SUMO化增强瘢痕疙瘩中HIF-1/2和TGF-β/Smad通路内相关蛋白活性的作用及调控机制

Over-fibrosis and over-deposition of extracellular matrix due to the expression up-regulation of target genes mediated by hypoxia inducible factor-1 (HIF-1) and HIF-2 and transforming growth factor-beta (TGF-β)/Smad signaling pathways are the major mechanism of keloid formation. However, recent founding revealed that the activity enhancement of the pathways is also involved in pathogenesis of keloid, but its mechanism is unknown. Several literatures recently reported that hypoxia could induce the expression of small ubiquitin-like odifier (SUMO) in human cells (e.g. embryo kidney cells) and the combination of SUMO with HIF-1α/2α, TβR-I and Smad4 (i.e. sumoylation) could increase their transcription activity and signal transduction. We found that the expression of SUMO-1/2/3 was up-regulated while the expression of SENPs for de-sumoylation was down-regulated in keloid tissue. All the data made us to suppose that the sumoylation has an ability to amplify the roles of HIF-1/HIF-2 and TGF-β/Smad signaling pathways. In this present project, we plan to determine that the combination of SUMO-1/2/3 with HIF-1α/2α, TβR-I and Smad4 in fibroblasts of keloid for sumoylation, the sumoylation increases the activities of HIF-1/2 transcription and TGF-β/Smad signaling, and both the expression down-regulation of SENPs and expression up-regulation of RSUME maintain the high sumoylation levels of the target proteins. Subsequently, the rat model and human keloid samples will be used to verify the results in vitro. All the results will illuminate the role and mechanism of the sumoylation during formation of keloid, which has high creativeness and medical significance.

缺氧诱导因子HIF-1/2和TGF-β/Smad通路参与上调靶基因表达所致过度纤维化和胞外基质沉积是瘢痕疙瘩形成主要机制，但近年发现上述通路中蛋白活性增强也参与发病过程，机制不明。现报道缺氧诱导表达的人胚肾等细胞小泛素蛋白样修饰蛋白SUMO修饰HIF-1α/2α、TβR-I和Smad4可增强转录活性和信号传导。我们发现瘢痕疙瘩中SUMO-1/2/3表达升高，但去SUMO化酶SENPs表达降低，推测SUMO化具有放大上述通路的效应。本项目拟确定瘢痕疙瘩成纤维细胞中SUMO-1/2/3使HIF-1α/2α和TβR-I及Smad4发生SUMO化；SUMO化增强HIF-1/2转录活性和TGF-β/Smad信号传导；SENPs表达下调和RSUME表达上调使靶蛋白保持高水平SUMO化。然后采用大鼠模型和人瘢痕疙瘩标本验证上述体外结果，以期阐明SUMO化在瘢痕疙瘩形成中的作用及分子机制，具有较高创新性。

项目背景：.缺氧诱导因子HIF-1/2和TGF-β/Smad通路参与上调靶基因表达所致过度纤维化和胞外基质沉积是瘢痕疙瘩形成主要机制，但近年发现上述通路中蛋白活性增强也参与发病过程，机制不明。我们发现瘢痕疙瘩中SUMO-1/2/3表达升高，但去SUMO化 酶SENPs表达降低，推测SUMO化具有放大上述通路的效应。但其具体的作用机制仍未明确。.主要研究内容：.从组织标本、细胞分子以及动物模型不同方面研究：SUMO化增强瘢痕疙瘩中HIF-1/2和TGF-β/Smad通路内相关蛋白活性的作用。.重要结果：.1.在瘢痕疙瘩组织中，SUMOs 蛋白呈高表达，SENPs 蛋白呈低表达，SUMO化正向调控HKFs的生长功能。.2.抑制HKFs中的SUMO化水平，可以抑制HIF-1/2和TGF-β/Smad通路内相关蛋白水平；提高HKFs中的SUMO化水平，可以提高HIF-1/2和TGF-β/Smad通路内相关蛋白水平；说 明SUMO化在瘢痕疙瘩内对HIF-1/2和TGF-β/Smad通路内起正向调控作用。.3.缺氧后HKFs中SUMO蛋白含量水平增加，SENPs含量水平降低。缺氧后HIF-1α的SUMO化程度增高，HIF-1α在瘢痕疙瘩内SUMO化位点为391和477号赖氨酸。.4.SUMO1的水平与TGF-β/Smad通路内Smad4蛋白含量水平及转录活性相关，SUMO1与Smad4在细胞核中共定位，以蛋白质直接结合的形式以增强Smad4蛋白的稳定性。.5.SUMO1与RanGAP1在瘢痕疙瘩中相互作用，且其之间的共价连接与TGF-β/Smad通路内Smad4蛋白核出口相关，RanGAP1-SUMO1的含量后可介导Smad4蛋白与核出口转运蛋白结合增多而导致Smad4核内滞留，对HKFs进行负向调控。.结论：.在瘢痕疙瘩成纤维细胞中，类泛素化通过修饰HIF-1/2和TGF-β/Smad通路内的相关蛋白对瘢痕疙瘩进行正向调控。类泛素化修饰HIF-1/2通路中HIF-1α来竞争性地与泛素降解蛋白结合位点，维持HIF-1α的稳定性进而引起下游胶原的过度合成与沉积。针对TGF-β/Smad通路，在瘢痕疙瘩中类泛素化修饰Smad4蛋白增强其稳定性，与RanGAP1-SUMO1形成复合物调控Smad4蛋白核定位持续激活信号通路。