CAMP-dependent Protein Kinase Enhances CYP17 Transcription via MKP-1 Activation in H295R Human Adrenocortical Cells

Abstract

Steroid hormone biosynthesis in the adrenal cortex is controlled by adrenocorticotropin (ACTH), which increases intracellular cAMP, resulting in the activation of cAMP-dependent protein kinase(PKA) and subsequent increase in steroidogenic gene transcription. We have found that a dual-specificity phosphatase is essential for conveying ACTH/cAMP-stimulated transcription of several steroidogenic genes in the human adrenal cortex. In the present study, the role of mitogen-activated protein kinase phosphatase-1 (MKP-1), a nuclear dual-specificity phosphatase, in the transcriptional activation of human CYP17 (hCYP17) in H295R human adrenocortical cells is established. Stimulation of H295R cells with dibutyryl-cAMP (Bt(2)cAMP) induces MKP-1 mRNA and protein expression within 30 min of exposure. In transient-transfection studies, transcriptional activity of an hCYP17 promoter-reporter construct was increased by Bt(2)cAMP and by overexpression of PKA or MKP-1. Furthermore, PKA phosphorylated an MKP-1-glutathione S-transferase fusion protein in in vitro assays and Bt(2)cAMP increased (32)P associated with MKP-1 that was immunoprecipitated from H295R cells. Finally, silencing MKP-1 expression using antisense oligonucleotides attenuated cAMP-stimulated hCYP17 expression, whereas silencing of ERK1/2 increased hCYP17 expression. These findings demonstrate integral roles for MKP-1 and ERK1/2 via regulation of the phosphorylation state of steroidogenic factor-1 (SF-1) in mediating ACTH/cAMP-dependent transcription of hCYP17, thereby maintaining the balance between transcriptional activation and repression.