PMID- 15001547 OWN - NLM STAT- MEDLINE DCOM- 20040624 LR - 20161124 IS - 0013-7227 (Print) IS - 0013-7227 (Linking) VI - 145 IP - 6 DP - 2004 Jun TI - Requirement for proximal putative Sp1 and AP-2 cis-deoxyribonucleic acid elements in mediating basal and luteinizing hormone- and insulin-dependent in vitro transcriptional activation of the CYP17 gene in porcine theca cells. PG - 2760-6 AB - The cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (CYP17) enzyme catalyzes the first committed step in androgen biosynthesis. In primary cultures of immature swine theca cells, LH and insulin induce CYP17 mRNA and incompletely processed heteronuclear RNA supraadditively over 2-6 h. To monitor in vitro transcriptional control by these two physiological signals, we cloned a -976 to +31-bp 5'-upstream region of the homologous CYP17 gene and fused it to a cytoplasmically targeted firefly luciferase minigene (CYP17/luc). LH and insulin individually stimulated transcriptional activity of transiently transfected CYP17/luc in theca cells by 2.7 +/- 0.31- and 2.5 +/- 0.24-fold over basal, respectively, at an optimal concentration (both 100 ng/ml) and time (6 h; both P < 0.01). Combined peptidyl agonists stimulated CYP17/luc by 6.6 +/- 1.2-fold (P < 0.001). To identify possible LH- and insulin-sensitive cis-acting DNA regulatory regions, we prepared four deletional constructs, -839, -473, -174, and -75/+35 bp 5' upstream of the transcriptional start site. Deletion from -976 to -839 bp decreased basal transcriptional activity by 89% and that stimulated by LH, insulin, and both effectors by 82%, 91%, and 78%, respectively (each P < 0.01). Further deletion to -473 bp conferred partial responsiveness to combined hormone stimulation, suggesting an intervening inhibitory sequence. Truncation to -174 bp and more proximally reduced basal CYP17/luc activity and hormonal action by more than 95% (P < 0.001). The marked loss of combined LH and insulin stimulation caused by deleting the region between -473 and -175 bp suggested the possible relevance of partially overlapping specificity protein-1 (Sp1) and activating protein-2 (AP-2)-like binding sites located between -193 and -180 bp. Point mutation of the proximal Sp1-like element in full-length -976/+31 CYP17/luc impaired basal transcription minimally (by 21%; P = NS) and stimulation by LH (76%), insulin (67%), and combined peptides (54%) significantly (each P < 0.05 vs. wild type). Mutation of the AP-2 site alone decreased basal CYP17/luc activity nonsignificantly (by 25%), but repressed stimulated transcriptional responses prominently, viz. to LH (57%), insulin (77%), and both effectors (82%; each P < 0.025 vs. wild type). Mutation of both sites inhibited basal and hormonally stimulated CYP17/luc activity by more than 95% (P < 0.001). At the level of second messenger signaling, insulin did not potentiate LH-enhanced cAMP accumulation, whereas a stable cAMP analog mimicked LH action and augmented insulin's stimulation of full-length and deletional fragments of CYP17/luc. In summary, LH and insulin stimulate transcriptional activity of a -976/+31 bp 5'-upstream cis-acting region of the (porcine) CYP17 gene individually and jointly in primary cultures of theca cells. Maximal transcriptional responsiveness to these peptide hormones requires proximal Sp1 and AP-2-like sequences -193 to -180 bp 5' upstream of the transcriptional start site. Exogenous cAMP mimics transcriptional up-regulation by LH and interacts analogously with insulin. These data are consistent with convergent drive of CYP17 gene expression by cAMP-protein kinase A and insulinsignaling pathways in untransformed theca cells. FAU - Zhang, Gongqiao AU - Zhang G AD - Division of Endocrinology and Metabolism, Department of Internal Medicine, Mayo Medical and Graduate Schools of Medicine, General Clinical Research Center, Mayo Clinic, Rochester, Minnesota 55905, USA. FAU - Veldhuis, Johannes D AU - Veldhuis JD LA - eng GR - R01-HD-16393/HD/NICHD NIH HHS/United States GR - U54-HD-28934/HD/NICHD NIH HHS/United States PT - Journal Article PT - Research Support, U.S. Gov't, P.H.S. DEP - 20040304 PL - United States TA - Endocrinology JT - Endocrinology JID - 0375040 RN - 0 (DNA-Binding Proteins) RN - 0 (Insulin) RN - 0 (Sp1 Transcription Factor) RN - 0 (Transcription Factor AP-2) RN - 0 (Transcription Factors) RN - 9002-67-9 (Luteinizing Hormone) RN - 9007-49-2 (DNA) RN - EC 1.14.14.19 (Steroid 17-alpha-Hydroxylase) SB - IM MH - Animals MH - Cells, Cultured MH - DNA/*physiology MH - DNA-Binding Proteins/*genetics MH - Female MH - Insulin/*physiology MH - Luteinizing Hormone/*physiology MH - Mutation MH - Point Mutation MH - Sp1 Transcription Factor/*genetics MH - Stereoisomerism MH - Steroid 17-alpha-Hydroxylase/chemistry/*genetics MH - Swine MH - Theca Cells/*physiology MH - Transcription Factor AP-2 MH - Transcription Factors/*genetics MH - Transcriptional Activation/*physiology EDAT- 2004/03/06 05:00 MHDA- 2004/06/25 05:00 CRDT- 2004/03/06 05:00 PHST- 2004/03/06 05:00 [pubmed] PHST- 2004/06/25 05:00 [medline] PHST- 2004/03/06 05:00 [entrez] AID - en.2003-1545 [pii] AID - 10.1210/en.2003-1545 [doi] PST - ppublish SO - Endocrinology. 2004 Jun;145(6):2760-6. doi: 10.1210/en.2003-1545. Epub 2004 Mar 4.