瘢痕疙瘩中DAB-1抑制E3连接酶SIAH1对TIEG1泛素化介导TGF-β/Smads信号通路的研究

The sustained activation of TGF-β1/Smads signal transduction pathway is the most important mechanism for the formation of keloids. Our study found that the expression of Smad7, which is the most important negative feedback signaling molecule was significantly down regulated by the transcription inhibitive factor TIEG1, which is up-regulated in keloid. However, the mechanism of TIEG1 up-regulated is not clear. Our study found that: Only the TIEG1 protein is up-regulated, the TIEG1 mRNA level stay in the same; We screened a new E3 ubiquitin ligase SIAH1 mediating the ubiquitination degration of TIEG1; Using Yeast-two hybrid technique and generating SIHA-1 plasmid as bait protein, we screened a interacted protein DAB-1 in the human fetal liver cDNA library; DAB-1 is up regulated in keloid. TIEG1 is up regulated by silencing DAB-1 gene via siRNA. As a result, we supposed that DAB-1 affects the activity of SIAH-1 through protein-protein interaction in keloid, suppressing the ubiquitination of TIEG1, resulting in the up-regulation of TIEG1.and the sustained activation of TGF-β1/Smads signal transduction pathway. In this study, we will take some molecular biology technique, such as RT-PCR, western blotting, immunofluorescence, GST pull-down to acquire evidence that DAB-1 inhibits the activity of ubiquitination of SIAH-1 through protein-protein interaction to suppress the degradation of TIEG1, and to observe the biological behaviour of keloid under DAB-1 control in vivo and vitro. So we could provide a new target for preventing and curing keloids, moreover, provide a new clue and theory.

TGF-β1/Smads与瘢痕疙瘩形成密切相关。我们既往工作证实高水平的TIEG直接抑制Smad7的表达，介导了TGF-β1/Smads通路的激活。然而，TIEG1上调的机制不清楚。前期工作观察到仅TIEG1蛋白水平上调，而mRNA无变化；进一步证实泛素降解系统异常导致TIEG1上调，并筛选到介导TIEG1泛素化的E3连接酶SIAH1；运用酵母双杂交技术筛查到一个SIAH1相互作用蛋白DAB-1；DAB-1在瘢痕疙瘩中高表达，过表达DAB-1可拮抗TIEG1与SIAH1相互作用。因此，我们推测：瘢痕疙瘩中DAB-1通过蛋白相互作用拮抗了SIAH-1与TIEG1结合，导致TIEG1泛素降解异常而蛋白水平上调，从而介导TGF-β1/Smads通路。本课题将采用缺失突变、FRET及免疫共沉淀等技术，探讨DAB-1通过抑制SIAH1对TIEG1泛素化，介导TGF-β/Smads通路激活的具体机制。

瘢痕疙瘩的发病机制目前仍不清楚。TGF-β1/Smads信号通路的持续激活是瘢痕疙瘩形成的重要机制。课题组在之前的国家自然科学基金面上项目的资助下，既往工作证实，瘢痕疙瘩中高水平的转录抑制因子TIEG直接抑制Smad7的表达，从而介导TGF-β1/Smads信号通路的激活。进一步研究发现TIEG1 通过结合Smad7 启动子核心区域-1392 /-1382 bp 中的Sp1 位点，直接调控Smad7 的转录，抑制Smad7 的表达，从而介导TGF-β1/Smads 信号通路的激活和瘢痕疙瘩的形成。该研究结果发表在J Invest Dermatol,2017;137(5):1051-1059。.本课题按计划进行，首先体外培养的正常皮肤和瘢痕疙瘩成纤维细胞，用溶酶体抑制剂和蛋白酶体抑制剂MG132分别处理细胞，明确TIEG1蛋白降解是由泛素系统决定的。其次，通过siRNA技术以及腺病毒基因转染技术，调控DAB-1基因表达，在瘢痕疙瘩成纤维细胞及动物模型中证实DAB1对TGF-β1/Smads信号通路以及胶原表达的影响。基本上验证了课题组的假设，DAB-1 介导SIAH1 对TIEG1 泛素化调控，从而介导TGF-β/Smads 信号通路激活的新机制，为确立DAB-1 作为瘢痕疙瘩治疗的新靶点提供更充分的科学依据。.在本课题进行的同时，课题组发现S1PR5 特异性调节剂FTY720能够通过调控Akt/mTOR 信号通路抑制增生性瘢痕发生，相关成果发表在J Invest Dermatol.2017;137(7):1552-1561。在临床上，病理性瘢痕是创面异常愈合的产物，如果改善创面愈合微环境，有助于减少瘢痕形成。课题组应用自体表皮基底细胞治疗供皮区创面，可以促进创面愈合，减少瘢痕增生，相关成果发表在Brit J Surg. 2017;104:836-842。另外，应用脱细胞异体真皮可以改善创面愈合质量，减少瘢痕增生，相关成果发表在J Am Coll Surg.2016;222(6):1171-9。