MBNL1's function in myofibroblast transformation and fibrosis

描述（由申请人提供）：心脏纤维化已迅速成为迄今为止影响心脏病临床管理的最大问题之一，因为目前的治疗只能延迟而不是预防纤维化重塑和心力衰竭。纤维化由主要由肌成纤维细胞协调的不受限制的组织修复反应引起。肌成纤维细胞是高度特化的细胞，其特征在于混合成纤维细胞/平滑肌细胞表型，因为它们可以收缩、迁移和分泌大量的细胞外基质。健康的心脏通常缺乏肌成纤维细胞，但损伤诱导的机械和神经体液环境的改变诱导成纤维细胞转化为肌成纤维细胞。最初，肌成纤维细胞的功能对于修复过程是至关重要的，因为其收缩功能， 基质分泌为受损的心肌提供结构支持;然而，慢性肌成纤维细胞活性最终导致肥大性瘢痕形成和累积性纤维化，这不仅对心脏功能有害，而且产生高度致炎性底物。由于缺乏在体内特异性操纵成纤维细胞的遗传工具，成纤维细胞和肌成纤维细胞对组织修复和纤维化疾病的贡献尚未明确定义。该提案的特点是两个新开发的成纤维细胞特异性Cre基因敲入小鼠模型，用于描绘成纤维细胞在心肌修复和纤维化中的作用，并确定成纤维细胞依赖性纤维化反应的分子调节剂。目前，大多数研究肌成纤维细胞转化的调控网络只集中在TGF？由于其在启动肌成纤维细胞转化和纤维化中的中心作用，其在信号传导中起重要作用，提供了驱动纤维化过程的非常有限范围的调节网络。这促使我们对成纤维细胞向肌成纤维细胞转化的新分子调节因子进行全基因组筛选，从中我们鉴定出RNA结合蛋白肌盲样剪接调节因子1（MBNL 1）的基因。迄今为止，MBNL 1功能从未与组织修复或纤维化相关，但我们在原代成纤维细胞（心脏和MEF）中的初步数据表明，MBNL 1对于诱导成纤维细胞向肌成纤维细胞转化是必要的和足够的，这表明MBNL 1是纤维化疾病的主要介质。因此，该提议旨在（1）直接检查损伤诱导的MBNL 1对转录物丰度和可变剪接的调节变化改变成纤维细胞的蛋白质组以功能性转化为肌成纤维细胞的机制，以及（2）直接检查MBNL 1的程序性成纤维细胞转化在组织修复和纤维化疾病中的作用。通过定义引导成纤维细胞向肌成纤维细胞转化的调控网络，该提议将进一步描绘纤维化疾病的细胞和分子基础，这反过来应该产生用于开发靶向干预和药物发现的新发明点，因为这些分子调节剂中的许多应该适合于药理学补救。

DESCRIPTION (provided by applicant): Cardiac fibrosis has rapidly emerged as one of the biggest problems affecting the clinical management of heart disease to date because current treatments only delay rather than prevent fibrotic remodeling and heart failure. Fibrosis results from an unrestrained tissue repair response orchestrated predominantly by the myofibroblast. Myofibroblasts are highly specialized cells characterized by a hybrid fibroblast/smooth muscle cell phenotype, as they can contract, migrate, and secrete vast amounts of extracellular matrix. The healthy heart is normally devoid of myofibroblasts, but injury-induced alterations of the mechanical and neurohumoral environment induce fibroblasts to transform into myofibroblasts. Initially, myofibroblast function is critical to the repair process as its contractile function and matrix secretion provides structural support to the injured myocardium; however, chronic myofibroblast activity eventually causes hypertrophic scarring and cumulative fibrosis, which is not only deleterious to cardiac function but creates a highly arrhythmogenic substrate. Due to the lack of genetic tools for specifically manipulating the fibroblast in vivo, the contribution ofthe fibroblast and myofibroblast to tissue repair and fibrotic disease has not been clearly defined. This proposal features two newly developed fibroblast-specific Cre knockin mouse models for delineating the fibroblast's role in myocardial repair and fibrosis and to identify the molecular regulators of the fibroblast-dependent fibrotic response. Currently, most studies examining the regulatory networks in myofibroblast transformation have been solely focused on TGF? signaling due to its central role in initiating myofibroblast transformation and fibrosis, providin a very limited scope of the regulatory networks driving the fibrotic process. This prompted us to perform a genome-wide screen for new molecular regulators of fibroblast to myofibroblast conversion from which we identified the gene for the RNA-binding protein muscleblind-like splicing regulator 1 (MBNL1). To date MBNL1 function has never been linked to tissue repair or fibrosis, but our preliminary data in primary fibroblasts (cardiac and MEFs) demonstrates that MBNL1 is both necessary and sufficient for inducing fibroblast to myofibroblast transformation, suggesting that MBNL1 is a primary mediator of fibrotic disease. Thus, this proposal is designed (1) to directly examine the mechanism by which injury induced changes in MBNL1's regulation of transcript abundance and alternative splicing alters the fibroblast's proteome to functionally transform into a myofibroblast and (2) to directly examine the role programmed fibroblast transformation by MBNL1 has in tissue repair and fibrotic disease. By defining the regulatory networks directing fibroblast to myofibroblast transformation this proposal will further delineate the cellular and molecular underpinnings for fibrotic disease which in turn should yield new invention points for developing targeted interventions and drug discovery as many of these molecular regulators should be amenable to pharmacologic remediation.