Quirk C C, Lozada K L, Keri R A, Nilson J H
Department of Pharmacology Case Western Reserve University School of Medicine Cleveland, Ohio 44106, USA.
Mol Endocrinol. 2001 May;15(5):734-46. doi: 10.1210/mend.15.5.0628.
Reproduction depends on regulated expression of the LHbeta gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHbeta promoter and make essential contributions to its activity as well as mediate responsiveness to GNRH: Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHbeta gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LbetaT2), an LHbeta promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GNRH: In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHbeta promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHbeta promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.
生殖依赖于促黄体生成素β（LHβ）基因的调控表达。与早期生长反应蛋白1（Egr-1）和类固醇生成因子1（SF-1）结合的调控元件串联拷贝位于LHβ启动子的近端区域，对其活性做出重要贡献，并介导对促性腺激素释放激素（GnRH）的反应：在这些串联元件之间有一个能够结合同源结构域蛋白Pitx1的单一位点。从在异源细胞系中进行的过表达模式研究来看，Egr-1、SF-1和Pitx1似乎通过一种不需要Pitx1与其位点结合的机制进行协同相互作用。由于这些过表达研究的生理影响尚不清楚，我们使用同源细胞系和转基因小鼠重新评估了LHβ基因启动子中Pitx1元件的需求，这两者都消除了转录因子过表达的需要。我们的分析表明对Pitx1调控元件有显著需求。当使用促性腺激素细胞系（LβT2）通过瞬时转染进行检测时，携带突变Pitx1元件的LHβ启动子构建体显示出转录活性减弱，但仍保留对GnRH的反应：相比之下，对转基因小鼠中野生型和突变表达载体的分析表明，LHβ启动子活性完全依赖于功能性Pitx1结合位点的存在。事实上，转基因小鼠对完整Pitx1结合位点的依赖性非常严格，以至于对GnRH的反应也丧失了，这表明突变启动子是无活性的。总体而言，我们的数据强化了这样一个概念，即LHβ启动子的活性部分是通过SF-1、Egr-1和Pitx1之间高度协同的相互作用来决定的。虽然Egr-1可被视为GnRH的关键下游效应器，而Pitx1是激活SF-1的关键伙伴，但我们的数据确凿地表明，Pitx1元件在允许这些功能在体内发生方面起着至关重要的作用。
