Insulin stimulates androgen biosynthesis and the accumulation of CYP17 mRNA and heterogeneous nuclear (hn) RNA in primary cultures of immature swine theca cells. To further assess insulinomimetic transcriptional control, we subcloned 1.007 kilobases (kb) of the 5′-upstream region of the CYP17 gene (−976 to 31 base pairs [bp] to the transcriptional start site) into a firefly-luciferase reporter construct. Insulin drove transcriptional activity of this probe in a time- and dose-dependent fashion, with maximal stimulation of 2.7- to 3.2-fold after insulin exposure (100 ng/ml) for 6 h. Progressive deletional constructs −839, −473, −174, and −75/ 31 bp delineated expected reduction in responsiveness, except paradoxical gain of basal CYP17 promoter activity by the −473/ 31-bp sequence. The latter suggests a possible intervening inhibitory sequence. Elimination of all sequences 5′-upstream to −174 bp markedly reduced basal transcriptional activity and abolished insulin action. Point mutation of a presumptive Sp1-like element located within −193/−180 bp inhibited basal and insulin-stimulated luciferase activity of the full-length promoter fragment by 40% and 67%, respectively. Disruption of a contiguous presumptive AP-2 site produced a comparable outcome. Combined mutation of the Sp1 and AP-2-like elements eliminated basal and insulin-potentiated CYP17 promoter activity. By Western analysis, insulin augmented cognate receptor phosphoprotein concentrations by 31-fold within 10 min. Chemical inhibitors of MEK-activated ERK1/2 attenuated insulin-enhanced CYP17 transcriptional activity by 76–80%. In summary, insulin drives transcriptional activity of a 5′-upstream regulatory sequence (−976 to 31 bp) of the swine CYP17 gene in primary cultures of theca cells, under a minimal requirement for combined activity of proximal (−193/180 bp) Sp1 and AP-2-like elements.
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