Department of Joint Surgery, The Affiliated Hospital of Qingdao University, Qingdao, 266000, China.
Department of Pharmacy, The Affiliated Hospital of Qingdao University, Qingdao, 266000, China.
Mol Med. 2021 Aug 28;27(1):96. doi: 10.1186/s10020-021-00360-w.
Apoptosis of chondrocyte is involved in osteoarthritis (OA) pathogenesis, and mechanical stress plays a key role in this process by activation of Piezo1. However, the negative regulation of signal conduction mediated by mechanical stress is still unclear. Here, we elucidate that the critical role of G protein coupled estrogen receptor (GPER) in the regulation of mechanical stress-mediated signal transduction and chondrocyte apoptosis.
The gene expression profile was detected by gene chip upon silencing Piezo1. The expression of GPER in cartilage tissue taken from the clinical patients was detected by RT-PCR and Western blot as well as immunohistochemistry, and the correlation between GPER expression and OA was also investigated. The chondrocytes exposed to mechanical stress were treated with estrogen, G-1, G15, GPER-siRNA and YAP (Yes-associated protein)-siRNA. The cell viability of chondrocytes was measured. The expression of polymerized actin and Piezo1 as well as the subcellular localization of YAP was observed under laser confocal microscope. Western blot confirmed the changes of YAP/ Rho GTPase activating protein 29 (ARHGAP29) /RhoA/LIMK /Cofilin pathway. The knee specimens of osteoarthritis model were stained with safranin and green. OARSI score was used to evaluate the joint lesions. The expressions of GPER and YAP were detected by immunochemistry.
Expression profiles of Piezo1- silenced chondrocytes showed that GPER expression was significantly upregulated. Moreover, GPER was negatively correlated with cartilage degeneration during OA pathogenesis. In addition, we uncovered that GPER directly targeted YAP and broadly restrained mechanical stress-triggered actin polymerization. Mechanism studies revealed that GPER inhibited mechanical stress-mediated RhoA/LIMK/cofilin pathway, as well as the actin polymerization, by promoting expression of YAP and ARHGAP29, and the YAP nuclear localization, eventually causing the inhibition of Piezo1. YAP was obviously decreased in degenerated cartilage. Silencing YAP caused significantly increased actin polymerization and activation of Piezo1, and an increase of chondrocyte apoptosis. In addition, intra-articular injection of G-1 to OA rat effectively attenuated cartilage degeneration.
We propose a novel regulatory mechanism underlying mechanical stress-mediated apoptosis of chondrocyte and elucidate the potential application value of GPER as therapy targets for OA.
软骨细胞凋亡参与骨关节炎（OA）的发病机制，机械应激通过激活 Piezo1 在这个过程中起着关键作用。然而，机械应激介导的信号传导的负调控仍不清楚。在这里，我们阐明了 G 蛋白偶联雌激素受体（GPER）在调节机械应激介导的信号转导和软骨细胞凋亡中的关键作用。
通过基因芯片检测沉默 Piezo1 后的基因表达谱。通过 RT-PCR、Western blot 和免疫组织化学检测临床患者软骨组织中 GPER 的表达，并探讨 GPER 表达与 OA 的相关性。用雌激素、G-1、G15、GPER-siRNA 和 YAP（Yes-associated protein）-siRNA 处理暴露于机械应激的软骨细胞。测量软骨细胞的活力。激光共聚焦显微镜观察聚合肌动蛋白和 Piezo1 的表达以及 YAP 的亚细胞定位。Western blot 证实了 YAP/Rho GTPase 激活蛋白 29（ARHGAP29）/RhoA/LIMK/肌动蛋白丝解聚蛋白通路的变化。OA 模型的膝关节标本用番红和绿染色。用 OARSI 评分评估关节病变。用免疫组化检测 GPER 和 YAP 的表达。
沉默 Piezo1 的软骨细胞的表达谱显示，GPER 的表达明显上调。此外，GPER 与 OA 发病过程中的软骨退变呈负相关。此外，我们发现 GPER 直接靶向 YAP，并广泛抑制机械应激触发的肌动蛋白聚合。机制研究表明，GPER 通过促进 YAP 和 ARHGAP29 的表达以及 YAP 的核定位，抑制机械应激介导的 RhoA/LIMK/肌动蛋白丝解聚蛋白通路和肌动蛋白聚合，从而抑制 Piezo1。退变软骨中 YAP 明显减少。沉默 YAP 导致肌动蛋白聚合和 Piezo1 激活明显增加，软骨细胞凋亡增加。此外，关节内注射 G-1 可有效减轻 OA 大鼠的软骨退变。
我们提出了一个新的机械应激介导的软骨细胞凋亡的调节机制，并阐明了 GPER 作为 OA 治疗靶点的潜在应用价值。
