The unfolded protein response and TGF-Beta signaling in pulmonary fibrosis

项目摘要/摘要 特发性肺纤维化(IPF)是一种进行性间质纤维化的疾病，可导致严重的 虚弱，最终导致呼吸衰竭和死亡。最近的研究表明内质网 应激(内质网应激)及其引起的未折叠蛋白反应(UPR)在肺组织病理生理学中的作用 遗传性糖尿病患者的纤维化，包括UPR信号的增加和上皮细胞的凋亡 和散发性IPF。尽管有这些观察，但尚不清楚普遍定期审议的激活如何机械地导致 纤维化症。Feroz Papa博士(共同发起人)的实验室已经开发出一种高度特异的小分子KIRA8 IRE1α的抑制剂，它是UPR中保守程度最高的介质。在未发表的作品中，谢泼德和 PAPA实验室(分别是赞助商和共同赞助商)已经表明，KIRA8降低了 博莱霉素对小鼠的纤维化作用。转化生长因子β是组织纤维化的一个公认的驱动因素。缺乏整合素的小鼠 αvβ6，一种关键的胞外激活因子，潜伏的转化生长因子β，表现出UPR信号的减少和ARE 相应地防止纤维化。在MLE12肺上皮细胞系中KIRA8对IRE1α的抑制作用 减少Smad2的磷酸化，这是转化生长因子β信号通路的早期步骤。总而言之，这些数据 建议该提案的中心模型：转化生长因子β和UPR信号共同促进过度 胶原沉积和病理性纤维化。拟议的研究将通过以下方式剖析分子机制 其中UPR促进转化生长因子β信号转导和纤维化。这项研究的第一个目的是寻求确定和 研究博莱霉素组小鼠肺内ER应激反应的相关细胞类型 信使RNA和microRNA的免疫荧光染色及细胞类型特异性纯化 (MiRNA)来自上皮细胞和成纤维细胞。这项研究的第二个目标是探索 转化生长因子β信号转导与细胞系和小鼠的upr。因为已知ir1α可以调节 被认为是调节转化生长因子β信号通路的组成部分，对miRNA的调制可能是一种 UPR促进转化生长因子β信号转导的机制。在细胞系中，转化生长因子β信号将在 普遍定期审议已被化学和遗传技术激活或失活。MiRNAs水平，特别是 MiR-17、miR-200和miR-150将通过定量PCR和对候选miRNA的调节来检测 用双荧光素酶测定法评价靶点。这些结果将通过分析信使核糖核酸和 从上皮细胞和成纤维细胞中纯化的miRNA。拟议的研究将推进基本的 了解肺纤维化的发病机制，为肺纤维化的治疗提供新的靶点 疾病。

Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) is a disease of progressive interstitial fibrosis, which leads to severe debilitation and eventually respiratory failure and death. Recent studies have implicated endoplasmic reticulum stress (ER stress) and the resulting unfolded protein response (UPR) in the pathophysiology of pulmonary fibrosis, including findings of increased UPR signaling and epithelial cell apoptosis in patients with hereditary and sporadic IPF. Despite these observations, it remains unclear how UPR activation leads mechanistically to fibrosis. The laboratory of Dr. Feroz Papa (co-sponsor) has developed KIRA8, a highly specific small molecule inhibitor of IRE1α, the most deeply-conserved mediator of the UPR. In unpublished work, the Sheppard and Papa laboratories (sponsor and co-sponsor, respectively) have shown that KIRA8 decreases markers of fibrosis in mice exposed to bleomycin. TGFβ is a well-established driver of tissue fibrosis. Mice lacking integrin αvβ6, a critical activator of extracellular, latent TGFβ, exhibit decreased UPR signaling and are correspondingly protected from fibrosis. In the MLE12 lung epithelial cell line, KIRA8 inhibition of IRE1α decreases SMAD2 phosphorylation, an early step in the TGFβ signaling pathway. Together, these data suggest the central model of the proposal: that TGFβ and UPR signaling conspire to promote excessive collagen deposition and pathological fibrosis. The proposed research will dissect the molecular mechanisms by which the UPR enhances TGFβ signaling and fibrosis. The first aim of the study seeks to identify and characterize the relevant cell types undergoing ER stress in the lungs of mice exposed to bleomycin, using immunofluorescence staining and cell-type specific purification of messenger RNA (mRNA) and microRNA (miRNA) from epithelial cells and fibroblasts. The second aim of the study will probe interactions between TGFβ signaling and the UPR in cell lines and mice. Because IRE1α is known to modulate miRNAs that are thought to regulate components of the TGFβ signaling pathway, modulation of miRNAs is likely to be a mechanism by which the UPR enhances TGFβ signaling. In cell lines, TGFβ signaling will be evaluated after the UPR has been activated or inactivated by chemical and genetic techniques. Levels of miRNAs, particularly miR-17, miR-200, and miR-150, will be measured by quantitative PCR, and regulation of candidate miRNA targets evaluated by dual luciferase assay. These results will be extended in vivo by analyzing mRNA and miRNA purified from epithelial cells and fibroblasts. The proposed studies will advance the fundamental understanding of the mechanisms of pulmonary fibrosis and offer novel targets for therapy of this devastating disease.