Epithelial abnormalities in IPF: role of ER stress and GRP78/BiP

描述（由申请人提供）：特发性肺纤维化/通常间质性肺炎（IPF/UIP）是一种病因不明的破坏性间质性肺疾病，特征为（肌）成纤维细胞活化/增殖、细胞外基质（ECM）沉积和进行性肺破坏。肺泡上皮在IPF/UIP发病机制中的核心作用最近被假设。在这种模式下，受损的肺泡上皮细胞（AEC）通过释放促进成纤维细胞增殖和分化的纤维增殖介质积极参与纤维形成，这些介质反过来诱导AEC凋亡和/或通过上皮-间质转化（EMT）直接产生成纤维细胞。然而，AEC损伤/激活的机制以及连接上皮异常与纤维化的下游分子途径仍不清楚。内质网（ER）应激与散发性和家族性肺纤维化（与表面活性蛋白（SP）突变相关）之间的关系已被报道。我们最近发现内质网应激触发被称为未折叠蛋白反应（UPR）的保护性信号，并通过激活非受体膜相关酪氨酸激酶Src在体外诱导AEC细胞凋亡和EMT，支持内质网应激在肺纤维化功能失调AEC表型中的因果作用。初步数据表明，活性氧（ROS）和b-连环蛋白的激活/与共激活剂CBP的相互作用参与了src依赖性信号传导。内质网伴侣葡萄糖调节蛋白78 （GRP78）是内质网稳态的主要调节因子，因此是调节肺泡上皮内质网应激反应的理想靶点。本研究的总体目标是利用肺上皮细胞特异性缺失GRP78的小鼠，通过调节GRP78，研究内质网应激诱导的与纤维化发病机制相关的上皮异常的机制。我们假设(1)Grp78的缺失诱导和/或加剧了体内的实验性纤维化；(2) GRP78不足诱导的上皮损伤通过对上皮的直接影响（如凋亡/EMT）和/或异常上皮-成纤维细胞串扰导致纤维化；(3) GRP78不足对EMT/纤维化的影响是通过Src和/或ros依赖性的¿-catenin/CBP信号介导的。我们将使用AEC特异性Grp78敲除小鼠，结合临床相关突变体SP-C¿exon4，我们已经建立的初级AEC体外单层模型，以及ICG-001，一种新型的-catenin/CBP相互作用抑制剂，来解决以下具体目标：评估Grp78缺失介导的内质网应激对诱导aec特异性Grp78敲除小鼠肺纤维化的影响2. 探讨内质网应激在诱导AEC特异性Grp78敲除小鼠中与纤维化相关的AEC异常（凋亡vs EMT vs异常上皮-成纤维细胞串扰）中的作用；和3。确定内质网应激诱导AEC损伤/纤维化的机制。这些研究将为IPF的发病机制提供深入的见解，并有助于确定这种不治之症的新治疗靶点。

DESCRIPTION (provided by applicant): Idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP) is a devastating interstitial lung disease of unknown etiology, characterized by (myo)fibroblast activation/proliferation, extracellular matrix (ECM) deposition and progressive lung destruction. A central role for alveolar epithelium in IPF/UIP pathogenesis has recently been postulated. In this paradigm, injured alveolar epithelial cells (AEC) actively participate in fibrogenesis by releasing fibroproliferative mediators that promote proliferation an differentiation of fibroblasts that in turn induce AEC apoptosis and/or giving rise directly to fibroblasts through epithelial-mesenchymal transition (EMT). However, mechanisms underlying AEC injury/activation and downstream molecular pathways linking epithelial abnormalities to fibrosis remain unclear. Association between endoplasmic reticulum (ER) stress and both sporadic and familial pulmonary fibrosis associated with surfactant protein (SP) mutations has been reported. We recently found that ER stress triggers protective signaling known as the unfolded protein response (UPR) and induces both apoptosis and EMT in AEC in vitro via activation of the non-receptor membrane-associated tyrosine kinase Src, supporting a causal role for ER stress in the dysfunctional AEC phenotype in pulmonary fibrosis. Preliminary data suggest that reactive oxygen species (ROS) and b-catenin activation/interaction with the co-activator CBP are involved in Src-dependent signaling. The ER chaperone glucose-regulated protein 78 (GRP78) is a master regulator of ER homeostasis and thus represents an ideal target with which to modulate ER stress responses in alveolar epithelium. The overall goals of this proposal are to investigate mechanisms underlying ER stress-induced epithelial abnormalities implicated in the pathogenesis of fibrosis by modulating GRP78 utilizing mice with lung epithelial cell-specific deletion of Grp78. We hypothesize that (1) loss of Grp78 induces and/or exacerbates experimental fibrosis in vivo; (2) GRP78 insufficiency-induced epithelial injury contributes to fibrosis via direct effects on the epithelium (e.g., apoptosis/EMT) and/or abnormal epithelial-fibroblast crosstalk; and, (3) effects of GRP78 insufficiency on EMT/fibrosis are mediated via Src and/or ROS-dependent ¿-catenin/CBP signaling. We will use AEC-specific Grp78 knockout mice, in conjunction with clinically relevant mutant SP-C¿exon4, our well- established primary AEC in vitro monolayer model, and ICG-001, a novel inhibitor of ¿-catenin/CBP interaction, to address the following Specific Aims: 1. Evaluate effects of ER stress mediated by loss of Grp78 on pulmonary fibrosis in inducible AEC-specific Grp78 knockout mice; 2. Investigate the role of ER stress in AEC abnormalities (apoptosis vs EMT vs aberrant epithelial-fibroblast crosstalk) implicated in fibrosis in inducible AEC-specific Grp78 knockout mice; and 3. Determine mechanisms underlying ER stress-induced AEC injury/fibrosis. These studies will provide insights into mechanisms underlying the pathogenesis of IPF and facilitate identification of novel therapeutic targets for this incurable disease.