EICOSANOID METABOLISM IN CHOLESTEROL-ENRICHED ARTERIAL SMOOTH-MUSCLE CELLS - EVIDENCE FOR REDUCED POSTTRANSCRIPTIONAL PROCESSING OF CYCLOOXYGENASE-I AND REDUCED CYCLOOXYGENASE-II GENE-EXPRESSION

Eicosanoid biosynthetic activity by the cyclooxygenase pathway is reduced in smooth muscle cell-derived foam cells [Pomerantz, K. B., & Hajjar, D. P. (1989) J. LipidRes. 30,1219-1231; Pomerantz, K.B., & Hajjar, D.P.(1990) Biochemistry 29, 1892-1899]. The present study identifies those mechanisms which contribute to reduced production of cyclooxygenase products following cholesterol enrichment of arterial smooth muscle cells. Cyclooxygenase activity, asassessed by the conversion of exogenous arachidonate to 6-keto-PGF1alopha, was reduced approximately 8-fold in intact lipid-laden cells relative to untreated cells. Microsomes from cholesterol-enriched cells also converted less [H-3]arachidonic acid to 6-keto-PGF1alpha and PGE2 relative to microsomes from untreated cells. The reduction in cyclooxygenase activity paralleled the reduced mass of the constitutive form of cyclooxygenase (COX-1) and PGI2 synthase by approximately 80% and 33%, respectively. Northern blot hybridization analyses of COX-1 mRNA steady-state levels revealed no differences between normal and cholesterol-enriched cells under basal conditions, indicating that cholesterol enrichment did not alter COX-1 gene expression. Furthermore, cholesterol enrichment did not alter the relative levels of COX-1 mRNA expression over time following exposure of the cells to actinomycin D, indicating that cholesterol enrichment did not significantly alter the rate of COX-1 mRNA degradation. Recovery of PGI2 biosynthesis in untreated cells exposed to serum following the inactivation of COX occurred within 12 h, while the recovery of COX activity in lipid-enriched cells did not return to levels observed in untreated cells even after up to 48 h, suggesting that the induction of COX-2 (inducible form of cyclooxygenase) synthesis by growth factors or cytokines is impaired. Indeed, cholesterol enrichment attenuated IL-1beta-, PDGF-, and TN Fa-induced PGI2 synthesis relative to controls and was consistent with the results of in vitro labeling experiments demonstrating that cholesterol enrichment reduced the incorporation of [S-35]methionine into immunoprecipitable COX-1 and COX-2 following induction by PDGF. Cholesterol enrichment also reduced the induction of COX-2 mRNA steady-state levels following exposure to PDGF. Taken together, these data demonstrate that reduced eicosanoid synthesis in smooth muscle-derived foam cells is due, in part, to impaired transcription of mRNA for COX-1 and COX-2 as well as fatty acid remodeling in membrane phospholipids. These findings support the hypothesis that cholesterol enrichment alters posttranscriptional processing of COX-1 expression, as well as altering COX-2 gene expression.