The cytochrome P450 17 -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 hnRNA 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 > 95% (P < 0.001). The marked loss of combined LH and insulin stimulation caused by deleting the region between -473/-175 bp suggested possible relevance of partially overlapping Sp1 and AP-2-like binding sites located between -193 bp 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 > 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 bp 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-PKA and insulin-signaling pathways in untransformed theca cells.