HEART FIBROBLAST AND COLLAGEN TYPE III REGULATION

心肌病理性肥大的发展， 通常与细胞外基质的异常积聚有关 细胞外基质（ECM）（主要是胶原蛋白）。 异常 纤维状胶原的积聚导致心室肌纤维化， 僵硬，并最终有助于心肌的发展 失败 心脏胶原蛋白由I型和III型组成， 第四类和第五类的程度较小。已经证明， 早期心脏压力超负荷性肥大 I型胶原蛋白增加。 体外结果 用心脏成纤维细胞进行的实验也表明， 胶原蛋白基因表达更敏感（与I型相比）， 来自用生长因子或 机械刺激 然而， III型胶原在心脏成纤维细胞中受到调节， 未知 本提案的总体目标是审查 III型胶原蛋白基因的影响，这些因素被认为是发挥作用的因素。 在指导细胞外基质蛋白产生中的重要作用， 心肌 我们建议回答以下问题：1）是否 转化生长因子β-1、血管紧张素II（ANG II）和机械 变形导致III型胶原蛋白产生增加， 小鼠心脏成纤维细胞培养物中的沉积？ 如果是的话通过什么 机制？ 为此，我们将确定是否刺激小鼠 心脏成纤维细胞与这些因素的结果增加表达 使用北方印迹法检测III型胶原mRNA和蛋白质的产生， ELISA技术。 为了确定分子的性质 负责细胞外基质蛋白产生增加的机制 径流、mRNA稳定性和转录（CAT转染）测定 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/或伸展？ (a)使用结合分析，我们将探索， 使用上述方法刺激小鼠心脏成纤维细胞 操作调节血管紧张素II的表达（数量） 受体。 (b)生化技术将用于检测ANG是否 II和/或牵张增加TGF β-1基因表达水平， release. (c)使用目标#1中描述的技术，我们将检查 如果同时机械和生长因子刺激心脏 成纤维细胞导致III型胶原蛋白水平的提高，和; 3） 这些因子对III型胶原基因表达的调节依赖于 启动子区域内包含的特定顺式作用元件， 基因？ 利用基于CAT-胶原III型的转染测定 小鼠启动子构建体（和选择性缺失），我们希望 确定哪些是特定的顺式作用元件包含在 可能介导这些转录激活的基因 不同的因素（TGF β-1，ANG II和拉伸）。

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).