Translation of phenylalanine hydroxylase-specific mRNA in vitro: Evidence for pretranslational control by glucocorticoids (in vitro protein synthesis/wheat-germ lysate/gene expression/liver enzyme/rat hepatoma cells)

We have found that the induction of phenylala- nine hydroxylase by hydrocortisone and serum in confluent cul- tures of H4-II-E-C3 rat hepatoma cells is accompanied by an in- crease in polysomal mRNA specific for phenylalanine hydroxylase, as measured by translation in a cell-free protein-synthesizing sys- tem. Thus, the induction is mediated largely, if not entirely, by a pretranslational mechanism, possibly by stimulation of the tran- scription of the phenylalanine hydroxylase gene. It has been shown that two cultured rat hepatoma cell lines, H4- II-E-C3 (H4) and MH1C1, contain phenylalanine hydroxylase but at a very low level (1). Addition of hydrocortisone or dexa- methasone at submicromolar concentrations and of steroid-free blood serum, from diverse mammalian species, to confluent cultures of the H4 cells caused an increase in the hydroxylase content of the cells to levels comparable with those found in normal rat liver (1, 2). This increase resulted from an increase in the amount of hydroxylase protein and not from activation of an inactive (pro)enzyme (3). The stimulation by glucocorti- coids was shown to be mediated by an increase in the rate of the synthesis of the enzyme (4) without any change in the rate of its degradation (5). By the use of a cell-free protein-synthesizing system of wheat germ and specific antiserum against rat liver phenylalanine hy- droxylase, we now show that the induction of the hydroxylase by hydrocortisone and serum in H4 cells is associated with an increase of polysomal mRNA specific for phenylalanine hy-