泛素-蛋白酶体途径调控的PDGFR-β降解在血管内膜增生中的作用和机制研究

Intimal hyperplasia, which is the common step in the pathological development of several cardiovascular diseases including atherosclerosis, is mainly caused by abnormal proliferation, migration and extracellular matrix secretion of vascular smooth muscle cells (VSMC). Studies show that PDGFR-β plays a key role in the proliferation, migration and phenotypic transformation of VSMC. However, the degradation mechanism of PDGFR-β is still elusive. In the preliminary investigation, we find that inhibition of proteasome can significantly increase PDGFR-β protein level. PDGFR-β is observed to contain the recognition and binding site of E3 ligase subunit βTrCP. Co-IP reveals the interaction between PDGFR-β and βTrCP. Knockdown of βTrCP can significantly enhance PDGFR-β protein stability, VSMC proliferation and migration, and reduce the expression of VSMC markers. Further analysis shows the Ser residue in this recognition and binding site could be phosphorylated by NEK2. Based on these evidences, we hypothesize βTrCP and NEK2 could mediate PDGFR-β degradation through ubiquitin-proteasome system, participating in the regulation of VSMC proliferation, migration and phenotypic transformation. To test this hypothesis, we intend to carry out in vitro and in vivo experiments to investigate the mechanism of PDGFR-β degradation through ubiquitin-proteasome pathway, and the corresponding biological effects on VSMC proliferation, migration and phenotypic transformation. We will also test the possibility to inhibit intimal hyperplasia by targeting PDGFR-β degradation.

血管内膜增生是动脉粥样硬化等多种心血管疾病的重要病理过程，主要由VSMC异常增殖、迁移和分泌细胞外基质引起。PDGFR-β是介导VSMC增殖、迁移和表型转化的关键分子，但其降解机制仍未知。我们的前期研究发现蛋白酶体抑制剂能显著升高PDGFR-β的蛋白水平，PDGFR-β上存在E3连接酶亚基βTrCP的识别结合位点，二者存在相互作用，敲除βTrCP能增强PDGFR-β的蛋白稳定性和VSMC增殖、迁移能力，降低VSMC标记物的表达。进一步分析发现该位点中的Ser可能被NEK2磷酸化。因此，我们推测βTrCP和NEK2共同介导PDGFR-β通过泛素-蛋白酶体途径降解，参与调控VSMC增殖、迁移和表型转化。本课题拟在前期研究的基础上，从体内和体外两方面研究PDGFR-β通过泛素-蛋白酶体降解的机制及其在VSMC增殖、迁移和表型转化中的作用，并探讨靶向PDGFR-β降解在抑制血管内膜增生中的可行性。

血管内膜增生是动脉粥样硬化、高血压和血管重建术后再狭窄等心血管疾病发展过程中的共同特征和重要环节，严重影响威胁人类生命健康。血小板源性生长因子受体β（PDGFRβ）是促进血管内膜增生的重要效应分子，其介导的的信号级联反应在血管平滑肌细胞增殖、迁移和表型转化过程中发挥重要的生物学作用。作为一种膜受体，PDGFRβ通常会在配体与其结合后发生内化并快速降解，以防止受体被过度激活。但是，PDGFRβ的降解机制特别是在血管平滑肌细胞中的降解机制仍不清楚。在该研究中我们发现PDGFRβ在血管新生内膜中表达升高，其表达升高主要是由于PDGFRβ蛋白降解受到抑制。我们发现PDGF-BB刺激能诱导PDGFRβ通过泛素-蛋白酶体途径快速降解，抑制蛋白酶体则能提高PDGFRβ蛋白稳定性和表达水平，减弱PDGF-BB对于PDGFRβ蛋白降解的诱导作用。E3连接酶SCFβTrCP通过S1092序列介导PDGFRβ蛋白降解，敲低βTrCP能显著增强PDGFRβ蛋白水平和稳定性，提高PDGF-BB对于PDGFRβ信号通路的激活作用和对血管平滑肌细胞增殖、迁移和表型转化的促进作用。NEK2在新生内膜中表达上调，是调控PDGFRβ蛋白降解的激酶。敲低NEK2能有效增强PDGFRβ蛋白水平和稳定性，而过表达NEK2则显著逆转PDGF-BB对于PDGFRβ信号通路激活和血管平滑肌细胞增殖、迁移和表型转化的促进作用。我们的研究结果提示，损伤血管中NEK2表达降低使PDGFRβ蛋白保持较高的稳定性，在受到PDGF-BB刺激时不易被降解，从而导致PDGFRβ信号通路持续激活，促进平滑肌细胞增殖、迁移和表型转化，最终导致血管内膜增生。我们的研究揭示了PDGFRβ蛋白降解异常在血管内膜增生过程中的作用机制，为血管内膜增生的预防和治疗提供了新的靶点，具有一定的临床意义和应用价值。