Park Hyun-Jung, Gholam-Zadeh Malihatosadat, Yoon Sun-Young, Suh Jae-Hee, Choi Hye-Seon
Department of Biological Sciences (BK21 Program), University of Ulsan, Ulsan 680-749, Korea.
Department of Pathology, Ulsan University Hospital, Ulsan 682-714, Korea.
Antioxidants (Basel). 2021 Apr 17;10(4):619. doi: 10.3390/antiox10040619.
Loss of ovarian function is closely related to estrogen (E) deficiency, which is responsible for increased osteoclast (OC) differentiation and activity. We aimed to investigate the action mechanism of E to decrease bone resorption in OCs to protect from ovariectomy (OVX)-induced bone loss in mice. In vivo, tartrate-resistant acid phosphatase (TRAP) staining in femur and serum carboxy-terminal collagen crosslinks-1 (CTX-1) were analyzed upon E injection after OVX in mice. In vitro, OCs were analyzed by TRAP staining, actin ring formation, carboxymethylation, determination of reactive oxygen species (ROS) level, and immunoprecipitation coupled with Western blot. In vivo and in vitro, E decreased OC size more dramatically than OC number and Methyl-piperidino-pyrazole hydrate dihydrochloride (MPPD), an estrogen receptor alpha (ERα) antagonist, augmented the OC size. ERα was found in plasma membranes and E/ERα signaling affected receptor activator of nuclear factor κB ligand (RANKL)-induced actin ring formation by rapidly decreasing a proto-oncogene tyrosine-protein kinase, cellular sarcoma (c-Src) (Y416) phosphorylation in OCs. E exposure decreased physical interactions between NADPH oxidase 1 (NOX1) and the oxidized form of c-Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), leading to higher levels of reduced SHP2. ERα formed a complex with the reduced form of SHP2 and c-Src to decrease c-Src activation upon E exposure, which blocked a signal for actin ring formation by decreased Vav guanine nucleotide exchange factor 3 (Vav3) (p-Y) and Ras-related C3 botulinum toxin substrate 1 (Rac1) (GTP) activation in OCs. E/ERα signals consistently inhibited bone resorption in vitro. In conclusion, our study suggests that E-binding to ERα forms a complex with SHP2/c-Src to attenuate c-Src activation that was induced upon RANKL stimulation in a non-genomic manner, resulting in an impaired actin ring formation and reducing bone resorption.
卵巢功能丧失与雌激素（E）缺乏密切相关，雌激素缺乏会导致破骨细胞（OC）分化和活性增加。我们旨在研究E减少OC骨吸收的作用机制，以预防小鼠卵巢切除（OVX）诱导的骨质流失。在体内，对OVX后的小鼠注射E后，分析股骨中的抗酒石酸酸性磷酸酶（TRAP）染色和血清羧基末端胶原交联物-1（CTX-1）。在体外，通过TRAP染色、肌动蛋白环形成、羧甲基化、活性氧（ROS）水平测定以及免疫沉淀结合蛋白质印迹法分析OC。在体内和体外，E比OC数量更显著地减小OC大小，而雌激素受体α（ERα）拮抗剂甲基哌啶基吡唑水合物二盐酸盐（MPPD）则增大OC大小。在质膜中发现了ERα，并且E/ERα信号通过快速降低OC中原癌基因酪氨酸蛋白激酶细胞肉瘤（c-Src）（Y416）的磷酸化来影响核因子κB受体激活剂配体（RANKL）诱导的肌动蛋白环形成。E暴露减少了NADPH氧化酶1（NOX1）与含Src同源2（SH2）结构域的蛋白酪氨酸磷酸酶2（SHP2）氧化形式之间的物理相互作用，导致还原型SHP2水平升高。ERα与还原型SHP2和c-Src形成复合物，在E暴露时降低c-Src激活，这通过降低OC中Vav鸟嘌呤核苷酸交换因子3（Vav3）（p-Y）和Ras相关C3肉毒杆菌毒素底物1（Rac1）（GTP）的激活来阻断肌动蛋白环形成信号。E/ERα信号在体外持续抑制骨吸收。总之，我们的研究表明，E与ERα结合形成SHP2/c-Src复合物，以非基因组方式减弱RANKL刺激诱导的c-Src激活，导致肌动蛋白环形成受损并减少骨吸收。
