Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger 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 pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased 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 failed to show a similar stimulatory effect. A significant increase in GHF-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 action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase 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 activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (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 cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-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 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)
Soto
J L
JL
Department of Physiology, Faculty of Medicine, University of Santiago de Compostela, Spain.
Castrillo
J L
JL
Dominguez
F
F
Dieguez
C
C
D000262:Adenylyl Cyclases / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D013006:Growth Hormone / Q000032:analysis / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008297:Male; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013004:Somatostatin / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; C494186:Pou1f1 protein, rat; D012333:RNA, Messenger; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D003609:Dactinomycin; D005576:Colforsin; D013004:Somatostatin; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D003513:Cycloheximide; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate