ITGB4BP调控TGF-β1翻译的分子机制及其在增生性瘢痕形成中作用的研究

hypertrophic scar is the result of wound healing in skin tissues response to injury. However the mechanisms and the regulations that dictate the balance of cell regeneration and fibrogenesis are not well understood. The fibroblast is considered as the key cell in the fibrosis process which could secrete transforming growth factor-β1 (TGF-β1). TGF-β1 has been demonstrated by many studies to play an essential role in induction and maintenance of the hypertrophic scar. Here we previously report that ITGB4BP was a negative regulator in hypertrophic scar formation, downregulating the expression of TGF-β1 and its downstream signals, inhibiting myofibroblast functions and the formation of fibrosis. Meanwhile ITGB4BP could inhibit the formation of heavy polysome. However ITGB4BP did not extent the wound healing time. Accoding to the researches, we found that ITGB4BP was an anti-association factor able to prevent the formation of 80s and binds nucleolar pre-60s. ITGB4BP phosphorylation on Ser-235 site promotes its release from 60s and regulates the translation. Therefore we get the hypothesis that the dephosphorylation of ITGB4BP on Ser-235 site associating with 60s inhibtes the formation of polysomes binding to TGF-β1 mRNA, which can reduce the rate of translation, resulting in the fibrosis suppression. On this background we firstly detect the effect of ITGB4BP on TGF-β1 mRNA binding polysomes via polysomal profile by up/down-regultating the expression of ITGB4BP. Then we test the roles of phosphorylate/dephosphorylate ITGB4BP on the polysomes. We further mutate the Ser-235 into Ala-235 to detect the effects on the polysomes. Finally we detect the effect of ITGB4BP on the fibrosis-related molecules according to the in vivo and in vitro models. According to our researches we might find the new function of ITGB4BP in hypertrophic scar, and the potential target to control the hypertrophic scar.

增生性瘢痕的形成及其调控机制研究一直是烧创伤基础领域的研究焦点之一，我们课题组前期研究发现ITGB4BP是增生性瘢痕形成的相关负调基因，在增生性瘢痕中呈低表达，显著抑制TGF-β1及其下游信号分子表达、抑制成纤维细胞功能和纤维形成，抑制多聚核糖体形成，但不影响创面愈合。ITGB4BP主要通过其235位的非磷酸化丝氨酸与核糖体60s亚基结合而抑制核糖体80s亚基形成，在翻译水平选择性调控蛋白合成。因此结合文献和课题组前期研究我们提出“非磷酸化ITGB4BP-235Ser与核糖体60s结合，抑制TGF-β1mRNA上多聚核糖体的分布，抑制TGF-β1 mRNA翻译效率，从而抑制TGF-β1蛋白合成，达到抑制增生性瘢痕形成的作用”设想。本研究拟通过多聚核糖体分析、磷酸化/去磷酸化、点突变和RNA干扰等方法研究ITGB4BP对TGF-β1翻译调控的影响，了解ITGB4BP在瘢痕形成中的作用及机制。

增生性瘢痕的形成及其调控机制研究一直是烧创伤基础领域的研究焦点之一。在前期研究中我们发现ITGB4BP（也称eIF6）在增生性瘢痕中呈低表达，参与抑制增生性瘢痕的形成。经过本项目的研究，我们首先通过多聚核糖体分析发现ITGB4BP抑制TGF-β1的mRNA稳定性。通过基因强制高表达/低表达发现ITGB4BP高表达后，TGF-β1及其下游分子Smad2、Smad3、Smad4以及α-SMA和I型胶原的表达（包括RNA水平和蛋白水平）均下降；当ITGB4BP低表达后，TGF-β1及其下游分子Smad2、Smad3、Smad4以及α-SMA和I型胶原的表达（包括RNA水平和蛋白水平）均上升。当ITGB4BP的235位点的丝氨酸突变为丙氨酸后，其对上述分子的抑制作用消失。并且当加入TPA磷酸化ITGB4BP的235位点后，其对上述分子的抑制作用仍然消失。因此证明ITGB4BP对TGF-β1的作用为其235位非磷酸化的丝氨酸。FACS检测ITGB4BP上调后细胞周期的S期比例增加，下调后S期比例下降。通过划痕实验发现ITGB4BP对细胞迁移无统计学差异，但磷酸化ITGB4BP后，细胞迁移速度加快，当ITGB4BP的235位点的丝氨酸突变为丙氨酸后，其促进细胞迁移的作用消失。在FPCL模型中，ITGB4BP高表达后抑制胶原的收缩，ITGB4BP敲降后，胶原收缩加强。在动物皮肤创面愈合模型中，ITGB4BP抑制创面创面纤维化，且促进创面愈合。通过本项研究，我们证明ITGB4BP通过其235位点的非磷酸化丝氨酸参与抑制TGF-β1 mRNA的稳定性，抑制纤维化相关分子，从而达到抑制增生性瘢痕的目的，为后期纤维化疾病的防治提供新的研究方向和作用靶点。