MBNL1's function in myofibroblast transformation and fibrosis

描述(申请人提供)：心脏纤维化已经迅速成为迄今为止影响心脏病临床治疗的最大问题之一，因为目前的治疗方法只能延缓而不是预防纤维化重塑和心力衰竭。纤维化是由主要由肌成纤维细胞调控的不受约束的组织修复反应引起的。肌成纤维细胞是一种高度特化的细胞，具有成纤维细胞和平滑肌细胞的混合表型，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，但损伤引起的机械和神经体液环境的改变会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是修复过程的关键，因为它的收缩功能和 基质分泌为受损心肌提供结构性支持；然而，慢性肌成纤维细胞活动最终会导致肥厚性瘢痕和累积性纤维化，这不仅对心脏功能有害，而且会造成高度心律失常的底物。由于缺乏在体内特异操作成纤维细胞的基因工具，成纤维细胞和肌成纤维细胞在组织修复和纤维性疾病中的作用尚不清楚。这项建议以两个新开发的成纤维细胞特异性Cre敲击小鼠模型为特色，以描述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的纤维化反应的分子调节因子。目前，对肌成纤维细胞转化调控网络的研究大多集中在转化生长因子？由于其在启动肌成纤维细胞转化和纤维化过程中的核心作用，信号转导在推动纤维化过程的调控网络中提供了非常有限的范围。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控因子进行了全基因组筛选，从中我们确定了RNA结合蛋白肌肉样剪接调节因子1(MBNL1)的基因。到目前为止，MBNL1的功能从未与组织修复或纤维化有关，但我们在原代成纤维细胞(心脏和MEF)中的初步数据表明，MBNL1是诱导成纤维细胞向肌成纤维细胞转化的必要条件和充分条件，表明MBNL1是纤维化疾病的主要介质。因此，本研究的目的是(1)直接研究损伤诱导MBNL1‘S转录丰度调节和选择性剪接改变成纤维细胞蛋白质组功能转化为肌成纤维细胞的机制；(2)直接研究MBNL1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，这项提议将进一步描绘纤维性疾病的细胞和分子基础，这反过来将产生新的发明点，用于开发靶向干预和药物发现，因为许多这些分子调控因子应该服从于药物补救。

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.