PKCepsilon-Related Proteins in Lung Fibrosis

肺纤维化中的 PKCepsilon 相关蛋白

• 批准号: 6922076
• 负责人: STANLEY R HOFFMAN
• 金额: $ 36.5万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/6922076
• 关键词: Lentivirus; apoptosis; calponin; caveolins; cell surface receptors; clinical research; collagen; enzyme activity; enzyme inhibitors; enzyme mechanism; fibroblasts; fibrosis; gene expression; human tissue; isozymes; laboratory mouse; lung disorder; morphology; postmortem; protein binding; protein kinase C; protein structure function; protein transport; scleroderma; tenascin; tissue /cell culture; transfection /expression vector

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（由申请人提供）：该RFA要求研究人员识别在纤维化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。我们已经确定了一组相互关联的新目标，并部分验证了一个目标。我们的中心目标是蛋白激酶C（PKC-C）的e亚型。从硬皮病患者的纤维化肺组织培养的细胞具有改变的PKC β信号传导。细胞外基质蛋白生腱蛋白在硬皮病肺成纤维细胞中过表达，这是由于改变了PKC信号传导。改变的PKC-β信号传导也通过以下事实揭示：1）来自印度香料姜黄的姜黄素抑制硬皮病肺成纤维细胞中的胶原蛋白表达，然后导致细胞经历凋亡，而正常肺成纤维细胞不受姜黄素影响;和2）硬皮病肺成纤维细胞可以通过PKC-β过表达而产生姜黄素抗性，而PKC-β耗竭使正常细胞对姜黄素敏感。此外，PKC-β的三个“结合伴侣”（钙调蛋白、小窝蛋白-1和RACK 2）在硬皮病肺成纤维细胞中的表达水平和亚细胞定位发生了改变。在硬皮病患者的肺组织切片中已经证实了腱生蛋白和钙调蛋白在体内的过表达。基于这些观察结果，我们将检验改变PKC-β、腱生蛋白或PKC-β结合伴侣的表达或功能将抑制肺纤维化的假设。实验将使用：1）来自硬皮病患者和匹配的正常受试者的培养的肺成纤维细胞和2）使用博来霉素诱导肺纤维化的小鼠进行。具体来说，我们将：1）优化通过慢病毒将潜在治疗药物输送到小鼠肺部的过程。2)使用姜黄素、使用增强或抑制PKC β表达的病毒以及使用阻断PKC β易位和功能的肽干扰PKC β表达和功能。这些实验将包括确定为什么硬皮病成纤维细胞对姜黄素特别敏感。3)使用病毒干扰PKC β结合配偶体的表达。4)使用病毒抑制腱生蛋白表达，并测试腱生蛋白是操纵PKC β表达时观察到的效应的下游介导物的想法。这些扰动的影响将在细胞培养物中的胶原蛋白表达和姜黄素诱导的细胞凋亡方面以及博来霉素处理的小鼠中的存活率、肺组织形态和胶原蛋白水平方面读出。这些方法将使我们能够验证我们已经确定的几种靶蛋白在肺纤维化中的作用。