PKCepsilon-Related Proteins in Lung Fibrosis

肺纤维化中的 PKCepsilon 相关蛋白

基本信息

批准号：
6663559
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
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（由申请人提供）：该RFA要求研究人员确定在纤维发生中起作用的新目标，然后验证改变靶标的表达或功能的确确实会抑制肺纤维化。我们已经确定了一组相互联系的新目标，并已部分验证了一个目标。我们的中心靶标是蛋白激酶C（PKC-EPSILON）的E同工型。从硬皮病患者的纤维化肺组织培养的细胞已改变PKC-EPSILON信号传导。由于PKC- EPSILON信号转导改变，细胞外基质蛋白替氏蛋白在硬皮病肺成纤维细胞中过表达。 PKC- EPSILON信号的改变也表明：1）姜黄素，来自印度香料姜黄的姜黄素抑制硬皮病性肺肺成纤维细胞中的胶原蛋白表达，然后使细胞导致细胞凋亡，而正常的肺成核母细胞不受姜黄素的影响； 2）硬皮病性肺成纤维细胞可以通过PKC-EPSILON过表达使姜黄素抗性，而PKC-EPSILON耗竭使正常细胞对姜黄素敏感。此外，PKC-EPSILON（Calponin，Caveolin-1和Rack2）的三个“结合伴侣”改变了硬化症肺成纤维细胞中表达水平和亚细胞局部定位。在硬皮病患者的肺组织中，已经证实了替氏菌和钙蛋白的过表达。基于这些观察结果，我们将检验以下假设：PKC-EPSILON的PKC-EPSILON，TENASCIL或结合伴侣的表达或功能将抑制肺纤维化。将使用以下方式进行实验：1）来自硬皮病患者的培养的肺成纤维细胞并与正常受试者相匹配，以及2）小鼠，其中肺纤维化在使用博来霉素诱导的小鼠中。具体而言，我们将：1）优化通过慢病毒到小鼠肺部的潜在治疗方法。 2）使用姜黄素加强或抑制PKC-EPSILON表达的病毒，并使用阻断PKC- EPSILON易位和功能的肽。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）扰动使用病毒的PKC-EPSILON结合伴侣的表达。 4）使用病毒抑制Tenascin的表达，并测试Tenascin是操纵PKC- EPSILON表达时观察到的效果的下游介体的想法。这些扰动的作用将以细胞培养物中的胶原蛋白表达和姜黄素诱导的凋亡以及生存，肺组织形态和博来霉素治疗的小鼠的胶原蛋白水平的读数进行读取。这些方法将使我们能够验证我们已经确定的几种靶蛋白的肺纤维化中的作用。