Role of Heat Shock Protein 70 in Lung Fibrosis

描述（由申请人提供）：IPF是一种慢性进行性疾病，以广泛的肺纤维化为特征。IPF的发病机制尚不完全清楚，但包括TGFb-1激活成纤维细胞和胶原基质沉积，从而产生实质瘢痕。另外，HSP-70在真核细胞中被诱导以应对各种应激，并通过抗炎和抗凋亡作用提供细胞保护。我们实验室最近的实验已经确定了热休克蛋白70作为成纤维细胞活性的潜在调节剂。初步结果表明，当TGFb-1刺激时，MRC人胎儿肺成纤维细胞以时间依赖的方式诱导表达HSP-70。此外，在缺乏HSF-1 （HSP-70的转录因子）的敲除小鼠株中，当TGFb-1被给予肺成纤维细胞时，α -平滑肌肌动蛋白的体外表达增加，这是成纤维细胞激活的标志。我们将使用HSF-1 KO小鼠和野生型同窝对照，进一步描述HSF-1诱导和HSP-70表达响应TGFb-1刺激的机制。使用Western-和Northern-blot，我们将分析多种次生信号通路，如MAPK、JNK、CAMKII和ROS的产生。我们假设这些因子参与了tgfb -1诱导的HSF-1的激活，其与热休克启动子的结合，以及随后的HSP-70的上调。我们将通过评价过表达HSP-70的作用来探讨HSP-70的细胞保护机制。这将通过将HSP-70基因载体转化到HSF-1 KO小鼠的肺中，并观察HSP-70上调对博莱霉素诱导的肺损伤的影响。然后使用免疫组织化学和共聚焦激光显微镜来评估肺组织学的变化和成纤维细胞激活标记物的表达。

DESCRIPTION (provided by applicant): IPF is a chronic, progressive disease characterized by extensive lung fibrosis. The pathogenesis of IPF is incompletely described, but includes activation of fibroblasts by TGFb-1, and deposition of collagen-matrix which creates parenchymal scarring. HSP-70, alternatively, is induced in eukaryotic cells in response to a variety of stresses and provides cytoprotection via anti-inflammatory and antiapoptotic effects. Recent experiments in our lab have identified HSP-70 as a potential regulator of fibroblast activity. Preliminary results show that, when stimulated with TGFb-1, MRC human fetal lung fibroblasts show inducible expression of HSP-70 in a time-dependent fashion. Also, there is increased in vitro expression of alpha-Smooth Muscle Actin, a marker of fibroblast activation, when TGFb-1 is given to lung fibroblasts in knock-out strains of mice lacking HSF-1, the transcription factor for HSP-70. Using HSF-1 KO mice and wild-type littermate controls, we will further describe the mechanisms of HSF-1 induction and HSP-70 expression in response to TGFb-1 stimulation. Using Western- and Northern-blot, we will analyze a variety of secondary signaling pathways, such as MAPK, JNK, CAMKII, and the generation of ROS. We hypothesize these factors to be involved in TGFb-1-induced activation of HSF-1, its binding to the heat shock promoter, and subsequent upregulation of HSP-70. We will investigate mechanisms of HSP-70 cytoprotection, through evaluating the effects of over-expressing HSP-70. This will be done via gene-vector transformation of HSP-70 into the lungs of HSF-1 KO mice, and observing the effects of HSP-70 upregulation on bleomycin-induced lung injury. Immunohistochemistry and confocal laser microscopy will then be used to evaluate changes in lung histology and the expression of fibroblastic markers of activation.