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

描述（由申请方提供）：特发性肺纤维化/普通型间质性肺炎（IPF/UIP）是一种病因不明的破坏性间质性肺病，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。最近已经假定肺泡上皮在IPF/UIP发病机制中的中心作用。在这种模式中，受损的肺泡上皮细胞（AEC）通过释放促进成纤维细胞的增殖和分化的纤维增殖介质来积极参与纤维发生，所述成纤维细胞的增殖和分化继而诱导AEC凋亡和/或通过上皮-间充质转化（EMT）直接产生成纤维细胞。然而，AEC损伤/活化的潜在机制和将上皮异常与纤维化联系起来的下游分子途径仍不清楚。内质网（ER）应激与表面活性蛋白（SP）突变相关的散发性和家族性肺纤维化之间的关联已有报道。我们最近发现，ER应激触发保护性信号，称为未折叠蛋白反应（UPR），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ER应激在肺纤维化中功能失调的AEC表型中的因果作用。初步数据表明，活性氧（ROS）和β-连环蛋白激活/与共激活剂CBP的相互作用参与Src依赖性信号传导。ER伴侣葡萄糖调节蛋白78（GRP 78）是ER稳态的主要调节因子，因此代表了调节肺泡上皮ER应激反应的理想靶点。本提案的总体目标是通过利用具有肺上皮细胞特异性缺失的Grp 78的小鼠调节GRP 78来研究与纤维化发病机制有关的ER应激诱导的上皮异常的潜在机制。我们假设：（1）Grp 78的丧失诱导和/或加剧体内实验性纤维化;（2）GRP 78免疫诱导的上皮损伤通过对上皮的直接作用（例如，（3）GRP 78不足对EMT/纤维化的影响是通过Src和/或ROS依赖性β-连环蛋白/CBP信号传导介导的。我们将使用AEC特异性Grp 78敲除小鼠，结合临床相关的突变体SP-C <$外显子4，我们完善的原代AEC体外单层模型，和ICG-001，一种新型的<$-连环蛋白/CBP相互作用抑制剂，以解决以下具体目的：1.评价由Grp 78缺失介导的ER应激对诱导型AEC特异性Grp 78敲除小鼠中肺纤维化的影响; 2.在诱导型AEC特异性Grp 78敲除小鼠中研究ER应激在涉及纤维化的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ER应激诱导AEC损伤/纤维化的潜在机制。这些研究将深入了解IPF发病机制的潜在机制，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.