INTERDEPENDENCE OF CHOLESTEROL METABOLISM AND GLYCOPROTEIN ASSEMBLY IN CULTURED AORTIC SMOOTH MUSCLE CELLS

Abstract

The pathways of cholesterol biosynthesis and dolichyl(pyro)-phosphate-mediated glycoprotein assembly were found to be coordinately regulated in the cultured calf aortic smooth muscle cell. Such interdependent mode of regulation was deduced from several types of experiments utilizing specific inhibitors of steroidogenesis.;Treatment of the cells with the oxygenated steroid, 25-hydroxycholesterol, resulted in the diminished incorporation of acetate (80-90%), but not mevalonate, into dolichyl(pyro)phosphate and cholesterol, suggesting that HMG-CoA reductase catalyzed a reaction that was rate-limiting for the syntheses of both compounds. The inhibition occurred within 4-5 hours and was followed some 8-14 hours later by an inhibition (40-50%) of glucose incorporation into dolichyl(pyro)phosphate- and peptide-bound oligosaccharides.;A 24-hour exposure of the cells to 25-hydroxycholesterol resulted in a generalized and severe diminution of glycoprotein biosynthesis as monitored by {lcub}('14)C{rcub}glucosamine incorporation. Glycoproteins, susceptible to this treatment, were isolated from control cells by preparative SDS-polyacrylamide gel electrophoresis and hot phenol-aqueous urea extraction, and were found to contain asparagine-linked carbohydrate units which were similar in size to calf thyroglobulin saccharide units A and B. Tunicamycin treatment specifically inhibited dolichyl saccharide formation and glycoprotein synthesis in a manner similar in magnitude and specificity to that observed with the oxygenated sterol. Because of the specificity of tunicamycin, the parallel results obtained with the oxygenated sterol suggested that the latter was indeed modulating and limiting the availability of phosphorylated dolichols. Microsomes prepared from sterol-treated cells were unable to efficiently carry out dolichylphosphate-dependent mannosyltransferase reactions. However this situation was reversed when exogenous dolichylphosphate was included in reactions.;In addition to depleting endogenous pools of dolichylphosphate, treatment of the cells with 25-hydroxycholesterol resulted in a significant (40-50%) loss of cellular cholesterol. Mevalonolactone was found to rescue dolichyl saccharide synthesis completely and glycoprotein assembly partially, but did not prevent the loss of cellular cholesterol.;Triparanol, a general inhibitor of (DELTA)('24)-sterol reductase, inhibited the synthesis of cholesterol at low concentrations and of cholesterol and glycoproteins, but not dolichyl saccharides, at higher concentrations. As in the case of the oxygenated sterol, triparanol treatment resulted in a loss of cellular cholesterol which was more severe at higher concentrations of the inhibitor.;The competitive inhibitor of HMG-CoA reductase, compactin, inhibited the synthesis of cholesterol, dolichyl saccharide, and glycoproteins more rapidly than did 25-hydroxycholesterol; however, the effects of the former were more readily reversible than those of the latter.;Taken together, the data suggest that there exists an interdependent regulation of the pathways for cholesterol and glycoprotein biosynthesis. The pathways of cholesterol, dolichylphosphate, and glycoprotein synthesis appear to comprise both shared and independent regulatory sites, while those of cholesterol and glycoproteins possess additional regulatory sites which are independent of dolichylphosphate synthesis.