REGULATION OF THE PITUITARY-SPECIFIC TRANSCRIPTION FACTOR GHF-1 PIT-1MESSENGER-RIBONUCLEIC-ACID LEVELS BY GROWTH HORMONE-SECRETAGOGUES IN RAT ANTERIOR-PITUITARY-CELLS IN MONOLAYER-CULTURE/

Pituitary-specific expression of the GH gene is dependent on a pituita ry-specific transcription factor GH factor-1 (GHF-1), a homeodomain pr otein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 mess enger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of sec ond messenger signaling systems, we studied the effects of different h ormones, all of which are known to be involved in the regulation of so matotroph cell function. We found that GH-releasing hormone (GHRH) inc reased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH fa iled to show a similar stimulatory effect. A significant increase in G HF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The a ction of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 mu g/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 mu g/ml) alone increased GHF-1 mRNA levels ( 6-fold increase after treatment for 12 h, P < 0.01), as well as potent iating GHRH-induced increase in GHF-1 mRNA concentration (9-fold incre ase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct ac tivators of second messenger signaling systems such as forskolin (10(- 5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetat e (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5 ) M), were also able to increase GHF-1 mRNA levels. Treatment of the c ells with somatostatin (10(-6) M) for either 2 or 48 h failed to modif y basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment o f the cells with insulin-like growth factor-1 (5 nM) inhibited basal G HF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRN A levels. The effect of GHRH can be mimicked by pituitary adenylate cy clase activating polypeptide-38 but not by GH-releasing peptide-6. Alt hough somatostatin does not seem to play a major role in the regulatio n of GHF-1 mRNA levels, insulin-like growth factor-1 exerts a powerful inhibitory effect.