Regulation of transfected glycoprotein hormone α-gene expression in primary pituitary cell cultures

Abstract

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 α-gene in primary pituitary cells and subsequently investigated the regulation of α-promoter transcription. Expression vectors driven by either the rous sarcoma virus-chloramphenicol acetyl transferase (RSVCAT) or the human α-gene (αCAT) promoters were transfected into cultures of dispersed female rat pituitary cells using calcium phosphate (CaPO4), diethylaminoethyl-dextran, lipofection, and electroporation procedures. CAT ectivity was optimal using the CPO4 technique, resulting in 511 ± 49% and 57 ± 5% conversion/100 μg protein/4 h for RSVCAT and αCAT, respectivly. Immunohistochemical analyses of αCAT expression using anti-CAT monoclonal antibodies demonstrated that the α-gene promoter is expressed in pituitary cells, predominantly if not exclusively, in gonadotropes and thyrotropes. Hormonal regulation of α-promoter activity was assessed using both the CAT and the luciferase (LUC) receptor systems. α-Promoter activity was significantly (P < 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 α-promoter directs expression specifically in pituitary cells, predominantly gonadotropes and thyrotropes. α-Gene transcription is stimulated by GnRH, TRH, and 8-bromo-cAMP.