title: Expression and dexamethasone regulation of the human corticotropin-releasing hormone gene in a mouse anterior pituitary cell line.
abstract: The factors controlling the expression of corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide involved in the regulation of ACTH secretion, are poorly understood partly because a suitable in vitro model is lacking. To study the regulation of CRH gene expression, an 8-kilobase (kb) DNA fragment containing the entire human CRH gene as well as approximately 6 kb of 5' sequence and 0.8 kb of 3' sequence was isolated from a lambda Charon 4A human genomic library and introduced into a mouse anterior pituitary cell line, AtT-20, by CaPO4 transfection with a neomycin-selectable marker. Approximately 10% of the neomycin-resistant lines stably expressed the CRH gene and secreted radioimmunoassay-detectable CRH into culture media at levels greater than 100 pg/ml. By Southern blot analysis the 8-kb DNA fragment containing the CRH gene had been incorporated intact into the AtT-20 genome. In each CRH-producing strain, but not in the parent AtT-20 cell line, we detected by Northern blot analysis an RNA species that hybridized to two radioactive cRNA probes specific for either the 5' or 3' portion of CRH mRNA, and that co-migrated with placental CRH mRNA. Dexamethasone treatment for 24-96 h caused a specific decrease in CRH mRNA and peptide levels of 40-50% in the five CRH-producing cell lines with half-maximal suppression at approximately 10(-9) M dexamethasone, indicating that CRH gene expression is negatively regulated by glucocorticoids. Thus, we have established an in vitro model suitable for studying in detail those cis- and trans-acting factors which regulate CRH gene expression.
authors.affiliation: Neuroendocrine Genetics Laboratory, Howard Hughes Medical Institute, Boston, Massachusetts.
authors.forename: G K
authors.initials: GK
authors.lastname: Adler
authors.forename: C M
authors.initials: CM
authors.lastname: Smas
authors.forename: J A
authors.initials: JA
authors.lastname: Majzoub
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics*; D004274:DNA, Recombinant / Q000302:isolation & purification; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D051379:Mice; D009693:Nucleic Acid Hybridization; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin; D003907:Dexamethasone; D003346:Corticotropin-Releasing Hormone