Sudarshan C, Yaswen L, Kulkarni A, Raghow R
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis, USA.
J Cell Physiol. 1998 Jul;176(1):67-75. doi: 10.1002/(SICI)1097-4652(199807)176:1<67::AID-JCP8>3.0.CO;2-6.
The profound effects of transforming growth factor beta1 (TGF-beta1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-beta1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-beta1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-beta1 null mice (TGF-beta-1(-/-)). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-beta1(+/+)) and TGF-beta1 null mice were compared by cell counting, by 3H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-alpha1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-beta1(+/+) and TGF-beta1(-/-) embryonic fibroblasts. We report that TGF-beta1(-/-) cells proliferated at about twice the rate of TGF-beta1(+/+) cells. Further, TGF-beta1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-alpha1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-beta1(+/-) and TGF-beta1(-/-) cells could be eliminated by treatment of TGF-beta1(+/+) cells with a neutralizing antibody of TGF-beta1. Thus, our results are consistent with the hypothesis that TGF-beta1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts.
通过对胚胎发育过程中转化生长因子β1（TGF-β1）基因敲除小鼠的详细分析，已证实TGF-β1对免疫系统、心脏发生、卵黄囊造血以及内皮细胞分化具有深远影响。我们使用来自TGF-β1基因敲除小鼠（TGF-β-1(-/-)）的胚胎成纤维细胞，系统地研究了TGF-β1在细胞增殖以及调节细胞外基质（ECM）特定成分基因表达能力方面的自分泌和旁分泌作用。通过细胞计数、3H胸苷掺入以及利用荧光激活细胞分选（FACS）测量细胞周期G1、S和G2/M期的细胞比例，比较了正常小鼠（TGF-β1(+/+)）和TGF-β1基因敲除小鼠胚胎成纤维细胞的增殖速率。同时，通过对TGF-β1(+/+)和TGF-β1(-/-)胚胎成纤维细胞总mRNA进行杂交，也对原α1（I）型胶原蛋白、纤连蛋白和纤溶酶原激活物抑制剂-1（PAI-1）的表达进行了定量分析。我们报告称，TGF-β1(-/-)细胞的增殖速率约为TGF-β1(+/+)细胞的两倍。此外，TGF-β1基因敲除的成纤维细胞积累并合成的原α1（I）型胶原蛋白、纤连蛋白和PAI-1 mRNA的基础水平较低。用TGF-β1中和抗体处理TGF-β1(+/+)细胞，可以消除TGF-β1(+/-)和TGF-β1(-/-)细胞在增殖速率和ECM基因表达方面的定量差异。因此，我们的结果与以下假设一致：TGF-β1在胚胎成纤维细胞中作为生长的负性自分泌调节因子和ECM生物合成的正性自分泌调节因子发挥作用。
