Abstract
Gonadotropin-releasing hormone (GnRH) acts via 7 transmembrane region receptors on gonadotrophs to stimulate synthesis and secretion of the luteinizing hormone and follicle-stimulating hormone. It is secreted in pulses, and its effects depend on pulse frequency, but decoding mechanisms are unknown. Here we have used (nuclear factor of activated T-cells 2 (NFAT2)-emerald fluorescent protein) to monitor GnRH signaling. Increasing [Ca2+]i causes calmodulin/calcineurin-dependent nuclear NFAT translocation, a response involving proteins (calmodulins and NFATs) that decode frequency in other systems. Using live cell imaging, pulsatile GnRH caused dose- and frequency-dependent increases in nuclear NFAT2-emerald fluorescent protein, and at low frequency, translocation simply tracked GnRH exposure (albeit with slower kinetics). At high frequency (30-min intervals), failure to return to basal conditions before repeat stimulation caused integrative tracking, illustrating how the relative dynamics of up- and downstream signals can increase efficiency of GnRH action. Mathematical modeling predicted desensitization of GnRH effects on [Ca2+]i and that desensitization would increase with dose, frequency, and receptor number, but no such desensitization was seen in HeLa and/or LβT2 cells possibly because pulsatile GnRH did not reduce receptor expression (measured by immunofluorescence). GnRH also caused dose- and frequency-dependent activation of αGSU, luteinizing hormone β, and follicle-stimulating hormone β luciferase reporters, effects that were blocked by calcineurin inhibition. Pulsatile GnRH also activated an NFAT-responsive luciferase reporter, but this response was directly related to cumulative pulse duration. This together with the lack of desensitization of translocation responses suggests that NFAT may mediate GnRH action but is not a genuine decoder of GnRH pulse frequency.
Highlights
Gonadotropin-releasing hormone (GnRH) acts via 7 transmembrane region receptors on gonadotrophs to stimulate synthesis and secretion of the luteinizing hormone and follicle-stimulating hormone
When HeLa cells were transduced with NFAT2-EFP, the reporter was largely cytoplasmic in unstimulated cells, but GnRH caused a pronounced translocation to the nucleus (Fig. 1B and supplemental Fig. 1)
Gonadotropin-releasing hormone is secreted from hypothalamic neurones to control synthesis and secretion of the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH)
Summary
GnRH, gonadotropin-releasing hormone ( known as GnRH I ϭ pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2); GnRHR, GnRH receptor; mGnRHR, mouse GnRHR; LH, luteinizing hormone; FSH, follicle-stimulating hormone; NFAT, nuclear factor of activated T cells; ERK, extracellular signal-regulated kinase; Ad, recombinant adenovirus; pfu, plaque-forming units; EFP, emerald fluorescent protein; DMEM, Dulbecco’s modified Eagle’s medium; RE, response element; Ctrl, control; GSU, gonadotropin subunit; BFP, blue fluorescent protein; NLS, nuclear localization sequence; ANOVA, analysis of variance; HA, hemagglutinin; N:C, nuclear:cytoplasmic; pEC50, negative log EC50. Where downstream responses have slower inactivation kinetics, responses may not have returned to the basal level before repeat stimulation, and this can cause cumulative (or saw-tooth) responses [5, 20, 21] This process of integrative tracking can amplify signaling but cannot alone explain the bell-shaped frequency-response relationships seen in many systems. GnRH increases expression of a number of dual-specificity phosphatases including DUSP1 and DUSP4 (mitogen-activated protein kinase phosphatases 1 and 2), both of which dephosphorylate (and inactivate) ERKs [43, 44] Any of these processes could generate negative feedback loops reducing Ca2ϩ signaling or ERK activity, thereby contributing to the frequency dependence of transcriptional regulation. GnRH effects on an NFAT-responsive luciferase reporter were dependent upon cumulative pulse duration, and together these data argue against a major role for negative feedback circuits in shaping frequency-response relationships through the Ca2ϩ/calmodulin/calcineurin pathway
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