GnRH Regulation of Jun and Atf3 Requires Calcium, Calcineurin, and NFAT

Abstract

GnRH binds to its receptor on gonadotropes and activates multiple members of the MAPK signaling family that in turn regulates the expression of several immediate early genes (IEGs) including Jun, Fos, Atf3, and Egr1. These IEGs confer hormonal responsiveness to gonadotrope-specific genes including Gnrhr, Cga, Fshb, and Lhb. In this study we tested the hypothesis that GnRH specifically regulates the accumulation of Jun and Atf3 mRNA through a pathway that includes intracellular Ca²⁺, calcineurin, and nuclear factor of activated T cells (NFAT). Our results indicate that pretreatment of murine LβT2 cells with 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, a Ca²⁺ chelator, reduced the expression of all the IEGs to varying degrees, whereas treatment with thapsigargin, an intracellular Ca²⁺ protein pump inhibitor, increased the expression of the IEG. Furthermore, cyclosporin A, a calcineurin-specific inhibitor, reduced the ability of GnRH to regulate accumulation of Jun and Atf3 mRNA and to a lesser extent Fos. In contrast, Egr1 mRNA was unaffected. NFATs are transcription factors regulated by calcineurin and were detected in LβT2 cells. GnRH increased luciferase activity of an NFAT-dependent promoter reporter that was dependent on intracellular Ca²⁺ and calcineurin activity. Additionally, although small interfering RNA specific for Nfat4 only marginally reduced GnRH regulation of Jun, Fos, and Atf3 mRNA accumulation, activity of an activator protein-1-responsive reporter construct was reduced by 48%. Together these data suggest that calcineurin and NFAT are new members of the gonadotrope transcriptional network that confer hormonal responsiveness to several key genes required for gonadotropin synthesis and secretion.