Role of TGF-beta and CTGF Signaling Transgenic Mouse Models of Scleroderma

硬皮病或系统性硬化症（SSc）是一种结缔组织疾病， 系统.这种疾病是复杂的，其特点是过度积累的胶原蛋白和其他 细胞外基质成分在皮肤和内部器官。因为TGF-β信号的增加 我们最近建立了一种新的小鼠模型，其中TGF-β受体1 在出生后的成纤维细胞中被组成性激活（TBR 1CA; Col 1a 2-CreER）。这些小鼠概括了 人SSc的主要特征，显示出真皮的显著和广泛的纤维化，较薄的表皮， 毛囊脱落，以及肺和肾中小血管壁的纤维化增厚。原发性皮肤 这些小鼠的成纤维细胞显示出下游TGF-β靶点的表达升高，再现了 标志性生化表型的干燥SSc真皮成纤维细胞。特别是有一个明显的 结缔组织生长因子（CTGF）表达增加。由于CTGF表达增加， 在疾病过程中起关键作用，我们产生了过度表达 通过使用来自pro-D 2的成纤维细胞特异性启动子/增强子在成纤维细胞中的CTGF（Col 1a 2-CTGF）（1） 胶原基因这些动物表现出严重的脱毛。皮肤活检的初步组织学检查 结果显示，真皮纤维化，表皮增厚，炎性细胞浸润， 皮肤纤维化的区域。小鼠胚胎成纤维细胞的初步分析 转基因小鼠显示I型胶原和Timp-3的表达升高。 我们建议表征Col 1a 2-CTGF小鼠以及它们的皮肤成纤维细胞。到 了解成纤维细胞中CTGF表达增加导致纤维化疾病的机制， 我们还将比较这些小鼠皮肤成纤维细胞的分子表型， TBR 1CA; Col 1a 2-CreER小鼠的那些和特定的人硬皮病患者的那些。进一步 研究这些机制，我们建议减弱两种皮肤成纤维细胞的纤维化表型， 通过特定信号传导途径的药理学抑制剂的硬皮病转基因小鼠模型，或 siRNA。我们还将尝试通过杂交来抑制这些转基因小鼠体内的纤维化表型 这些小鼠中SmadS或整联蛋白D 6的无效突变。最后，我们将研究 导致TBR 1CA; Col 1a 2-CreER小鼠对博来霉素诱导的肺纤维化的敏感性增加， 在Col 1a 2-CTGF小鼠中是否发生类似的敏感性。这些研究应该提供关于 TGF-β和CTGF在引起纤维化疾病中的相对重要性。

Scleroderma or systemic sclerosis (SSc) is a disorder of the connective tissues affecting various organ systems. The disease is complex and is characterized by excessive accumulation of collagen and other extracellular matrix components in the skin and internal organs. Because increased signaling by TGF-beta has been implicated in this disease we recently established a novel mouse model in which the TGF-beta Receptor1 is constitutively activated in fibroblasts post-natally (TBR1CA; Col1a2-CreER). These mice recapitulated the major features of human SSc, showing pronounced and generalized fibrosis of the dermis, thinner epidermis, loss of hair follicles, and fibrotic thickening of small blood vessel walls in lung and kidney. Primary skin fibroblasts of these mice showed elevated expression of downstream TGF-beta targets, reproducing the hallmark biochemical phenotype of explanted SSc dermal fibroblasts. In particular there was a marked increase in connective tissue growth factor (CTGF) expression. Since increased expression of CTGF has been implicated to play a key role in the disease process, we generated transgenic mice that over-express CTGF in fibroblasts (Col1a2-CTGF) by using a fibroblast-specific promoter/enhancer from the pro-D2(l) collagen gene. The animals exhibit a severe loss of hair. Initial histological examination of skin biopsies showed pronounced and generalized fibrosis of the dermis, thicker epidermis and inflammatory infiltrates in the area of the skin fibrosis. Preliminary analysis of mouse embryonic fibroblasts derived from these transgenic mice showed elevated expression of collagen type I and Timp-3. We propose to characterize the Col1a2-CTGF mice as well as their explanted skin fibroblasts. To understand the mechanisms by which increased expression of CTGF in fibroblasts causes a fibrotic disease, we will also perform a comparison of the molecular phenotypes of the skin fibroblasts of these mice with those of TBR1CA; Col1a2-CreER mice and with those of specific human scleroderma patients. To further examine these mechanisms we propose to attenuate the fibrotic phenotypes of the skin fibroblasts of the two transgenic mouse models of scleroderma by pharmacological inhibitors of specific signaling pathways or siRNAs. We also will attempt to inhibit the fibrotic phenotypes of these transgenic mice in vivo by crossing null mutations in either SmadS or integrin D6 in these mice. Finally, we will examine the mechanisms that cause increased sensitivity to bleomycin-induced lung fibrosis in TBR1CA; Col1a2-CreER mice and test whether a similar sensitivity occurs in Col1a2-CTGF mice. These studies should give information about the relative importance of TGF-beta and CTGF in causing fibrotic diseases.