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Ovarian Actions of Tumor Necrosis Factor- (TNF): Pleiotropic Effects of TNF on Differentiated Functions of Untransformed Swine Granulosa Cells^*

JOHANNES D. VELDHUIS, JAMES C. GARMEY, RANDALL J. URBAN, LAURENCE M. DEMERS and BHARAT B. AGGARWAL

Department of Internal Medicine, University of Virginia Health Sciences Center Charlottesville, Virginia 22908
Department of Pathology, Milton S. Hershey Medical Center, Pennsylvania State University Hershey, Pennsylvania 17033
Department of Protein Biochemistry, Genentech, Inc. South San Francisco, California 94080

Address all correspondence and requests for reprints to: Dr. Johannes D. Veldhuis, Box 202, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908.

Abstract

We have examined interactions between tumor necrosis factor- (TNF), a product of the immune system, and ovarian cells using serum-free monolayer cultures of untransformed swine granulosa cells. Recombinant human TNF, a potent cytoactive product of activated macrophages, bound specifically and with high affinity to intact granulosa cells. Binding sites had an apparent K_d of 0.17 nM (95% confidence interval, 0.065–0.31), and a binding capacity of 80 nmol/µg DNA (95% confidence interval, 52–110). The binding capacity of granulosa cells for TNF (but not the binding affinity) was increased approximately 2-fold by treatment with FSH and insulin. The biological effects of TNF on pig granulosa cells were expressed after 48 and 96 h in culture. At the latter time, TNF significantly suppressed insulin- and insulin- plus FSH-stimulated progesterone accumulation, with respective ID₅₀ values of 0.08 ± 0.008 and 0.06 ± 0.014 nM, but did not affect basal progesterone accumulation or DNA content. TNF also significantly attenuated the stimulatory effect of combined treatment with FSH and insulin on cAMP generation during 48–96 h of culture. TNF inhibited the stimulatory effects of forskolin, cholera toxin, and the cAMP analog 8-bromo-cAMP on progesterone accumulation, indicating multiple sites of action of this immune modulator. Inhibition of progestin biosynthesis was observed even in the presence of 25-hydroxycholesterol, a soluble oxygenated sterol substrate for the cholesterol side-chain cleavage reaction, and was accompanied by decreased concentrations of specific cellular mRNA encoding cholesterol side-chain cleavage enzyme. There were no changes in the amounts of a constitutively expressed enzyme, phosphoglyceraldehyde dehydrogenase. Inhibitory actions of TNF were specific to de novo steroid hormone biosynthesis, since nanomolar concentrations of this cytokine stimulated accumulation of prostaglandin E₂ and prostaglandin F₂ basally and during treatment with FSH, cholera toxin, or 8-bromo-cAMP. In contrast, prostaglandin accumulation was not enhanced by interferon- or interleukin-2.

In summary, untransformed porcine granulosa cells exhibit specific, high affinity, low capacity saturable binding sites for TNF, and the number of such binding sites can be regulated by combined treatment with insulin and FSH. Granulosa cells are susceptible to the inhibitory actions of TNF on FSH- and insulin-supported progesterone biosynthesis and cAMP accumulation. One important locus of TNF action is blockade of hormonally stimulated increases in specific mRNA encoding the cholesterol side-chain cleavage cytochrome P450 enzyme. Suppressive effects of TNF on steroidogenesis are specific, since they occur concurrently with TNF's stimulation of prostaglandin E₂ and F₂ accumulation. We conclude that an array of pleiotropic actions of TNF can be identified in normal ovarian (granulosa) cells.

Footnotes

^* This work was supported in part by Clinical Associate Physician Award NIH 3-MO1-RR-00847-1491 and a Diabetes Center pilot/feasibility grant from the University of Virginia Diabetes and Endocrinology Research Center (Grant DK-38942 to R.J.U.); Research Career Development Award KO4-HD-00634 and NIH Grants RO1-HD-16393 and HD-16806 (to J.D.V.); and the Clayton Foundation for Research and the New Program Development Funds from the University of Texas M. D. Anderson Cancer Center (to B.B.A.).

Current address: Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77550.

Current address: Department of Clinical Immunology and Biological Therapy, Cytokine Research Laboratory, M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030.

Received February 22, 1991.

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