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PKCepsilon-Related Proteins in Lung Fibrosis

肺纤维化中的 PKCepsilon 相关蛋白

基本信息

批准号：
7108637
负责人：
STANLEY R HOFFMAN
金额：
$ 35.64万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-01 至 2007-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): This RFA asks investigators to identify novel targets that play a role in fibrogenesis, then to validate that altering the expression or function of targets does indeed inhibit pulmonary fibrosis. We already have identified a set of interconnected novel targets and have partially validated one target. Our central target is the e isoform of protein kinase C (PKC-epsilon). Cells cultured from the fibrotic lung tissue of scleroderma patients have altered PKC-epsilon signaling. The extracellular matrix protein tenascin is overexpressed in scleroderma lung fibroblasts due to altered PKC-epsilon signaling. Altered PKC-epsilon signaling is also revealed by the facts that: 1) Curcumin, from the Indian spice turmeric, inhibits collagen expression in scleroderma lung fibroblasts then causes the cells to undergo apoptosis while normal lung fibroblasts are unaffected by curcumin; and 2) Scleroderma lung fibroblasts can be made curcumin-resistant by PKC-epsilon overexpression while PKC-epsilon depletion makes normal cells sensitive to curcumin. Moreover, three "binding partners" for PKC-epsilon (calponin, caveolin- 1, and RACK2) have altered levels of expression and subcellular localizations in scleroderma lung fibroblasts. The overexpression of tenascin and calponin in vivo has been confirmed in sections of lung tissue from scleroderma patients. Based on these observations, we will test the hypothesis that altering the expression or function of PKC-epsilon, of tenascin, or of binding partners for PKC-epsilon will inhibit pulmonary fibrosis. Experiments will be performed using: 1) Cultured lung fibroblasts from scleroderma patients and matched normal subjects and 2) Mice in which lung fibrosis in induced using bleomycin. Specifically we will: 1) Optimize the delivery of potential treatments via lentivirus into the lungs of mice. 2) Perturb PKC-epsilon expression and function using curcumin, using virus that enhance or inhibit PKC-epsilon expression, and using a peptide that blocks PKC-epsilon translocation and function. These experiments will include a determination of why scleroderma fibroblasts are particularly sensitive to curcumin. 3) Perturb the expression of PKC-epsilon binding partners using virus. 4) Inhibit tenascin expression using virus and test the idea that tenascin is a downstream mediator of the effects observed when PKC-epsilon expression is manipulated. The effects of these perturbations will be read out in terms of collagen expression and curcumin-induced apoptosis in cell cultures and in terms of survival, lung tissue morphology, and collagen levels in bleomycin-treated mice. These approaches will allow us to validate the role in lung fibrosis of the several target proteins that we have already identified.

描述(申请人提供)：这项RFA要求研究人员确定在纤维化形成中发挥作用的新靶点，然后验证改变靶点的表达或功能确实抑制肺纤维化。我们已经确定了一系列相互关联的新目标，并部分验证了一个目标。我们的中心靶点是蛋白激酶C的e亚型(PKC-epsilon)。硬皮病患者纤维化肺组织培养的细胞改变了PKC-epsilon信号。由于PKC-epsilon信号的改变，细胞外基质蛋白tenascin在硬皮病肺成纤维细胞中过表达。从印度香料姜黄素中提取的姜黄素可抑制硬皮病肺成纤维细胞的胶原表达，导致细胞发生凋亡，而正常肺成纤维细胞不受姜黄素的影响；2)PKC-epsilon的过表达可使硬皮病肺成纤维细胞对姜黄素产生抗性，而PKC-epsilon的缺失使正常细胞对姜黄素敏感。此外，PKC-epsilon的三个“结合伙伴”(钙粘蛋白、小窝蛋白-1和RACK2)改变了硬皮病肺成纤维细胞的表达水平和亚细胞定位。在硬皮病患者的肺组织切片中，已经证实在体内过表达Tenascin和Calponin。基于这些观察，我们将检验一种假设，即改变PKC-epsilon、Tenascin或PKC-epsilon的结合伙伴的表达或功能将抑制肺纤维化。实验将使用：1)硬皮病患者和匹配的正常人的肺成纤维细胞培养；2)博莱霉素诱导肺纤维化的小鼠。具体地说，我们将：1)优化通过慢病毒进入小鼠肺部的潜在治疗方法。2)用姜黄素、用能增强或抑制PKC-epsilon表达的病毒、用能阻断PKC-epsilon转位和功能的多肽干扰PKC-epsilon的表达和功能。这些实验将包括确定硬皮病成纤维细胞对姜黄素特别敏感的原因。3)利用病毒干扰PKC-epsilon结合伙伴的表达。4)用病毒抑制Tenascin的表达，并验证Tenascin是PKC-epsilon表达被操纵时观察到的效应的下游介体的想法。这些扰动的影响将通过细胞培养中胶原蛋白的表达和姜黄素诱导的细胞凋亡以及博莱霉素处理的小鼠的存活率、肺组织形态和胶原水平来读出。这些方法将使我们能够验证我们已经确定的几个靶蛋白在肺纤维化中的作用。