Ferris Heather A, Walsh Heidi E, Stevens Jonathan, Fallest Patricia C, Shupnik Margaret A
Department of Physiology, University of Virginia Medical School, Charlottesville, Virginia 22903, USA.
Biol Reprod. 2007 Dec;77(6):1073-80. doi: 10.1095/biolreprod.107.064139. Epub 2007 Aug 15.
Rat luteinizing hormone beta (Lhb) gene transcription is stimulated by hypothalamic gonadotropin-releasing hormone 1 (GnRH1), and this response may be modulated by other signaling pathways such as cAMP. Here we characterize the ability of cAMP, alone or with GnRH1, to stimulate Lhb gene transcription in mouse pituitary and clonal gonadotroph cells. Both cAMP and pituitary adenylyl cyclase-activating peptide increase GnRH1 stimulation of luciferase activity in pituitaries of mice expressing the rat Lhb-luciferase transgene, suggesting cAMP and GnRH1 pathways interact in vivo. cAMP stimulation of the Lhb-luciferase transgene was similar between females in metestrus and proestrus, but GnRH1 stimulation was greater at proestrus. Additive effects with combined treatments were observed at metestrus and proestrus. Elevated intracellular cAMP stimulated Lhb promoter activity in LbetaT2 clonal gonadotroph cells, alone and with GnRH1. In LbetaT2 cells, cAMP stimulation of the Lhb promoter was eliminated by inhibition of protein kinase A (PKA); GnRH1 stimulation was partially suppressed by either PKA or protein kinase C inhibitors. Only the proximal GnRH1-responsive region of the promoter was required for cAMP stimulation, and mutation of the 3' NR5A1 site diminished the response. Regulation of primary mRNA transcripts from the endogenous Lhb gene by cAMP and GnRH1 correlated with results from the Lhb-luciferase transgene or transfected promoter. Occupancy of the endogenous promoter by EGR1 was increased by GnRH1 with or without forskolin, but forskolin alone had little effect. Thus, cAMP stimulation of Lhb promoter activity, and enhancement of GnRH1 stimulation, occurs in multiple physiological states independent of steroid status, via a PKA-dependent mechanism.
大鼠促黄体生成素β(Lhb)基因转录受下丘脑促性腺激素释放激素1(GnRH1)刺激,并且这种反应可能受其他信号通路如cAMP的调节。在此,我们描述了cAMP单独或与GnRH1一起刺激小鼠垂体和克隆促性腺激素细胞中Lhb基因转录的能力。cAMP和垂体腺苷酸环化酶激活肽均可增强表达大鼠Lhb-荧光素酶转基因的小鼠垂体中GnRH1对荧光素酶活性的刺激,提示cAMP和GnRH1通路在体内相互作用。在动情后期和发情前期的雌性小鼠中,cAMP对Lhb-荧光素酶转基因的刺激作用相似,但GnRH1在发情前期的刺激作用更强。在动情后期和发情前期观察到联合处理具有相加效应。细胞内cAMP升高单独或与GnRH1一起刺激LbetaT2克隆促性腺激素细胞中的Lhb启动子活性。在LbetaT2细胞中,抑制蛋白激酶A(PKA)可消除cAMP对Lhb启动子的刺激;GnRH1刺激可被PKA或蛋白激酶C抑制剂部分抑制。cAMP刺激仅需要启动子近端的GnRH1反应区域,3' NR5A1位点的突变会减弱反应。cAMP和GnRH1对内源性Lhb基因初级mRNA转录本的调节与Lhb-荧光素酶转基因或转染启动子的结果相关。无论有无福司可林,GnRH1均可增加内源性启动子上早期生长反应蛋白1(EGR1)的结合,但单独使用福司可林几乎没有影响。因此,cAMP对Lhb启动子活性的刺激以及对GnRH1刺激的增强,通过一种依赖PKA的机制,在多种生理状态下发生,与类固醇状态无关。