclerosis has become increasingly evident, primarily because of the finding that such modified lipoproteins were found associated widi the atherosclerotic plaques (Morton et al., 1986). Second, when Watanabe heritable hyperlipidemic rabbits were exposed to "Probucol" (a potent antioxidant), this prevented the atherosclerotic process (Carew et al, 1987; Kita et al, 1987). Although these lipoproteins could potentially cause injury to the aortic wall, very little is known regarding its biochemical mechanism of action. Our laboratory was the first to demonstrate that Ox-LDL stimulates the proliferation of aortic smooth muscle cells (Chatterjee, 1992). In a subsequent study, we reported that Ox-LDL can specifically stimulate the synt h e s i s of l a c t o s y l c e r a m i d e by a c t i v a t i n g a U D P Gal:glucosylceramide pi^4 galactosyltransferase (GalT-2; Chatterjee and Ghosh, 1996). Moreover, large amounts of OxLDL and LacCer were found to be associated witii aortic plaque intima from patients who died of atherosclerosis compared to unaffected intima (Chatterjee et al, 1997). In other studies, we reported that exogenously supplied lactosylceramide (LacCer) stimulates the proliferation of smooth muscle cells by stimulating Ras GTP loading, kinases (MEK, Raf), p44 mitogen-activated protein kinase (MAPK), and c-fos expression (Bhunia et al., 1996; Chatterjee and Ghosh, 1996). Accordingly, we rationalized mat if LacCer serves as a signaling molecule in Ox-LDL mediated cell proliferation, then inhibition of GalT-2 should abrogate this signaling phenomena. In this article, we present evidence that Ox-LDL specifically activates GalT-2 and the phosphorylation of tyrosine and tfireonine residues in p44 MAPK in cultured human aortic smooth muscle cells. This phenomena is abrogated by D - P D M P , an inhibitor of GalT-2. The best known mitogen-activated protein (MAP) kinases are p44 MAPK (extracellular signal regulated kinase, ERK,) and p42 MAPK (ERK2). These are a group of serine/threonine protein kinases that constitute an activation process triggered by a variety of growth stimuli (Boulton et aL, 1991; Rossomando et al, 1992; Davis, 1994; Cano and Mahadevan, 1995). Such protein kinases have been suggested to phosphorylate and activate transcriptional factors such as c-myc (Seth et al., 1991; Traverse et al, 1992), c-fos (Deng and Karin, 1994), and p62TCF(GUle etal., 1992; Marais et aL, 1993), which regulates the expression of genes essential for cell proliferation (Davis, 1993). In mis article, we present evidence that upstream activators, such as Ras, are involved in Ox-LDL mediated activation of p44 MAPK The latter, in turn, specifically stimulates c-fos proto-oncogene expression that eventually leads to cell proliferation. Effects of concentration of Ox-LDL, LDL and Ox-LDL antibody on the phosphorylation/activity of p44 MAPK Ox-LDL exerted a concentration-dependent stimulation in the phosphorylation as shown in Western immunoblot assays (Figures 2, 3). The maximum stimulation (-3-fold) in die activity Fig. 1. Effects of Ox-LDL and LDL concentrations on MAP kinase activity in cultured human aortic smooth muscle cells. Cells were treated with various concentrations (0-50 u.g/ml) of Ox-LDL and LDL for 10 min and lysed in lysis buffer. The MAP Irinases were immunoprecipitated from the cell lysate with anti-MAP-kinase antibody conjugated with protein A/agarose and MAPK activity was measured in the immunoprecipitates. Each point is the mean SD of three individual experiments. D, Ox-LDL; , LDL of MAPK occurred with 10 ^g/ml Ox-LDL (Figure 1). At a higher concentration of Ox-LDL (50 (xg/ml), MAPK activity was somewhat decreased, compared to the control. In contrast, MAPK Western immunoblot assays revealed that incubation of cells with similar concentrations of LDL (Figures 1, 2B,D) did not appreciably stimulate the phosphorylation of p44 MAPK. The antibody against Ox-LDL abrogated the Ox-LDL mediated increase in the phosphorylation of p44 MAPK (Figure 2B,D). Interestingly, Ox-LDL did not alter the level of nonphosphorylated form of p44 MAPK (middle band in Figure 2A) and nonphosphorylated p 4 2 MAPK (bottom band in Figure 2), whereas the mass of phosphorylated form of p44 MAPK (indicated by an arrow in Figure 2) was altered. Effects of time of incubation with Ox-LDL on the activation ofp44MAPK We found that, within 5 min of incubation of cells with OxLDL (10 u,g/ml), a significant increase (2-fold) in the activation of p MAPK occurred (Figure 3). Maximum stimulation (3-fold) in the activation of p MAPK, as compared to the control, occurred 10 min after the incubation of cells with Ox-LDL. Thereafter, the activation of p44 MAPK decreased continuou (...truncated)