雌激素受体GPER调节HIF-1α蛋白促进肌成纤维细胞表型转化参与BPH进展

Benign Prostate Hyperplasia (BPH) is one of most common progressive diseases among aging man. Estrogen has been recognized as one of the most important factors that may influence BPH progression by promoting the proliferation of mesenchymal cells. Recently, fibrosis-like alteration and higher expression of G protein-coupled estrogen receptor (GPER), a newly discovered transmembrane estrogen receptor, were detected in the prostate tissues of early-onset symptomatic BPH patients. Meanwhile the agonist of GPER could induce myofibroblast differentiation of fibroblast and upregulate the protein level of Hypoxia Inducible Factor-1α (HIF-1α). HIF-1α is a key molecular that may regulate myofibroblast-differentiation-related genes. Therefore, we hypothesized that estrogen could activate GPER to aler HIF-1α protein modification. Then HIF-1α may regulate other differentiation related genes and induce myofibroblast differentiation to promote BPH progression. In this project, first, we will analyze the concentration of estrogen and the expression of GPER, HIF-1α in the prostate of BPH patients with different progression stages. Then, we would verify the impacts of GPER and HIF-1α on myofibroblast differentiation of prostate fibroblast. Last, we may investigate the mechanism that GPER regulate HIF-1α protein modification and HIF-1α regulate differentiation related genes. The project will illustrate the role of GPER and HIF-1α in the myofibroblast differentiation of prostate fibroblast and the detailed mechanism. It might provide a theoretical basis of dealing with BPH progression.

良性前列腺增生BPH是老年男性常见进展性疾病，雌激素可促进前列腺间质增生，是BPH进展的重要因素。我们前期发现，早期进展BPH患者前列腺呈纤维样改变，雌激素受体GPER表达增高，GPER激动剂可促进肌成纤维细胞表型转化、分泌胶原纤维，并上调肌成纤维细胞表型转化关键分子—缺氧诱导因子HIF-1α。故假设：雌激素通过GPER调节HIF-1α，进而调控表型转化相关靶基因，促进肌成纤维细胞表型转化，加速BPH进展。本课题拟①分析不同进展阶段BPH患者前列腺内雌激素水平、GPER及HIF-1α等分子的表达；②明确GPER、HIF-1α促进肌成纤维细胞表型转化的作用；③探究GPER调控HIF-1α蛋白表达及HIF-1α调节肌成纤维细胞表型转化关键靶基因的分子机制。本课题拟阐明GPER调控HIF-1α促进肌成纤维细胞表型转化在BPH进展中的作用机制，为临床控制BPH进展提供理论依据。

雌激素可促进前列腺间质增生，是BPH进展的重要因素。我们前期发现，早期进展BPH中GPER表达增高，GPER激动剂可促进肌成纤维细胞表型转化分泌胶原纤维，并上调其表型转化关键分子HIF-1α。故假设雌激素通过GPER调节HIF-1α，进而调控肌成纤维细胞表型转化，加速BPH进展。MTT实验发现雌激素（E2或G1）能促进前列腺间质细胞（WPMY-1和Prsc）增殖，敲除 GPER能显著抑制这一作用，说明雌激素通过激活 GPER促进间质细胞的增殖。以往研究提示可能和Gαi/EGFR/ERK信号通路有关，分别加入GPER、Gαi、EGFR、ERK的抑制剂，均能在不同程度上逆转E2或G1诱导的间质细胞增殖，同时Western blot证实上述抑制剂均能逆转E2或G1诱导的ERK1/2 磷酸化，表明雌激素通过激活 GPER/Gαi/EGFR/ERK信号通路促进间质细胞增殖。进一步研究雌激素/GPER/Gαi信号通路上调HIF-1α蛋白表达的机制。qPCR实验发现雌激素并不能增加间质细胞HIF-1α的mRNA水平，提示HIF-1α蛋白水平的改变可能和转录后调控有关。通过加入放线菌酮（真核细胞新生蛋白合成抑制剂）检测WPMY-1细胞在有或没有G1的处理下HIF-1α蛋白降解速率的差异，结果显示G1可增强HIF-1α蛋白的稳定性，减缓其降解速率。此外，在用G1处理WPMY-1细胞的同时加入蛋白酶体抑制剂MG132能挽救因敲除GPER所导致的HIF-1α蛋白的降解，而在没有敲除GPER的对照组中MG132并不能上调HIF-1α蛋白的表达，说明G1激活GPER对HIF-1α蛋白的上调是通过抑制蛋白酶体进而增加HIF-1α的稳定性而实现的。既往文献提示GPER通过PI3K/AKT/MDM2信号通路调节HIF-1α蛋白的稳定性，我们通过Western blot证实E2或G1均能促进WPMY-1细胞中AKT的磷酸化从而上调MDM2的磷酸化，促进HIF-1α蛋白的表达，最终增加其下游纤维化蛋白α-SMA, Collagen I、Lox的表达；而加入AKT的抑制剂能显著逆转以上效应。以上实验结果提示，雌激素/GPER/Gαi通过激活PI3K/AKT/MDM2信号通路抑制蛋白酶体，增加HIF-1α蛋白稳定性，最终促进前列腺间质细胞增殖。