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

转化生长因子β增加成纤维细胞中I型胶原的水平 这种刺激的最小部分是由转录调节的 机械装置。纤维化疾病的特征之一，包括全身性 硬化症是一种I型胶原和其他 皮肤和一些内部的细胞外基质(ECM)成分 器官，但这种异常蓄积的机制很差 明白了。据推测，过量的转化生长因子β可能介导了 夸大了ECM成分的积累。类似的异常情况 I型胶原和其他ECM成分的积聚在 常染色体显性遗传紧致皮肤突变小鼠(Tsk1)，在该突变小鼠中，部分in- 原纤维蛋白1基因的框架复制直接与 异常表型。我们的实验室最近发现了一种有效的 小鼠前α2(I)胶原基因的远上游增强子 在转基因小鼠中引导报告基因的高水平表达 尤其是皮肤和内脏器官的成纤维细胞。因为一个 存在着空间和时间上的相关性 细胞外转化生长因子β与I型胶原基因的激活 这些组织在胚胎发育过程中，我们推测Colla2 上游增强子可能包含对转化生长因子β有反应的元件。 进一步探讨转化生长因子β增加I型转化生长因子的机制 胶原蛋白的合成及其导致的异常堆积 在tsk1突变小鼠的I型胶原中，我们建议以下特异性 目标。(A)描绘在Colla2远上游增强子中的序列 在体内对转化生长因子β有反应。(B)划定 Colla2远上游增强子和/或近端启动子 对导致I型胶原合成增加的信号的反应 Tsk1突变小鼠。(C)产生和鉴定转基因小鼠，在其中 成纤维细胞中转化生长因子β信号通路的活性 增加或减少。(D)审查对该组织活动的影响 显性负性和结构性活性的Colla2上游增强子 成纤维细胞中转化生长因子β途径信号成分的突变 文化。(F)测量转化生长因子β信号的活性水平 正常和tsk1小鼠成纤维细胞中的信号转导途径。

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.