br Signaling pathways activated by
Signaling pathways activated by pulsatile GnRH In the pituitary, GnRH acts by binding to the G protein-coupled GnRHR on the cell surface of the gonadotrope, inducing interaction of the receptor with heterotrimeric G proteins and catalyzing GTP-GDP exchange on the G protein α subunit (Lambert, 2008, Oldham and Hamm, 2008). Four Gα subfamilies have been identified in the mammalian genome: Gαs, Gαq/11, Gα12/13 and Gαi/o (Simon et al., 1991). It is well established that the GnRHR interacts with Gα proteins to activate a variety of distinct signaling pathways. However, questions remain about precisely which G proteins are involved in GnRHR signaling in the gonadotrope, how each G protein contributes to signaling, and how the GnRH pulses are decoded to activate signaling cascades that preferentially induce FSH or LH production. Initially, even before gonadotrope-derived cell lines became available, it was suggested that the GnRHR couples to a pertussis toxin-insensitive G protein (Gp, later renamed as Gq), based on studies performed in cultured rat pituitary Protease Inhibitor Cocktail (EDTA-Free, 200X in DMSO) (Naor et al., 1986). Subsequently, studies in αT3-1, CHO-K1 and COS-7 cells enhanced the hypothesis that the GnRHR initiates signaling pathway(s) by coupling exclusively to Gαq/11 (Hsieh and Martin, 1992, Grosse et al., 2000). On the other hand, studies in LβT2 cells indicated that both Gαq/11 and Gαs were involved in GnRHR signaling (Liu et al., 2002a, Liu et al., 2002b). In addition, studies from the same laboratory noticed a differential desensitization of Gαs and Gαq/11 in response to pulsatile GnRH stimulation (Tsutsumi et al., 2010). In a more recent study, the expression and role of individual G proteins were investigated in LβT2 cells using siRNA and bacterial toxins. These studies indicated that GnRH signaling in gonadotrope cells involved primarily Gαq/11 and Gαs (and to a lesser extent Gα12/13 as well) and, more interestingly, that their depletion differentially affected GnRH-stimulated gonadotropin gene expression (Choi et al., 2012). Gαq/11 knockdown reduced GnRH-stimulated Fshb mRNA levels, while Gαs knockdown reduced GnRH-stimulated Lhb mRNA levels. However, these results were not entirely consistent with the effects on the promoter activity of Fshb and Lhb. Although the Gαi/o subfamily is expressed in LβT2 cells, it was not found to be involved in regulation of gonadotropin gene expression (Choi et al., 2012, Krsmanovic et al., 2003).
Regulation of gonadotropin gene expression by pulsatile GnRH The signaling pathways described previously affect Cga, Fshb and Lhb gene expression. These genes are differentially regulated by varying GnRH pulse frequencies, although the α-subunit is produced in excess, regardless of GnRH pulse frequency (Haisenleder et al., 1991, Dalkin et al., 1989). Therefore, the control of FSH and LH synthesis is closely correlated with the production of the distinct β-subunits. This section of this review will focus on transcription factors that are activated by pulsatile GnRH and mediate Fshb and Lhb expression.
Conclusions Normal reproductive function and fertility require the precise regulation of LH and FSH. Identifying the molecular mechanisms that regulate gonadotropin synthesis may help us to better understand the ovulatory and menstrual cycles, puberty, and even the pathophysiology of reproductive disorders such as polycystic ovarian syndrome. Several signaling pathways have been implicated in both LH and FSH synthesis. It appears that the GnRHR differentially activates multiple distinct signaling pathways in response to varying GnRH pulse frequencies. Considerable progress has been made during the past decade, but much more remains to be elucidated to fully understand the enigma of the GnRH pulse frequency decoder. More in vivo studies are necessary to confirm and refine results obtained in immortalized gonadotropic cell lines. In vivo studies using mice with gonadotrope-specific deletion of Gαs or Gαq/11 will help to further elucidate the role of each G protein in gonadotropin regulation. Furthermore, additional studies of the GnRHR are needed to understand the differential G protein coupling of the GnRHR upon pulsatile GnRH stimulation. In addition, other factors regulating gonadotropins, including inhibin, activin, follistatin, sex steroids, and other neuropeptides, as well as epigenetic contributions to regulation, need to be integrated into this model in order to fully understand the regulation of gonadotropins in health and disease.