蛋白酶体抑制通过UPR-BMP2环路诱导骨肉瘤细胞成骨分化的分子机制研究

Osteosarcoma is a malignant bone tumor with high mortality and disability, characterized by early distant metastasis. The recent studies indicate that proteasome inhibition can suppress the proliferation and induce apoptosis of osteosarcoma cells. However, we found that low dose of proteasome inhibitor can induce osteogenic differentiation of osteosarcoma cells, meanwhile, enhance unfolded-protein response (UPR) of the endoplasmic reticulum (ER) and upregulate the expression level of BMP2. Furthermore, we found that BMP2 stimulation alone also can induce osteogenic differentiation of osteosarcoma cells. Thus, we propose here that the UPR-BMP2 loop is involved in the regulating of osteogenic differentiation of osteosarcoma cells by proteasome inhibition. In this proposal, we will investigate into the mechanism underling osteogenic differentiation of osteosarcoma cells induced by proteasome inhibition in vitro and in vivo, mainly focusing on the molecular mechanism of UPR-BMP2 loop in this process, to elucidate the transcriptional regulation of ATF4 and XBP1s to BMP2 by systematically using the techniques for determining transcriptional regulation, signaling transduction pathway, and gene function. The anticipated results will enhance our understanding of the mechanisms underlying osteogenic differentiation and provide novel strategies for treatment of osteosarcoma.

骨肉瘤是一种恶性骨肿瘤，死亡率及致残率高，目前临床尚缺乏有效治疗手段。尽管有报道称蛋白酶体抑制能抑制骨肉瘤细胞增殖并诱导其凋亡，但我们的进一步研究发现低浓度蛋白酶体抑制剂能诱导骨肉瘤细胞成骨分化，并且其作用的发挥与UPR反应增强及BMP2上调表达密切相关。此外，我们还发现BMP2也能诱导骨肉瘤细胞发生成骨分化和内质网UPR反应增强。生物信息学预测发现BMP2启动子区存在内质网UPR通路关键分子ATF4和XBP1s多个结合位点。据此，我们认为蛋白酶体抑制介导的UPR-BMP2环路可能是诱导骨肉瘤成骨分化的重要原因。本研究将通过体内外实验证实蛋白酶体抑制对骨肉瘤细胞成骨分化的促进作用，通过靶向抑制UPR通路活性或敲除ATF4、XBP1s来明确UPR-BMP2环路调控骨肉瘤细胞成骨分化的分子机制。预期结果不仅能加深我们对于成骨分化相关分子机制的认识，更有望为骨肉瘤的治疗提供重要的理论与实验依据。

骨肉瘤是一种恶性骨肿瘤，死亡率及致残率高，目前临床尚缺乏有效治疗手段。有报道称蛋白酶体抑制能抑制骨肉瘤细胞增殖并诱导其凋亡，但我们的进一步研究发现蛋白酶体抑制能诱导骨肉瘤细胞成骨分化，并且其作用的发挥与UPR通路的活化密切相关。本项目基于多种骨肉瘤细胞系，通过茜素红染色实验和Gomori法显色证实蛋白酶体抑制在体外可以有效诱导骨肉瘤细胞成骨分化。通过免疫印迹实验证实蛋白酶体抑制能激活UPR通路PERK/eIF2α/ATF4和IRE1α/XBP1s支路。使用小分子抑制剂阻断PERK/eIF2α/ATF4 和 IRE1α/XBP1s支路能逆转蛋白酶体抑制诱导的骨肉瘤细胞成骨分化，抑制蛋白酶体抑制介导的成骨分化关键分子的上调表达。通过构建XBP1s过表达稳转骨肉瘤细胞株，在体外证实过表达XBP1s能诱导骨肉瘤细胞成骨分化，上调成骨分化相关分子的表达。通过构建骨肉瘤原位动物模型在体证实蛋白酶体抑制能诱导骨肉瘤细胞成骨分化，对骨肉瘤具有潜在的治疗效果。本项目结果加深了对于成骨分化相关分子机制的认识，为骨肉瘤的治疗提供重要的理论与实验依据。