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Submitted on November 12, 2001
Accepted on September 12, 2002
1 Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington School of Medicine, Seattle, WA (C.A.P.); Department of Medicine and of Biochemistry, Biophysics and Genetics, Program in Molecular Biology and Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, CO (N.M. and A.G.H.); Department of Molecular Cell Physiology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (Y.A.)
* To whom correspondence should be addressed. E-mail: cpickett{at}u.washington.edu.
Epidermal growth factor (EGF) and TRH(TRH) both produce enhanced PRL(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 non-selective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rPRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of DAG, while Go 6976, a selective inhibitor of Ca2+-dependent PKC's, 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 PKC's 
, 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, while nPKC may also be able to transmit the EGF response. Inhibitor data suggests that the EGF response may also involve Ca2+-dependent isozymes while the TRH response appears to be more dependent on DAG.
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