Insulin Drives Transcriptional Activity of the CYP17 Gene in Primary Cultures of Swine Theca Cells1

Abstract

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.

Direct Insulin and Leptin Action on Pro-opiomelanocortin Neurons Is Required for Normal Glucose Homeostasis and Fertility

“…The gene encoding the enzyme upstream of 3beta-HSD in the testosterone synthesis pathway, CYP17, showed a trend toward increased expression as well (controls: 1.000 ± 0.3606 N=10 vs. double knockout: 2.412 ± 0.5784 N=10, p = 0.0530). Notably, insulin drives transcriptional activity of the CYP17 gene in primary cultures of theca cells (Zhang and Veldhuis, 2004), and increases 3β-HSD expression in human granulosa cells (McGee et al, 1995). Thus, the reproductive deficits seen in Pomc -Cre, Lepr flox/flox IR flox/flox mice may reflect inappropriate regulation of fertility secondary to peripheral insulin resistance and hyperandrogenemia.…”

Characteristics of Polycystic Ovarian Syndrome and Relationship with Ghrelin in Adolescents

“…18 One of the mechanisms is that insulin acts like IGF1 which increases LH effect on ovaries via cross reaction with IGF1 receptors. 19 The other ones are that it may increase free IGF1 levels, 20 stimulate cytochrome p450c17 enzyme both in ovaries and in adrenal gland, 21,22 and decrease sex hormone binding globulin levels. 23 Hyperandrogenism plays a key role in occurring clinical appearance.…”

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

“…Insulin induces rapid autophosphorylation of the ␤-subunit of the cognate membrane-associated receptor in porcine granulosa and theca cells (11,12). Insulin may also stimulate ovarian cells in part via the inositolglycan pathway (13,14).…”

“…The putative CYP17 sequence region was ligated into the BglII-restricted pGL3-Basic plasmid according to the protocol provided by the manufacturer. DNA sequencing was used to delineate orientation and identity of a Ϫ976/ϩ31 bp sequence 5Ј upstream of the transcriptional start site (11).…”

“…1), three pairs of primers were designed to contain one mutation site each (10,11). The preparation of mutants was carried out using the QuikChange SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA) based on the manufacturer's procedure.…”