Targeting myofibroblast activation in chronic fibrotic disease

愈合伤口中肌成纤维细胞的慢性积累与过度收缩、间质胶原的过度沉积和破坏性的组织重塑有关。血管平滑肌α -肌动蛋白（-SMA）是一种由分化的肌成纤维细胞短暂表达的收缩蛋白，用于产生闭合开放性伤口所需的张力。在慢性纤维化疾病中，肌成纤维细胞分化是功能失调的，我们发现这些细胞中激活(-SMA和I型胶原基因所需的分子信号也提供负反馈，可能在伤口愈合和破坏性重塑过程中限制过度活跃的肌成纤维细胞的募集。本提案中概述的研究有望揭示DNA和mrna结合蛋白YB-1、Pur（和Pur）与（- sma）和I型胶原促进因子之间功能相互作用的新形式，并阐明这些蛋白如何受到TGF(1)和凝血酶等促纤维化因子的影响，如果不加以控制，这些因子可能导致肌成纤维细胞进展为肥厚性瘢痕。实验旨在启动、放大或减弱肌成纤维细胞分化，以更好地了解在转录和翻译水平上控制-SMA和I型胶原基因输出的策略，并揭示可能有助于减少异常伤口愈合结果的新干预策略。目的1将检查TGF(1)调控的YB-1和Pur蛋白阻遏物与-SMA和胶原启动子DNA以及转录激活因子Sp1、SRF和Smads 2、3的相互作用，描绘这种功能相互作用所需的阻遏物多肽链区域，并尝试使用肽诱饵和小分子药物抑制剂破坏复合物的形成并使病理肌成纤维细胞分化失效。目的2将确定凝血酶是在翻译控制水平上增强肌成纤维细胞分化，从而作为TGF(1)的补充，还是通过诱导抗纤维化转录调节蛋白Egr-1阻断转录和肌成纤维细胞募集来拮抗该生长因子。目的3研究将探索另一种不依赖smad的肌成纤维细胞分化和纤维化机制。基于药理抑制TGF(1/Smad激酶-或磷脂酰肌醇-3激酶(PI3K)/Akt激酶信号传导的功能丧失方法将被用于评估它们对体外肌成纤维细胞活化和缺血/再灌注损伤小鼠心脏纤维化的可能抑制作用。TGF(1)和凝血酶利用YB-1和Pur蛋白独特的DNA、RNA和蛋白质结合特性的能力，为肌成纤维细胞分化过程中基因表达的控制提供了一个新的动态视角，可能为慢性纤维化疾病的治疗管理提供最佳策略。

Chronic accumulation of myofibroblasts in healing wounds is associated with hypercontractility, excessive deposition of interstitial collagens, and destructive tissue remodeling. Vascular smooth muscle alpha-actin ((-SMA) is a contractile protein transiently expressed by differentiated myofibroblasts for generating tensile force required to close open wounds. In chronic fibrotic disease, myofibroblast differentiation is dysfunctional and we discovered that molecular signaling required for activation of both the (-SMA and type I collagen genes in these cells also provides negative feedback that could potentially limit the recruitment of hyperactive myofibroblasts during wound healing and destructive remodeling. Studies outlined in this proposal are expected to reveal novel forms of functional interplay of the DNA- and mRNA-binding proteins YB-1, Pur (, and Pur ( with the (-SMA and type I collagen promoters and clarify how these proteins are affected by pro- fibrotic agents such as TGF(1 and thrombin that, if unchecked, may cause myofibroblast progression to hypertrophic scarring. Experiments are designed to initiate, amplify, or attenuate myofibroblast differentiation to better understand strategies for controlling (-SMA and type I collagen gene output at the transcriptional and translation levels as well as reveal novel interventional strategies that might be useful for minimizing aberrant wound healing outcomes. Aim 1 will examine TGF(1-regulated interaction of YB-1 and Pur protein repressors with (-SMA and collagen promoter DNA and the transcriptional activators Sp1, SRF, and Smads 2,3, delineate regions of repressor polypeptide chains required for this functional interplay, and attempt to disrupt complex formation and disable pathobiologic myofibroblast differentiation using peptide decoys and small molecule pharmacologic inhibitors. Aim 2 will determine if thrombin potentiates myofibroblast differentiation at the level of translational control thus functioning as a TGF(1 supplement or instead antagonizes this growth factor by blocking transcription and myofibroblast recruitment by inducing the anti-fibrotic transcriptional regulatory protein, Egr-1. Aim 3 studies will explore alternative, Smad-independent mechanisms of myofibroblast differentiation and fibrosis. Loss-of-function approaches based on pharmacologic inhibition of TGF(1/Smad kinase- or phosphatidylinositol-3-kinase (PI3K)/Akt kinase signaling will be used to evaluate their possible suppressive effect on myofibroblast activation in vitro and cardiac fibrosis in mice after ischemia/reperfusion injury. The ability of TGF(1 and thrombin to exploit the unique DNA-, RNA-, and protein-binding properties of YB-1 and Pur proteins adds a new dynamic perspective to control of gene expression during myofibroblast differentiation that may reveal optimum strategies for therapeutic management of chronic fibrotic diseases.