Epidermal Growth Factor Increases Cortisol Production and Type II 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4-Isomerase Expression in Human Adrenocortical Carcinoma Cells: Evidence for a Stat5-Dependent Mechanism

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3βHSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3βHSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from −110 to −118 in the human type II 3βHSD promoter. A Stat5 expression construct was cotransfected with a 3βHSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides −301→+45 of the human type II 3βHSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3βHSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.