Transient elevation of cAMP by prostaglandins triggers subsequent up-regulation of LDL receptor activity in cultured human cells

Abstract

Previous work [ 1.2.31 in this laboratory has shown that increased cell cAMP has dual effects on expression of the LDL receptor (LDLR) in human exlrahepatk cells (hSF and hVSMC). i) CAMP upregulates JDLR activity by increasing the rate of receptor protein synthesis; this is not secondary to sterol regulation as the effect persists in the presence of 25-hydroxycholesterol and is unaffected by the HMGCoA reductase inhibitor mevinolin. ii) CAMP also stimulates lysosomal cholesterol ester hydrolase [4], releasing free cholesterol which can inhibit LDLR expression. We have shown 151 that PGE1, E2 and the stable PGI2 analogue Iloprost increase LDLR activity in hSF and hVSMC. This is a true up-regulation, requiring at least 8h preincubation. and depending on mRNA and protein synthesis. We now consider the mechanism of this PG-mediated upregulation. To simplify interpretation of the data, we carried out the investigations in the presence of the lysosomal inhibitors CQ or WCl to prevent down-regulation of the LDLR due to mobilization of cholesterol from lysosomal esters as explained in ii) (above). To show that PG caused real up-regulation of the LDLR, all experiments were carried out on cells in which LDLR number was already up-regulated by 24h preincubation in LPDM. Cells were then incubated with P G s and lysosomal inhibitors for up to 24h in SFM before removing these agents for LDLR measurements. Cell surface expression of the LDLR was measured by the specific binding of [lZr]LDL at 4OC [l]. Cell CAMP was assayed according to [6] m d expressed as % of acid soluble &nine n:icltmtides. While it is well established that PGs can act through elevation of CAMP, the mechanism can differ according to cell type. Accordingly we determined whether PG receptors were functionally linked to adenylate cyclase in the cells used for our LDLR measurements. Forskolin, a direct activator of AC. was used as a positive control. cAMP content was measured after l h with each PG. Table 1 shows that those PG which elevate LDLR activity after 24h also increase cell cAMP content (measured after lh). P G s which were without effect on CAMP content did not affect LDLR expression.