CONTROL OF TYPE I COLLAGEN GENES IN WILD TYPE AND TIGHT SKIN 1 MICE

TGF β增加成纤维细胞中I型胶原的水平， 这种刺激至少有一部分是由转录因子介导的。 机制等纤维化疾病的标志之一，包括全身性 硬化是I型胶原蛋白和其他胶原蛋白的异常积聚。 细胞外基质（ECM）成分在皮肤和一些内部 器官，但这种异常积累的机制很差 明白据推测，过量的TGF β可能介导了 ECM成分过度积聚。一个类似的异常 I型胶原蛋白和其他ECM成分的积累被发现在 常染色体显性遗传的Tight Skin突变小鼠（tsk 1），其中部分in- 编码蛋白1基因的框内重复直接与 异常表型。我们的实验室最近发现了一种 小鼠pro α 2（I）胶原基因中的远上游增强子， 指导转基因小鼠中报告基因的高水平表达 特别是皮肤和内脏器官的成纤维细胞。由于 空间和时间相关性之间存在的存在 细胞外TGF β和I型胶原基因的激活， 这些组织在胚胎发育过程中，我们假设，Colla 2 上游增强子可含有对TGF β有反应的元件。 为了进一步探索TGF β增加I型胶原的机制， 胶原蛋白合成和那些导致异常积累的 tsk 1突变小鼠的I型胶原，我们提出以下具体的 目标。(a)描绘Colla 2远上游增强子中的序列， 在体内对TGF β有反应。(b)描绘出 Colla 2远上游增强子和/或近端启动子， 对引起I型胶原合成增加的信号有反应， tsk 1突变小鼠。(c)产生并表征转基因小鼠，其中 成纤维细胞中TGF β信号通路的活性是 增加或减少。(d)检查对活性的影响， Colla 2上游显性负性增强子和组成型活性 成纤维细胞中TGF β途径信号成分的突变体， 文化(f)测量TGF β信号传导的活性水平 在正常和TSK 1小鼠的成纤维细胞中的信号通路。

TGFbeta increases the levels of type I collagen in fibroblasts and at least part of this stimulation is mediated by transcriptional mechanisms. One of the hallmarks of fibrotic diseases including Systemic Sclerosis is an abnormal accumulation of type I collagen and other extracellular matrix (ECM) components in the skin and some internal organs, but the mechanisms of this abnormal accumulation are poorly understood. It has been postulated that excess TGFbeta might mediate the exaggerated accumulation of ECM components. A similar abnormal accumulation of type I collagen and other ECM components is found in the autosomal dominant Tight Skin mutant mice (tsk1) in which a partial in- frame duplication of the fibrillin 1 gene has been directly linked to the abnormal phenotype. Our laboratory has recently identified a potent far-upstream enhancer in the mouse pro alpha2(I) collagen gene which directs high levels of expression of reporter genes in transgenic mice specifically in fibroblasts of the skin and visceral organs. Since a spatial and temporal correlation exists between the presence of extracellular TGFbeta and the activation of type I collagen genes in these tissues during embryonic development, we postulate that the Colla2 upstream enhancer may contain elements that are responsive to TGFbeta. To further explore the mechanisms by which TGFbeta increases type I collagen synthesis and those leading to the abnormal accumulation of type I collagen in tsk1 mutant mice, we propose the following Specific Aims. (a) Delineate sequences in the Colla2 far-upstream enhancer that are responsive to TGFbeta in vivo. (b) Delineate sequences within the Colla2 far-upstream enhancer and/or proximal promoter which are responsive to signals that cause increased type I collagen synthesis in tsk1 mutant mice. (c) Generate and characterize transgenic mice in which the activity of the TGFbeta signaling pathway in fibroblasts is increased or decreased. (d) Examine the effects on the activity of the Colla2 upstream enhancer of dominant negative and constitutively active mutants of signaling components of the TGFbeta pathway in fibroblasts in culture. (f) Measure the levels of activity of the TGFbeta signaling pathway in fibroblasts of normal and tsk1 mice.