Sites of inhibition of steroidogenesis by activation of protein kinase-C in swine ovarian (Granulosa) cells

J. A. Flores, J. C. Garmey, Johannes D Veldhuis, J. D. Veldhuis

Research output: Contribution to journalArticle

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Abstract

We tested the hypothesis that low density lipoprotein (LDL) metabolism and cellular concentrations of gene transcripts of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc mRNA) are sites of significant protein kinase-C (PKC) action in the long term (48-h) inhibitory modulation of steroid hormone biosynthesis in ovarian granulosa cells. To this end, we used 12-O-tetradecanoylphorbol-13-acetate (TPA) as an activator of PKC and a monolayer culture system of immature swine granulosa cells responsive to insulin and lipoprotein under serum-free conditions. Insulin-regulated LDL metabolism was identified as a major site of TPA-mediated inhibition of steroidogenesis in granulosa cells. Treatment with TPA (30 ng/ml), but not inactive phorbol base, effectively decreased insulin-stimulated [125I]iodo-LDL binding by 75%, internalization by 90%, and degradation by 75%, as well as delivery and utilization of the [3H]cholesterol moiety of LDL in progesterone biosynthesis by intact granulosa cells. Cellular concentrations of P450scc mRNA, as measured by Northern blot hybridization with a 32P-labeled 1-kilobase porcine cDNA clone, were significantly increased by insulin. This insulin effect was virtually abolished by cotreatment with TPA (30 ng/ml). In contrast, accumulation of mRNA transcripts of a nonsteroidogenic gene, 3-phosphoglyceraldehyde dehydrogenase, but not 18S ribosomal RNA, was enhanced by TPA. In summary, major inhibitory actions of PKC activation on granulosa cell steroidogenesis are expressed at specific loci of LDL metabolism, including LDL receptor number, internalization, and degradation, as well as the delivery and utilization of the [3H]cholesterol moiety of LDL to intact granulosa cells. Moreover, a PKC activator suppresses the intracellular accumulation of insulin-stimulated P450scc mRNA, but not that of phosphoglyceraldehyde dehydrogenase or 18S ribosomal RNA. The results obtained in this in vitro study suggest that the inhibition by TPA at these different sites along the steroidogenic pathway may be similar to that which occurs via hormones that work through the PKC system, such as prostaglandin F(2α).

Original languageEnglish (US)
Pages (from-to)1983-1990
Number of pages8
JournalEndocrinology
Volume132
Issue number5
DOIs
StatePublished - 1993
Externally publishedYes

Fingerprint

Granulosa Cells
Tetradecanoylphorbol Acetate
Protein Kinase C
Swine
Insulin
LDL Lipoproteins
18S Ribosomal RNA
Messenger RNA
Glyceraldehyde-3-Phosphate Dehydrogenases
LDL Cholesterol
Cytochrome P-450 Enzyme System
Glyceraldehyde 3-Phosphate
Cholesterol Side-Chain Cleavage Enzyme
Hormones
LDL Receptors
Prostaglandins F
Northern Blotting
Genes
Lipoproteins
Progesterone

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Sites of inhibition of steroidogenesis by activation of protein kinase-C in swine ovarian (Granulosa) cells. / Flores, J. A.; Garmey, J. C.; Veldhuis, Johannes D; Veldhuis, J. D.

In: Endocrinology, Vol. 132, No. 5, 1993, p. 1983-1990.

Research output: Contribution to journalArticle

Flores, J. A. ; Garmey, J. C. ; Veldhuis, Johannes D ; Veldhuis, J. D. / Sites of inhibition of steroidogenesis by activation of protein kinase-C in swine ovarian (Granulosa) cells. In: Endocrinology. 1993 ; Vol. 132, No. 5. pp. 1983-1990.
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