靶向调节EGFR-Gαi1/3-Gab1通路抗激素诱导成骨细胞凋亡作用及机制研究

Dexamethasone (Dex) is known to induce osteoblast cell apoptosis, causing femoral head necrosis. Our previous studies have shown that epiregulin and pleiotrophin activate EGFR-AKT-mTOR signaling in cultured osteoblasts, and exert pro-survival roles against Dex. Our preliminary studies have indentified two potential adaptor proteins for Epn/Ptn signaling: including inhibitory heterotrimeric G proteins α subunit 1 and 3 (Gαi1/3) and Grb2 associated bind 1 (Gab1). We found that Ptn induced EGFR-Gαi1/3-Gab1 association, which was required for downstream signaling transduction. Sh-RNA silencing of Gαi1/3 or Gab1 inhibited Ptn-induced AKT/mTOR activation in osteoblasts, and reduced its pro-survival activity against Dex. We propose that EGFR-Gαi1/3-Gab1 signaling might be a novel signaling target in osteoblasts against Dex-mediated damages. In this proposal, by using in vivo and in vitro models, we aim to further understand the expression and association of EGFR-Gαi1/3-Gab1 signaling axis in human femoral head necrosis tissues, and compared them with surrounding normal femoral head tissues. Research focus will be put on how EGFR-Gαi1/3-Gab1 axis mediates Epn/Ptn-induced PI3K/AKT/mTOR activation, and the defective roe of PI3K/AKT/mTORC1/C2 signaling in Epn/Ptn-induced anti-Dex activity in cultured osteoblasts. MiRNA strategy will also be utilized to alter EGFR expression and to inhibit Dex-induced osteoblast apoptosis. The results of this project will provide theoretical and experimental support for using EGFR ligands (i.e. Ptn and Epn) in the treatment of steroid-induced femoral head necrosis.

糖皮质激素诱导成骨细胞凋亡致股骨头坏死。前期研究结果证实上皮调节蛋白（Epn）和多效蛋白（Ptn）激活成骨细胞的EGFR-AKT-mTOR通路，拮抗地塞米松诱导的成骨细胞损伤。预实验结果显示Epn/Ptn诱导成骨细胞EGFR-Gαi1/3-Gab1信号复合物耦联，而基因敲除或shRNA敲减Gαi1/3或Gab1则抑制Epn/Ptn诱导的AKT-mTOR的活化和成骨细胞保护作用。本项目中拟应用体内、外激素性股骨头坏死模型，系统观察Epn/Ptn抗激素成骨细胞损伤的作用，解析EGFR-Gαi1/3-Gab1复合物激活PI3K-AKT-mTOR通路在其中的作用和分子机制。同时观察该复合物在人激素性股骨头坏死组织中的表达和活化水平。最后通过miRNA-7靶向调节成骨细胞EGFR水平，达到抑制激素性成骨细胞损伤的目的。拟证实EGFR-Gαi1/3-Gab1通路是成骨细胞抗激素损伤的新靶点。

长期或大剂量临床应用地塞米松等皮质激素会导致一系列骨科的并发症，如骨质疏松症、股骨头坏死等。激素过量使用已成为非创伤性股骨头坏死首要危险因素。实验和临床研究资料表明骨组织细胞的凋亡及坏死是股骨头坏死发生与演变的细胞学基础。成骨细胞（Osteoblasts）在骨的发育、重建、修复中发挥着重要作用，在骨重建系统中主导骨生成，并诱导破骨细胞分化成熟促进骨吸收。本研究通过应用细胞和小鼠激素性股骨头坏死模型，系统研究Ptn或Epn对激素诱导成骨细胞凋亡的保护作用，阐明了其相关信号转导机制。研究结果显示，人激素性股骨头坏死组织中EGFR蛋白和mRNA的表达水平和正常骨组织相比显著下调，而其抑制性miRNA-7则高表达。EGFR通路被Epn和Ptn激活后，均能促进成骨细胞的存活。通过多种手段抑制EGFR 后，均可逆转成骨细胞保护作用。通过免疫共沉淀方法发现Epn和Ptn诱导EGFR-Gαi1/3-Gab1信号复合物耦联，而shRNA敲减Gαi1/3或Gab1，则显著抑制Epn和Ptn诱导的下游AKT/mTOR的活化。EGFR是人类成骨细胞中miR-7的主要靶点，在miRNA靶向调节EGFR通路促进成骨细胞存活中，miR-7抑制剂通过激活EGFR信号传导来保护人类成骨细胞免受激素的损伤。证实EGFR-Gαi-Gab1信号复合物介导PI3K/AKT/mTOR通路的活化是Ptn或Epn抗激素性成骨细胞凋亡的核心分子机制。为临床运用Ptn、Epn等EGFR激动剂治疗激素性股骨头坏死提供实验依据；并为基因治疗激素性股骨头坏死提供有益探索。