Winters Stephen J, Ghooray Dushan, Fujii Yasuhisa, Moore Joseph P, Nevitt Jennifer R, Kakar Sham S
Division of Endocrinology & Metabolism, University of Louisville, Louisville, KY 40202, United States.
Mol Cell Endocrinol. 2007 Jun 15;271(1-2):45-54. doi: 10.1016/j.mce.2007.03.006. Epub 2007 Apr 4.
GnRH applied continuously or in pulses of high frequency increases follistatin, and thereby differentially regulates FSH and LH. This study was conducted in alphaT3-1 and LbetaT2 gonadotroph cells to begin to understand the signaling pathways through which GnRH stimulates follistatin synthesis. GnRH increased follistatin expression and stimulated a follistatin-LUC reporter in LbetaT2 cells, but was inactive in alphaT3-1 cells. GnRH also increased cAMP levels and stimulated a cAMP-responsive promoter only in LbetaT2 cells. Forskolin stimulated follistatin in both cell lines. GnRH activation of follistatin was blocked by the PKA inhibitor H89 and by over-expression of a dominant-negative inhibitor of CREB (A-CREB). Activation was also suppressed by PKC depletion, and was reduced by the PKC inhibitor bisindolylmaleimide. The MEK inhibitor PD98059 blocked activation by GnRH or forskolin implying that MAPK contributes to cAMP/PKA-mediated activation of follistatin. When LbetaT2 cells were transfected with follistatin-LUC together with A-CREB, and perifused with GnRH, activation was blocked during continuous GnRH, but stimulation by hourly GnRH pulses was unaffected. These experiments provide evidence that GnRH stimulates follistatin through multiple signaling pathways, and that cAMP-CREB activation is obligatory when GnRH is applied continuously. The finding that follistatin transcription was CREB-dependent with continuous but not pulsatile GnRH implies that the mode of ligand activation of GnRH receptors modifies the transcriptional response by changing the signaling network. These results provide a mechanism linking GnRH pulsatility to the differential control of FSH-beta and LH-beta gene expression through follistatin.
持续应用或高频脉冲式应用促性腺激素释放激素（GnRH）可增加卵泡抑素，从而对促卵泡生成素（FSH）和促黄体生成素（LH）进行差异性调节。本研究在αT3-1和LβT2促性腺激素细胞中开展，以初步了解GnRH刺激卵泡抑素合成的信号通路。GnRH可增加LβT2细胞中卵泡抑素的表达并刺激卵泡抑素荧光素酶报告基因，但在αT3-1细胞中无活性。GnRH还仅在LβT2细胞中增加环磷酸腺苷（cAMP）水平并刺激cAMP反应性启动子。福斯高林可刺激两种细胞系中的卵泡抑素。GnRH对卵泡抑素的激活被蛋白激酶A（PKA）抑制剂H89以及cAMP反应元件结合蛋白（CREB）的显性负性抑制剂（A-CREB）的过表达所阻断。激活也被蛋白激酶C（PKC）耗竭所抑制，并被PKC抑制剂双吲哚马来酰亚胺所降低。丝裂原活化蛋白激酶（MEK）抑制剂PD98059可阻断GnRH或福斯高林的激活，这意味着丝裂原活化蛋白激酶（MAPK）参与cAMP/PKA介导的卵泡抑素激活。当LβT2细胞与A-CREB一起转染卵泡抑素荧光素酶报告基因并灌注GnRH时，持续给予GnRH期间激活被阻断，但每小时给予GnRH脉冲的刺激不受影响。这些实验提供了证据表明GnRH通过多种信号通路刺激卵泡抑素，并且当持续应用GnRH时cAMP-CREB激活是必需的。卵泡抑素转录在持续而非脉冲式GnRH作用下依赖于CREB这一发现意味着GnRH受体的配体激活模式通过改变信号网络来修饰转录反应。这些结果提供了一种机制，将GnRH的脉冲性与通过卵泡抑素对FSH-β和LH-β基因表达的差异性控制联系起来。
