Inhibition of protein synthesis blocks the response to 25-hydroxycholesterol by inhibiting degradation of hydroxymethylglutaryl-CoA reductase.

The activity of the rate-limiting enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase in Chinese hamster ovary (CHO) cells decreased more rapidly in cells treated with 25-hydroxycholesterol alone (t 1/2 = 1.5 h) than in those incubated with cycloheximide alone (t 1/2 = 5 h). The inhibitory action of 25-hydroxycholesterol on reductase activity was reduced when the sterol and cycloheximide were added together, and was totally abolished when cells were preincubated with cycloheximide for 30 min before the addition of 25-hydroxycholesterol. The effect of puromycin was similar to that of cycloheximide. Treatment of cells with an inhibitor of RNA synthesis, i.e., actinomycin D or cordycepin, had little effect on hydroxymethylglutaryl-CoA reductase activity; however, preincubation of cells with these reagents also decreased the ability of 25-hydroxycholesterol to suppress the reductase activity. These data are consistent with a model which suggests (a) that 25-hydroxycholesterol inhibits the activity of hydroxymethylglutaryl-CoA reductase by repressing its synthesis, (b) that cycloheximide and puromycin affect hydroxymethylglutaryl-CoA reductase activity by blocking the de novo synthesis of the enzyme and by reducing the degradation of the preexisting enzyme, (c) that actinomycin D and cordycepin affect the supply of message for the continuous synthesis of at least one component of a system which degrades hydroxymethylglutaryl-CoA reductase, and (d) that one component of the degradative system has a half-life shorter than 0.5 h.