Calcineurin Mediates the Gonadotropin-Releasing Hormone Effect on Expression of Both Subunits of the Follicle-Stimulating Hormone through Distinct Mechanisms

Abstract

Gonadotropin-releasing hormone (GnRH) regulates the expression of all three gonadotropin genes, encoding the common ␣ subunit (␣GSU) and hormone-specific ␤ subunits, through the activation of several signal transduction pathways. We have shown that GnRH also upregulates calcineurin, and we hypothesized that calcineurin mediates the effects of GnRH on the transcription of the ␣GSU and follicle-stimulating hormone ␤ (FSH␤) genes through two of its targets: nuclear factor of activated T cells (NFAT) and CREB-regulated transcription coactivator (TORC). We show that calcineurin is essential for GnRH-induced expression of both genes but that NFAT and TORC1 play quite distinct roles in activating each gene. GnRH induces calcineurindependent nuclear import of NFAT3, which activates the ␣GSU promoter, while TORC1 also mediates GnRH activation of this promoter, but not through CREB. GnRH initially stimulates the degradation of TORC1 but protects the N terminus of the newly synthesized protein to enhance its activity. Calcineurin induces Nur77 expression, likely via NFAT3, and Nur77 interacts synergistically with TORC1 and CREB to increase FSH␤ promoter activity. Although TORC plays a role in the basal activity of the FSH␤ promoter, it does not interact with phosphorylated CREB and probably does not play a major role in direct GnRH signaling to this gene. TORC may be part of an alternatively regulated pathway, possibly involving cross talk with other stimulatory hormones.In the pituitary gonadotrope, the gonadotropin-releasing hormone (GnRH) plays a crucial role in activating the transcription of all three gonadotropin subunit genes: the common ␣ subunit (␣GSU) and the hormone-specific ␤ subunits encoding luteinizing hormone (LH␤) and follicle-stimulating hormone (FSH␤). Upon binding its G-protein-coupled receptor (GPCR), GnRH activates protein kinase C (PKC) and a number of well-characterized mitogen-activated protein kinase (MAPK) pathways, as well as elevating cyclic AMP (cAMP) levels to activate protein kinase A (PKA) (7,20,33,42,53). It also induces an increase in intracellular calcium levels by stimulating calcium release from intracellular stores as well as the influx of extracellular calcium through voltage-sensitive channels (7, 42). One of the effects of this increase in calcium levels is the activation of calmodulin and calmodulin-dependent kinases (CaMKs), which mediate various aspects of GnRH-induced signaling, including inhibition of histone deacetylases and regulation of extracellular signal-regulated kinase (ERK) activity (6,10,23,24,34,44,61). Calmodulin also activates the serine/threonine protein phosphatase calcineurin to regulate the expression of various genes (26). In the gonadotrope, calcineurin moderates the activity of the prolyl isomerase Pin1, facilitating its localization to the nucleus, where it plays an important role in GnRH signaling to several key transcription factors (36). Calcineurin also plays a role in the GnRH-induced derepression of FSH␤ gene expression in immature ␣T3-1 gonadotrope ...

RNA transcribed from a distal enhancer is required for activating the chromatin at the promoter of the gonadotropin α-subunit gene

“…Murine gonadotropederived αT3-1 cells were cultured and transfected as described (43), and some were treated with GnRH (100 nM) or forskolin (10 μM; Sigma). HA-and FLAGPitx1 expression constructs have been reported previously (44).…”

“…S7), Absolute Blue SYBRGreen ROX Mix (Thermo Fisher), and the Illumina Eco Real-Time PCR as reported in ref. 43. Amplicon levels were quantitated relative to standard curves comprising cDNA or genomic DNA and were normalized to Gapdh mRNA.…”

“…ChIP was carried out after formaldehyde cross-linking as described in ref. 43 and is detailed in SI Experimental Procedures. Immunoprecipitation and input levels were measured by real-time qPCR using standard curves comprised of sonicated genomic DNA, as above.…”

Gonadotropin gene transcription is activated by menin-mediated effects on the chromatin

“…The MI-2 hydrochloride (Cayman Chemicals, Michigan) was dissolved in absolute ethanol and added at b0.1% final volume. Transfections were carried out at 50-60% confluency using GenePorter2 Transfection Reagent (Genelantis), according to the manufacturer's instructions and as described [6].…”

“…The mechanisms through which these genes are activated in basal conditions and via induction of various signaling cascades and genespecific transcription factors have been described in some detail [1][2][3][4][5][6]. However, in order for these proteins to bind the gene promoters and transcription to be activated, the DNA must be made accessible through altering the structure of the chromatin.…”