CAMP-dependent protein kinase enhances CYP17 transcription via MKP-1 activation in H295R human adrenocortical cells.
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.
Sewer
Marion B
MB
School of Biology, Georgia Institute of Technology, Atlanta 30332-0230, USA. sewer@biology.gatech.edu
Waterman
Michael R
MR
D000302:Adrenal Cortex / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation; D020868:Gene Silencing; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D013254:Steroid 17-alpha-Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases