Walsh Heidi E, Shupnik Margaret A
University of Virginia, Charlottesville, Virginia 22908, USA.
Mol Endocrinol. 2009 Feb;23(2):237-50. doi: 10.1210/me.2008-0098. Epub 2008 Dec 18.
GnRH is the main modulator of LH secretion and transcription of the LH subunit genes in pituitary gonadotropes. The LHbeta gene is preferentially transcribed during pulsatile GnRH stimuli of one pulse/30 min and is thus carefully controlled by specific signaling pathways and transcription factors. We now show that GnRH-stimulated LHbeta transcription is also influenced by the ubiquitin-proteasome system. GnRH-stimulated activity of an LHbeta reporter gene was prevented by proteasome inhibitors MG-132 and lactacystin. Inhibition was not rescued by overexpression of two key transcription factors for LHbeta, early growth response-1 (Egr-1) and steroidogenic factor-1 (SF-1). Increased endogenous LHbeta transcription after GnRH treatment was also prevented by MG-132, as measured by primary transcript assays. To investigate possible mechanisms of LHbeta transcriptional inhibition by proteasome blockade, we employed chromatin immunoprecipitation to measure LHbeta promoter occupancy by transcription factors. Without GnRH, binding was low and unorganized. With GnRH, Egr-1 and SF-1 associations were stimulated, cyclic, and coincidental, with a period of approximately 30 min. MG-132 disrupted GnRH-induced Egr-1 and SF-1 binding and prevented phosphorylated RNA polymerase II association with the LHbeta promoter. Egr-1, but not SF-1, protein was induced by GnRH and accumulated with MG-132. Egr-1 and SF-1 were ubiquitinated in gonadotropes and ubiquitinated forms of these factors associated with the LHbeta promoter, suggesting their degradation may be key for LHbeta proteasome-dependent transcription. Together, these results demonstrate that degradation via the proteasome is vital to GnRH-stimulated LHbeta expression, and this occurs in part by allowing proper transcription factor associations with the LHbeta promoter.
促性腺激素释放激素（GnRH）是垂体促性腺细胞中促黄体生成素（LH）分泌及LH亚基基因转录的主要调节因子。LHβ基因在每30分钟一次脉冲式GnRH刺激期间优先转录，因此受到特定信号通路和转录因子的严格调控。我们现在发现，GnRH刺激的LHβ转录也受泛素-蛋白酶体系统影响。蛋白酶体抑制剂MG-132和乳胞素可抑制GnRH刺激的LHβ报告基因活性。两种LHβ关键转录因子早期生长反应因子-1（Egr-1）和类固醇生成因子-1（SF-1）的过表达无法挽救这种抑制作用。通过初级转录本分析测定，MG-132也可阻止GnRH处理后内源性LHβ转录增加。为研究蛋白酶体阻断抑制LHβ转录的可能机制，我们采用染色质免疫沉淀法测定转录因子与LHβ启动子的结合情况。无GnRH时，结合水平低且无规律。有GnRH时，Egr-1和SF-1的结合受到刺激，呈周期性且同步，周期约为30分钟。MG-132破坏GnRH诱导的Egr-1和SF-1结合，并阻止磷酸化的RNA聚合酶II与LHβ启动子结合。GnRH诱导Egr-1蛋白表达，但不诱导SF-1蛋白表达，且Egr-1蛋白在MG-132存在时积累。Egr-1和SF-1在促性腺细胞中被泛素化，这些因子的泛素化形式与LHβ启动子相关，表明它们的降解可能是LHβ蛋白酶体依赖性转录的关键。总之，这些结果表明，通过蛋白酶体降解对GnRH刺激的LHβ表达至关重要，这部分是通过使转录因子与LHβ启动子正确结合来实现的。
