Regulation of transfected glycoprotein hormone alpha-gene expression in primary pituitary cell cultures.
Studies of gene regulation are greatly facilitated by the ability to transfect DNA into cultured cells. We examined a variety of transfection techniques to optimize transient expression of the human glycoprotein hormone alpha-gene in primary pituitary cells and subsequently investigated the regulation of alpha-promoter transcription. Expression vectors driven by either the rous sarcoma virus-chloramphenicol acetyl transferase (RSVCAT) or the human alpha-gene (alpha CAT) promoters were transfected into cultures of dispersed female rat pituitary cells using calcium phosphate (CaPO4), diethylaminoethyl-dextran, lipofection, and electroporation procedures. CAT activity was optimal using the CaPO4 technique, resulting in 511 +/- 49% and 57 +/- 5% conversion/100 micrograms protein/4 h for RSVCAT and alpha CAT, respectively. Immunohistochemical analyses of alpha CAT expression using anti-CAT monoclonal antibodies demonstrated that the alpha-gene promoter is expressed in pituitary cells, predominantly if not exclusively, in gonadotropes and thyrotropes. Hormonal regulation of alpha-promoter activity was assessed using both the CAT and the luciferase (LUC) reporter systems. alpha-Promoter activity was significantly (P less than 0.001) stimulated by 8-bromo-cAMP (217% increase), GnRH (75% increase), GnRH agonist analog (141% increase), and TRH (75% increase). The expression of control plasmids (RSVLUC, TKLUC, pOLUC) was not affected by treatment with these agents. We conclude that CaPO4-mediated transfection allows analyses of transient gene expression in primary pituitary cells. The alpha-promoter directs expression specifically in pituitary cells, predominantly gonadotropes and thyrotropes. alpha-Gene transcription is stimulated by GnRH, TRH, and 8-bromo-cAMP.
Burrin
J M
JM
Thyroid Unit, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Jameson
J L
JL
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D007986:Luteinizing Hormone / Q000096:biosynthesis; D010902:Pituitary Gland / Q000378:metabolism*; D010906:Pituitary Hormone-Releasing Hormones / Q000502:physiology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011919:Rats, Inbred Strains; D013972:Thyrotropin / Q000096:biosynthesis; D014162:Transfection
D010906:Pituitary Hormone-Releasing Hormones; D007986:Luteinizing Hormone; D013972:Thyrotropin; D015500:Chloramphenicol O-Acetyltransferase