HEART FIBROBLAST AND COLLAGEN TYPE III REGULATION

心肌病理性肥大的发展已被 常与细胞外基质异常积聚有关 间质中的蛋白质(ECM)(主要是胶原蛋白)。不正常的人 纤维性胶原蛋白堆积导致脑室改变 僵硬，并最终可促进心肌的发展 失败了。心脏胶原蛋白由I型和III型组成， IV和V类的程度较小。已经证明，在 心脏压力超负荷性肥厚的早期阶段 III型到I型胶原增加。从体外获得的结果 对心脏成纤维细胞进行的实验也表明，III 胶原蛋白基因的表达(与I型相比)对 用生长因子或其他方法处理细胞所带来的挑战 机械刺激。然而，其分子机制 在心脏成纤维细胞中调节的是III型胶原 未知。这项提案的总体目标是审查该法规 III型胶原基因的影响因素被认为是一个 在指导细胞外基质蛋白生产中的重要作用 心肌。我们建议解决以下问题：1)是否 转化生长因子β-1、血管紧张素Ⅱ(Ang II)与机械 变形导致III型胶原产生增加和 小鼠心脏成纤维细胞培养中的沉积？如果是这样，通过什么 机械装置？为此，我们将确定对小鼠的刺激 心脏成纤维细胞在这些因子作用下的表达增加 用Northern印迹和免疫印迹技术检测III型胶原基因和蛋白的表达 酶联免疫吸附试验技术。为了确定分子的性质 核内ECM蛋白产量增加的机制 径流、信使核糖核酸稳定性和转录(CAT)检测 2)III型胶原蛋白表达增加 依赖于血管紧张素II受体、血管紧张素Ⅱ、 TGFbeta-1和/或Stretch？(A)使用约束性分析，我们将探讨是否 上述化合物对小鼠心脏成纤维细胞的刺激作用 手法调节血管紧张素II的表达(数量) 感受器。(B)将使用生化技术检查ANG是否 II和/或牵张增加TGFbeta-1基因表达水平和 放手。(C)使用目标1中描述的技术，我们将检查 如果同时机械刺激和生长因子刺激心脏 成纤维细胞导致III型胶原水平升高，并且；3) 这些因子对III型胶原基因表达的调节依赖于 的启动子区域内含有特定的顺式作用元件 基因呢？利用基于CAT-III型胶原蛋白的转基因技术 我们想要的小鼠启动子的构建(和选择性缺失) 确定哪些特定的顺式作用元素包含在 可能通过这些基因中的每一个来介导转录激活 不同的因子(TGFbeta-1、Ang II和Stretch)。

The development of pathological hypertrophy in the myocardium has been frequently associated with abnormal accumulation of extracellular matrix proteins (ECM) (mostly collagens) in the interstitium. The abnormal accumulation of fibrillar collagens results in an altered ventricular stiffness and can ultimately contribute to the development of myocardial failure. Cardiac collagens are comprised of types I and III and to a lesser extent of types IV and V. It has been demonstrated that during the early phases of cardiac pressure overload hypertrophy the ratio of type III to type I collagen increases. Results obtained from in vitro experiments performed with cardiac fibroblasts also suggest that III collagen gene expression is more responsive (vs. that of type I) to challenges derived from treating cells with either growth factors or mechanical stimulation. However, the molecular mechanisms by which collagen type III is regulated in the cardiac fibroblast are currently unknown. The overall aim of this proposal is to examine the regulation of the collagen type III gene by factors that are postulated to play an important role in directing extracellular matrix protein production in the myocardium. We propose to address the following questions: 1) Does transforming growth factor beta-1, angiotensin II (ANG II) and mechanical deformation result in an increase in type III collagen production and deposition in a culture of mouse cardiac fibroblasts? If so through what mechanisms? For this purpose, we would determine if stimulation of mouse cardiac fibroblast with these factors results in an increased expression of collagen type III mRNA and protein production using Northern blot and ELISA techniques. In order to determine the nature of the molecular mechanisms responsible for the increase in ECM protein production nuclear runoffs, mRNA stability and transcriptional (CAT-transfection) assays will be performed; 2) Does increased expression of collagen type III depend on the interaction between the angiotensin II receptor, ANG II, TGFbeta-1 and/or stretch? (a) Using binding assays we would explore if stimulation of mouse cardiac fibroblasts using the above described manipulations regulate the expression (number) of angiotensin II receptors. (b) Biochemical techniques would be used to examine if ANG II and/or stretch increases the levels of TGFbeta-1 gene expression and release. (c) Using the techniques described in Aim #1, we will examine if the simultaneous mechanical and growth factor stimulation of cardiac fibroblasts results in enhanced levels of collagen type III, and; 3) Does regulation of collagen type III gene expression by these factors depend on specific cis-acting elements contained within the promoter region of the gene? Utilizing transfection assays based on a CAT-collagen type III mouse promoter construct (and selective deletions) we would like to determine what are the specific cis-acting elements contained within the gene that might mediate the transcriptional activation by each of these different factors (TGFbeta-1, ANG II and stretch).