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.

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 in these cells. Previous studies have implicated the isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple duanyu1531 isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective duanyu1531 inhibitors and by expression of genes for wild-type and kinase-negative forms of the duanyu1531 isozymes. Multiple nonselective duanyu1531 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 Ca(2+)-dependent produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding alpha, betaI, betaII, delta, gamma, and lambda failed to enhance either EGF or TRH responses, whereas overexpression of enhanced the EGF response. Neither stable nor transient overexpression of 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 nduanyu1531delta construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple duanyu1531 isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, whereas nduanyu1531eta may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca(2+)-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.

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