Cyclic AMP-mediated hormonal control of LDL receptor activity in cultured human cells.

Previous work in this laboratory [ 1.2.31 showed that elevation of cyclic AMP has dual effects (A and B below) on the expression of the LDL receptor in human vascular smooth muscle cells (VSMC) and skin fibroblasts. A). Cyclic AMP elevating agents stimulate LDL receptor activity [ I ] in human cells by increasing the rate of receptor protein synthesis [2]. The stimulation is not secondary to the decrease of the regulatory pool of free cholesterol since it is unaffected, or even enhanced, by inhibition of cholesterol synthesis and esterification, or by inhibition of the conversion of cholesterol to its repressor metabolites [2]. The cyclic AMP mediated up-regulation of the receptor persists in the presence of repressors such as LDL and 25hydroxycholesterol but does not ovemde their effects [2]. B). Cyclic AMP-elevating agents also stimulate the hydrolysis of lysosomal cholesterol esters [4], thus increasing the cellular cholesterol pool and repressing the expression of the LDL receptor. This cholesterol-mediated repressive effect of cyclic AMP can be prevented by chloroquine (CQ) and m C 1 which inhibit lysosomal actions or by ketoconazole which inhibits conversion of free cholesterol to its repressor metabolite [2,3]. It is well established that the action of some prostaglandins are mediated through elevation of cyclic AMP. Here we report the effects of prostaglandins(PG) El, E2 and the stable PGI2 analogue, Iloprost, on regulation of the LDL receptor. In order to simplify interpretation of the data, we have carried out the investigations in the presence of the lysosomal inhibitors CQ and N h C l to prevent any down-regulation of the LDL receptor due to mobilization of cholesterol from lysosomal esters as explained in B (above). To show that PG caused real up-regulation of the LDL receptor, all experiments were carried out on cells in which the LDL receptor number was already maximally expressed by preincubation in LPDM for 24h. Cells were then incubated with PGs and lysosomal inhibitors for up to 24h before removing these agents for LDL receptor measurements. Cell surface expression of the LDL receptor was measured by the specific binding of [1251]LDL at 4OC [l]. Metabolism of LDL by the receptor pathway was determined (after removal of m C I ) by hydrolysis of LDL-borne cholesterylesters to give intracellular free cholesterol. This was assessed by its availability for esterification with [3H]oleate [3]. In fibroblasts, 24h preincubation with PGEl caused significant, concentration-related elevation of LDL binding; 2.8pM PGEl raised binding by 4 3 5 % (PcO.Ol), 14pM PGEl gave a 209f46% (Pd.01) increase above controls. PGE2 yielded similar results, but higher doses (14pM) were needed to ensure consistent effects. In VSMC, 2.8pM Iloprost gave a 5W7C (P4.002) elevation in LDL binding, rising to 128+12% (P<O.OOl) at 14pM. Iloprost effects on fibroblasts showed similar trends. LDL metabolism in VSMC was similarly stimulated by all 3 PGs. At 2.8pM, PGEl gave a 99f16% (P<O.Ol) increase, W E 2 gave a 55f3% (Pc0.02) increase and Iloprost gave a 75+4% (P<O.OOI) increase. PGEl was shown to have no influence on the esterification process itself, all effects being mediated through supply of free cholesterol via up-regulation of the LDL receptor. Study of the timecourse of action of Iloprost on LDL binding showed a marked time dependency with no significant stimulation being observed until after 8h with the agent (Fig. 1). There was a small, but significant, inhibition of binding at 4h.