TRPV5介导雌激素抑制破骨细胞骨吸收作用及机制研究

The precise molecular events underlying the inhibitory effect of estrogen (E2) on osteoclastic bone resorption remained elusive. Our previous studies showed that E2 inhibited osteoclastic resorption by increasing the expression of TRPV5 channel in osteoclasts. The estrogen receptor (ER) blocker significantly attenuated the stimulatory effect of estrogen on TRPV5 expression. The promoter sequence of the TRPV5 gene does not consist of estrogen response elements (ERE) which are required in the classical ERE genomic signaling pathway. However, the TRPV5 gene promoter contains several of these AP-1, SP1 and NF-κB sites, which can mediate the transcriptional activation of estrogen-liganded ER through non-classical genomic pathway. On the basis of this information, we presume that E2 regulates the expression of TRPV5 in the osteoclasts through the non-classical genomic signaling pathway. Therefore, our research is centralized with the regulation of the expression of TRPV5 in the osteoclasts. RNA interference(RNAi), chromatin immunoprecipitation(ChIP) and bioinformatics are applied. Firstly, TRPV5-mediated the inhibitory effect of E2 on the osteoclastic bone resorption will be confirmed. Sencondly, the regulation of E2 on the expression of TRPV5 in the osteoclasts by estrogen receptor (ER) will be clarificated. Finally, the regulatory effect of E2 on the TRPV5 gene activation and transcription is mediated through the ER dependent non-classical genomic pathway will be revealed. According to this study, the molecular mechanisms that TRPV5 mediates the inhibitory effect of E2 on the osteoclastic bone resorption will be found. More importantly, the application of E2 to prevent and treat osteoporosis will gain more scientific evidences from this study.

雌激素（E2）具有抑制破骨细胞骨吸收作用，但其确切机制仍不清楚。我们前期发现：E2通过上调破骨细胞TRPV5的表达，抑制其骨吸收能力。雌激素受体(ER)阻滞剂可抑制E2对破骨细胞TRPV5表达的刺激作用。TRPV5基因启动子区域无经典ERE信号通路必需的雌激素反应元件，而含有AP-1、SP1和NF-κB等非经典ERE信号通路转录调控因子结合位点。因此，推测E2通过非经典ERE信号通路调控破骨细胞TRPV5表达。本课题拟以破骨细胞TRPV5表达调控为线索，通过RNAi、ChIP、生物信息学技术等手段，首先，探讨并确定TRPV5介导E2抑制破骨细胞骨吸收作用；其次，阐明E2通过ER调控破骨细胞TRPV5表达；最后，明确雌激素受体非经典ERE信号通路在E2调控破骨细胞TRPV5基因转录激活中的作用。籍此阐释TRPV5介导E2抑制破骨细胞骨吸收作用及分子机制，进一步揭示E2防治骨质疏松的理论依据。

骨质疏松症已成为严重影响中老人的健康的慢性病之一，该病可发生于不同的年龄和性别，绝经后女性多见，雌激素（E2）在维持女性骨量和骨强度上具有重要作用，具有抑制骨吸收作用，但其确切作用机制不明确。TRPV5是破骨细胞上重要的钙离子通道，通过参与钙离子信号调控，影响破骨细胞的分化和骨吸收能力。本课题通过实验设计，探索E2，TRPV5，ER，以及转录结合因子AP-1、SP1和NF-κB在调控破骨细胞功能中的相互作用关系。应用了RNAi，ChIP，免疫共沉淀，western-blot等实验方法，研究并明确了：1.TRPV5介导E2抑制破骨细胞骨吸收作用；2.E2通过雌激素受体（ER）调控破骨细胞TRPV5表达；3. TRPV5基因启动子在500bp至2000bp范围内具备完整的启动子活性，其中基因启动子500bp范围内可能具备基因转录表达所需要的大部分转录结合位点；4.AP-1、SP1和NF-κB在雌激素调控TRPV5的转录激活中均起作用，其中NF-κB可能起主要作用。本课题研究成果为雌激素防治绝经后骨质疏松提供新的科学依据，为新型抗骨质疏松药物的研发提供新的思路。