ABSTRACT
The orphan nuclear receptor, steroidogenic factor 1 (SF-1), plays a major role in adrenal and gonadal development, as well as in sexual differentiation. It has been demonstrated that the expression of a number of genes regulated by SF-1 is inhibited by the transforming growth factor, (TGF-beta). To date, however, the influence of TGF-beta on the expression of SF-1 gene has not been reported. A Northern blot analysis with the use of a radiolabeled cDNA probe, and immunodetection with antibodies directed against SF-1, demonstrated that the Sf-1 transcript and the SF-1 protein levels were lowered by TGF-beta in Y-1 adrenocortical cells, both in untreated and adenylyl cyclase activator, forskolin-treated cells. An examination of the Sf-1 transcript stability in the presence of actinomycin D revealed no influence of TGF-beta on the rate of Sf-1 mRNA decay. Inhibition of Sf-1 expression by TGF-beta was abolished by cycloheximide, suggesting that the growth factor inhibitory effect requires ongoing protein synthesis. We conclude that in Y-1 cells TGF-beta inhibits the expression of SF-1 gene at a transcriptional level, and we postulate that the inhibitory effect of TGF-beta on steroid hormone synthesis in the adrenal cortex could be due to an attenuated transcription of Sf-1.
Subject(s)
Adrenal Cortex/metabolism , Homeodomain Proteins/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Transcription, Genetic/drug effects , Transforming Growth Factor beta/physiology , Adrenal Cortex Neoplasms , Animals , Blotting, Northern , Cell Line, Tumor , Colforsin/pharmacology , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Homeodomain Proteins/biosynthesis , Mice , RNA, Messenger/biosynthesis , Receptors, Cytoplasmic and Nuclear/biosynthesis , Steroidogenic Factor 1 , Time Factors , Transcription Factors/biosynthesis , Transforming Growth Factor beta/pharmacologyABSTRACT
The objective of our study was to investigate the effect of stimulation of the cAMP-dependent pathway on the expression of an orphan nuclear receptor, SF-1/Ad4BP in mouse adrenal tumour, Y-1 cells in culture. We evaluated the temporal pattern of the effects of corticotropin (ACTH) and the adenylyl cyclase activator forskolin on the level of SF-1 mRNA, and compared the time course of induction of SF-1 with that of CYP11A1. Forskolin, corticotropin and 8-Br-cAMP significantly elevated the level of the SF-1 transcript, after 1.5 h of incubation, with a concomitant increase of SF-1 protein level, observed after 6 h. The CYP11A1 transcript increased gradually over the incubation period, and reached the maximal level after 12 to 24 h. The steady-state level of the SF-1 transcript was unaffected by forskolin when the cells were incubated with actinomycin D, indicating that stimulation of the cAMP pathway results in enhanced transcription of the gene. The effect of forskolin was augmented by cycloheximide, suggesting that an inhibitory protein, whose synthesis was inhibited by cycloheximide, could be involved in negative regulation of SF-1 expression. It is concluded that SF-1 expression is positively regulated by the cAMP pathway at the transcriptional level, and can represent the primary event in cAMP-mediated induction of steroid hormone synthesis in Y-1 cells.
Subject(s)
Cyclic AMP/metabolism , Signal Transduction , Steroidogenic Factor 1/genetics , Steroidogenic Factor 1/metabolism , Adrenocorticotropic Hormone/pharmacology , Animals , Blotting, Northern , Blotting, Western , CREB-Binding Protein/genetics , Cell Line, Tumor , Cholesterol Side-Chain Cleavage Enzyme/genetics , Colforsin/pharmacology , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Histone Acetyltransferases/genetics , Mice , Nuclear Receptor Coactivator 1 , Phosphoproteins/genetics , Signal Transduction/drug effects , Transcription Factors/geneticsABSTRACT
Cytochrome P450c17, encoded by the CYP17 gene, is a component of the 17alpha-hydroxylase/17,20-lyase enzyme complex essential for production of adrenal glucocorticoids and androgens as well as gonadal androgens. The expression of CYP17 in adrenocortical cells is stimulated by corticotropin (ACTH) via the signal transduction pathway involving cAMP and protein kinase A (PKA). Thus, in addition to glucocorticoids, ACTH stimulates formation of adrenal androgens, which are known to induce transforming growth factor beta (TGF-beta) secretion. TGF-b in turn inhibits steroid hormone output by attenuating both basal and ACTH-dependent expression of CYP17. The present study revealed that treatment of bovine and human H295R adrenocortical cells with androgens resulted in a decrease in the basal level of CYP17 transcript and cortisol secretion, without affecting forskolin-stimulated levels. We also demonstrated that in H295R cells TGF-beta inhibited both basal and forskolin-stimulated accumulation of CYP17 mRNA. Determination of promoter activity, directing luciferase reporter gene expression in H295R cells transfected with deletion fragments of bovine CYP17 promoter, indicated that the -483 to -433 bp fragment of the promoter was necessary for the inhibitory action of TGF-beta on CYP17 expression. It is concluded that in bovine and human adrenocortical cells, androgens inhibit basal CYP17 expression probably at the transcriptional level and independently of the effect of TGF-beta.
