Denton C P, Zheng B, Shiwen X, Zhang Z, Bou-Gharios G, Eberspaecher H, Black C M, de Crombrugghe B
University of Texas, Houston, USA.
Arthritis Rheum. 2001 Mar;44(3):712-22. doi: 10.1002/1529-0131(200103)44:3<712::AID-ANR121>3.0.CO;2-1.
Reporter transgenes were introduced into the type 1 tight-skin (Tsk1/+) mouse model of scleroderma to test the hypothesis that fibroblast-specific genetic programs are activated in fibrosis.
Transgenes harboring upstream fragments of the 5' flanking region of the mouse proalpha2(I) collagen gene (Col1a2), linked to a 400-bp minimal Col1a2 promoter driving an Escherichia coli beta-galactosidase (LacZ) reporter gene, were introduced into Tsk1/+ mice by breeding. Expression of these transgenes, which function as lineage-specific markers of fibroblast differentiation, was compared between the Tsk-LacZ mice and non-Tsk littermates. Responsiveness of these constructs to the profibrotic cytokine, transforming growth factor beta1 (TGFbeta1), was investigated by transient transfection of reporter constructs in tissue-culture cells.
There was significant activation of reporter genes harboring the upstream enhancer in Tsk1/+ mice starting from 1 week of age. This was maximal at 6 weeks old (mean +/- SD 237 +/- 24% of non-Tsk controls; P= 0.001). Recombinant TGFbeta1 significantly activated reporter genes regulated by the upstream enhancer in transient transfection, and Tsk-LacZ fibroblasts showed elevated LacZ expression in tissue culture.
These data suggest that activating signals in Tsk1/+ mice may act via fibroblast-specific regulatory elements within the murine Col1a2 gene. Although TGFbeta has been implicated in the pathogenesis of fibrosis, and reporter genes regulated by the upstream enhancer appear to be TGFbeta responsive in vitro, our results suggest that fibroblast-specific pathways may also be involved.
将报告基因导入硬皮病1型紧皮（Tsk1 / +）小鼠模型，以检验成纤维细胞特异性遗传程序在纤维化过程中被激活这一假说。
通过杂交将携带小鼠原α2（I）型胶原基因（Col1a2）5'侧翼区上游片段的转基因导入Tsk1 / +小鼠，该片段与驱动大肠杆菌β - 半乳糖苷酶（LacZ）报告基因的400bp最小Col1a2启动子相连。比较Tsk - LacZ小鼠和非Tsk同窝小鼠中这些作为成纤维细胞分化谱系特异性标志物的转基因的表达情况。通过在组织培养细胞中瞬时转染报告构建体，研究这些构建体对促纤维化细胞因子转化生长因子β1（TGFβ1）的反应性。
从1周龄开始，Tsk1 / +小鼠中携带上游增强子的报告基因有显著激活。在6周龄时达到最大值（平均值±标准差为非Tsk对照的237±24％；P = 0.001）。重组TGFβ1在瞬时转染中显著激活由上游增强子调控的报告基因，并且Tsk - LacZ成纤维细胞在组织培养中显示出LacZ表达升高。
这些数据表明，Tsk1 / +小鼠中的激活信号可能通过小鼠Col1a2基因内的成纤维细胞特异性调控元件起作用。虽然TGFβ已被认为与纤维化的发病机制有关，并且由上游增强子调控的报告基因在体外似乎对TGFβ有反应，但我们的结果表明成纤维细胞特异性途径可能也参与其中。
