title: Evaluation of glucocorticoid-induced growth hormone gene expression in chicken embryonic pituitary cells using a novel in situ mRNA quantitation method.
abstract: We reported that corticosterone (CORT) can induce differentiation of growth hormone (GH) cells in vitro and in vivo during chick embryonic development. In the present study, a quantitative in situ hybridization plate assay (ISHPA) for GH mRNA was developed and used to assess the mechanism of glucocorticoid-induced GH gene expression directly in cell culture plates. Embryonic pituitary cells were treated with GH-releasing hormone (GHRH) alone, CORT alone and GHRH and CORT in combination. CORT increased levels of GH mRNA 22-fold, while GHRH acted synergistically with CORT to further augment GH mRNA levels (130-fold relative to control). GHRH alone induced only a 2.5-fold increase in GH mRNA. GH mRNA levels were increased after 8 h but not after 4 h of CORT treatment. In addition, synergistic effects of GHRH on CORT-induced GH mRNA were also observed after 8 h of treatment, however, GHRH alone for up to 24 h failed to increase GH mRNA levels, suggesting that embryonic pituitary cells do not respond substantially to GHRH in the absence of CORT. Cycloheximide (CHX) blocked CORT induction of GH mRNA, indicating that synthesis of some protein(s) is required for CORT induction of GH gene expression. Bypassing the GHRH receptor through treatment with forskolin and 3-isobutyl-1-methylxanthine (IBMX) and phorbol 12-myristate-13-acetate failed to increase GH mRNA levels, suggesting that a lack of GHRH receptors alone cannot account for the lack of GHRH responses in the absence of CORT. Treatment with inhibitors of protein kinase A (PKA; H-89), protein kinase C (PKC; calphostin C) and mitogen activated protein kinase (MAPK; PD098059) did not block induction of GH mRNA by CORT. In contrast, Manumycin, an inhibitor of Ras-GTPase, significantly suppressed the effect of CORT on GH mRNA. These results indicate that glucocorticoid induction of GH gene expression in embryonic pituitary cells requires active protein synthesis. The protein(s) involved in this induction is probably not a component of the PKA, PKC or MAPK signaling cascades but may involve Ras or a Ras-like compound. Current efforts in our laboratory are directed at identifying this intermediary protein(s).
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park 20742, USA.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Bossis
authors.forename: Tom E
authors.initials: TE
authors.lastname: Porter
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002642:Chick Embryo; D003345:Corticosterone / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004338:Drug Combinations; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000235:genetics*; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D017403:In Situ Hybridization; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D010902:Pituitary Gland / Q000187:drug effects* / Q000196:embryology* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors; D015347:RNA Probes; D012333:RNA, Messenger / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D004338:Drug Combinations; D004791:Enzyme Inhibitors; D015347:RNA Probes; D012333:RNA, Messenger; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020929:Mitogen-Activated Protein Kinase Kinases; D016283:Proto-Oncogene Proteins p21(ras); D003345:Corticosterone