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Role of Specific Protein Kinase C Isozymes in Mediating Epidermal Growth Factor, Thyrotropin-Releasing Hormone, and Phorbol Ester Regulation of the Rat Prolactin Promoter in GH4/GH4C1 Pituitary Cells

Department of Medicine (C.A.P.), Division of Metabolism, Endocrinology and Nutrition, University of Washington School of Medicine, Seattle, Washington 98195-6426; Department of Medicine and of Biochemistry, Biophysics and Genetics (N.M., A.G.-H.), Program in Molecular Biology and Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262; and Department of Molecular Cell Physiology (Y.A.), Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan

Address all correspondence and requests for reprints to: Cheryl A. Pickett, M.D., Ph.D., Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, Box 356426, University of Washington School of Medicine, 1959 Northeast Pacific Street, Seattle, Washington 98195-6426. E-mail: cpickett{at}u.washington.edu.

Epidermal growth factor (EGF) and TRH both produce enhanced prolactin (PRL) gene transcription and PRL secretion in GH4 rat pituitary tumor cell lines. These agents also activate protein kinase C (PKC) in these cells. Previous studies have implicated the PKC isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of PKC in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple PKC isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective PKC inhibitors and by expression of genes for wild-type and kinase-negative forms of the PKC isozymes. Multiple nonselective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rat PRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of diacylglycerol, whereas Go 6976, a selective inhibitor of Ca²⁺-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel nPKC isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding PKCs , ßI, ßII, , , and failed to enhance either EGF or TRH responses, whereas overexpression of nPKC enhanced the EGF response. Neither stable nor transient overexpression of nPKC produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Expression of a kinase inactive nPKC construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple PKC isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, nPKC, whereas nPKC may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca²⁺-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.

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