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Regulation of P450c17 expression in the early embryo depends on GATA factors.

ABSTRACT: The enzyme P450c17 is required for glucocorticoid, sex steroid, and some neurosteroid biosynthesis. Defective human P450c17 causes sexual infantilism and 46,XY sex reversal but is compatible with life, whereas ablation of the corresponding mouse gene causes embryonic lethality at around E7. Normal mouse embryos express P450c17 protein and activity in the embryonic endoderm at E7. Adult adrenal and gonadal steroidogenesis requires steroidogenic factor-1 (SF-1), but SF-1 is not expressed in the early mouse embryo. We show that P450c17 is expressed in differentiated mouse parietal and visceral endoderm lineages, in cultured mouse F9 embryonic carcinoma stem cells, in mouse embryonic stem cells, and in cultured mouse P19 stem cells. Bases -110 to -55 (which contain an SF-1 site and two potential GATA sites) of the rat cyp17 gene confer promoter activity in F9 cells. Overexpression of SF-1 has no effect, whereas overexpression of GATA4 in F9 cells increases transcription from -110/-55 fused to a reporter and increases endogenous P450c17 mRNA. Chromatin immunoprecipitation assays show that GATA4 binds to -215/+55 of mouse cyp17. Stimulating F9 cells with retinoic acid and cAMP differentiates them into visceral and parietal endoderm. Commensurate with cell differentiation, quantitative PCR showed increased GATA4 and GATA6 mRNAs, temporally followed by increased P450c17 mRNA. Small interfering RNA inhibition of GATA4 or GATA6 in undifferentiated or differentiated F9 cells diminished endogenous cyp17 expression. Thus, P450c17 is expressed in mouse embryonic stem cells, its expression increases upon differentiation to an early embryonic endoderm lineage, and GATA4/6 are responsible for activation of P450c17 gene expression at this early stage of embryonic development.

SUBMITTER: Shi Y

PROVIDER: S-EPMC2646537 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Regulation of P450c17 expression in the early embryo depends on GATA factors.

Shi Yimin Y Schonemann Marcus D MD Mellon Synthia H SH

Endocrinology 20081001 2

The enzyme P450c17 is required for glucocorticoid, sex steroid, and some neurosteroid biosynthesis. Defective human P450c17 causes sexual infantilism and 46,XY sex reversal but is compatible with life, whereas ablation of the corresponding mouse gene causes embryonic lethality at around E7. Normal mouse embryos express P450c17 protein and activity in the embryonic endoderm at E7. Adult adrenal and gonadal steroidogenesis requires steroidogenic factor-1 (SF-1), but SF-1 is not expressed in the ea ...[more]

PMID: 18832096

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Project description:Cytochrome P450c17 (P450c17) is the single enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme's affinity for its redox partner, P450 oxidoreductase. We searched for the relevant kinase(s) that phosphorylates P450c17 by microarray studies and by testing of kinase inhibitors. Microarrays show that 145 of the 278 known serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8-Br-cAMP. Key components of the ERK1/2 and MAPK/ERK kinase (MEK)1/2 pathways, which have been implicated in the insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various kinase inhibitors that probe the protein kinase A/phosphatidylinositol 3-kinase/Akt pathway and the calcium/calmodulin/MAPK kinase pathway had no effect on the ratio of 17,20 lyase activity to 17alpha-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the Rho-associated, coiled-coil containing protein kinase (ROCK)/Rho pathway suppressed 17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect 17,20 lyase activity. We conclude that members of the ROCK/Rho pathway act upstream from the kinase that phosphorylates P450c17 in a fashion that augments 17,20 lyase activity, possibly acting to catalyze a priming phosphorylation.

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Regulation of P450c17 expression in the early embryo depends on GATA factors.

Publications

Regulation of P450c17 expression in the early embryo depends on GATA factors.

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