The hypothalamic neuropeptide TRH, through G-protein-coupled transmembrane pituitary receptors, rapidly stimulates intracellular signaling events that, in turn, stimulate gene transcription. Our previous studies in transfected pituitary tumor cells indicated that TRH stimulation of thyrotropin (β-subunit (TSHβ) gene expression involves both calcium mobilization and protein kinase-C activation. To characterize the gene-proximal elements of the intracellular signaling pathways involved, we examined the effects of TRH, ionomycin, and phorbol ester (TPA) on cellular protooncogenes (c-jun and c-fos) known to be responsive to calcium mobilization and protein kinase-C activation. TRH stimulated a 3-fold increase in both c-jun and c-fos mRNA levels within 1 h, followed by a rapid decline in steady state mRNA levels. A secondary response to the single administration was noted, culminating in a 5-fold stimulation at 20 h. The increase in c-jun and c-fos mRNA levels occurred before the increased steady state mRNA levels of both PRL and TSHβ chimera in transfected pituitary GH3 cells. Furthermore, we examined the role of calcium in these effects using the ionophore ionomycin to elevate and TMB-8 to decrease intracellular calcium. We used the phorbol ester TPA to investigate the effects of increased protein kinase-C activity and H7 or pretreatment with TPA to monitor the decreased kinase activity. Our data indicate that calcium mobilization and protein kinase activation represent distinct components of the signaling events initiated by TRH resulting in increased c-jun and c-fos mRNA levels. Only c-fos mRNA is increased by all three factors, suggesting that c-fos may be a key element in mediating the intracellular processes reflecting TRH action.
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