Chronic GnRH Inhibits Activin Induction of the ovine FSH Beta Subunit: Involvement of cAMP Response Element Binding Protein (CREB) and Nitric Oxide Synthase typeI (NOSI)
SHAFIEE-KERMANI, FARIDEH. Chronic Gonadotropin-Releasing Hormone (GnRH) Inhibits Activin Induction of the Ovine Follicle Stimulating Hormone Beta-Subunit (oFSH?): Involvement of cAMP Response Element Binding Protein (CREB) and Nitric Oxide Synthase Type I (NOSI). (Under the direction of Dr. William L. Miller.)
Pituitary follicle stimulating hormone (FSH) is absolutely necessary for folliculogenesis and is important for spermatogenesis. It is primarily induced by activin and modulated by GnRH through its ?-subunit (FSH?). As an autocrine/paracrine factor, activin induces FSH?, but GnRH that is released from the hypothalamus in a pulsatile manner induces and inhibits FSH? based on its pulse amplitude and frequency. This study focuses on GnRH-mediated inhibition of activin-induced expression of FSH?, a situation found in vivo. Activin-treated primary murine pituitary cultures robustly express mut-oFSH?Luc-?AP1, a luciferase transgene driven by 4.7 kb of the ovine FSH? promoter. This promoter lacks two GnRH inducible AP-1 sites making it easier to observe GnRH mediated inhibition. Luciferase activity of this transgene was decreased 94% by 100 nM GnRH with an IC50 of 10-10 M and t½ of 4 h. The expression of follistatin that can inhibit activin was not increased by GnRH. Activators of cAMP and PKC such as forskolin and phorbol 12-myristate 13-acetate (PMA), respectively, mimicked the GnRH inhibitory effect. Kinetic studies of wild type oFSH?Luc in transformed gonadotropes, L?T2, showed a continuous induction by activin (5.5 fold) over 20 h. The induction by activin, up to 6 h, was not affected by GnRH, but was completely blocked thereafter. Cyclic AMP response element binding protein (CREB) was implicated in this inhibition because overexpression of its constitutively active mutant mimicked the inhibitory effect of GnRH and its inhibitor (ICERII) reversed the inhibition caused by GnRH, forskolin and PMA in L?T2 cells. In addition, GnRH, forskolin or PMA increased the expression of a CREB responsive promoter 6xCRE-Luc. Interstingly, inhibition of nitric oxide type I (NOSI) by 7-nitroindazole also reversed GnRH-mediated inhibition of wt-oFSH?Luc by 60%. It is known that GnRH and CREB induce expression of NOSI in gonadotropes and brain cortical cells, respectively. These data support the hypothesis that GnRH can inhibit activin-induced oFSH? expression by sequential activation of CREB and NOSI through cAMP and/or PKC pathway.
Advisor:William L. Miller; Robert R. Anholt; James A. Knopp; Dennis T. Brown
School:North Carolina State University
School Location:USA - North Carolina
Source Type:Master's Thesis
Date of Publication:04/17/2007