McKay N G, Khong T F, Haites N E, Power D A
Department of Medicine, University of Aberdeen, Scotland.
Exp Mol Pathol. 1993 Dec;59(3):211-24. doi: 10.1006/exmp.1993.1040.
Fibronectin is a multidomain glycoprotein which accumulates in mesangial proliferative glomerulonephritis (MPGN). Recent evidence has implicated transforming growth factor beta (TGF-beta) in the pathogenesis of experimental MPGN. We have, therefore, examined the influence of TGF-beta 1 on mesangial cell fibronectin synthesis. Considering, first, levels of mRNA, TGF-beta 1 increased steady-state fibronectin RNA in cultured mesangial cells by 1.9 times 24 hr after treatment of cycling mesangial cells and by 11.8 times in growth-arrested cells. There was, however, no alteration in fibronectin pre-mRNA splicing in either the EIIIA or IIICS regions. Fibronectin protein concentrations in cell culture supernatants, determined by immunoprecipitation of supernatants from cells labeled with [35S]methionine and by ELISA, were not increased by treatment with TGF-beta 1. Western blots and immunoprecipitation of metabolically labeled cells showed that fibronectin was increased, however, in the deoxycholate-insoluble extracellular matrix (ECM) of cells stimulated with TGF-beta 1. TGF-beta 1 altered the physicochemical properties of fibronectin in ECM and supernatant such that the isoelectric point of fibronectin, determined from Western blots of 2D SDS-PAGE gels, was reduced so that both became more acidic. These studies demonstrate, therefore, that in addition to increasing its synthesis, TGF-beta 1 increases incorporation of fibronectin into the ECM. Because fibronectin possesses binding sites for other ECM proteins, greater incorporation of fibronectin following TGF-beta 1 treatment may be an important pathogenetic mechanism in mesangial sclerosis. Moreover, the altered charge of fibronectin may increase localization of serum immunoglobulins to the mesangium.
纤连蛋白是一种多结构域糖蛋白，在系膜增生性肾小球肾炎（MPGN）中会蓄积。最近有证据表明，转化生长因子β（TGF-β）参与了实验性MPGN的发病机制。因此，我们研究了TGF-β1对系膜细胞纤连蛋白合成的影响。首先，考虑到mRNA水平，在处理处于增殖期的系膜细胞24小时后，TGF-β1使培养的系膜细胞中纤连蛋白RNA的稳态水平增加了1.9倍，而在生长停滞的细胞中增加了11.8倍。然而，EIIIA或IIICS区域的纤连蛋白前体mRNA剪接没有改变。通过对用[35S]甲硫氨酸标记的细胞上清液进行免疫沉淀和酶联免疫吸附测定（ELISA）确定，细胞培养上清液中的纤连蛋白蛋白浓度并未因TGF-β1处理而增加。蛋白质印迹法和对代谢标记细胞的免疫沉淀显示，在用TGF-β1刺激的细胞的脱氧胆酸盐不溶性细胞外基质（ECM）中，纤连蛋白增加了。TGF-β1改变了ECM和上清液中纤连蛋白的物理化学性质，使得从二维SDS-PAGE凝胶的蛋白质印迹法测定的纤连蛋白的等电点降低，两者都变得更酸。因此，这些研究表明，除了增加其合成外，TGF-β1还增加了纤连蛋白向ECM中的掺入。由于纤连蛋白具有与其他ECM蛋白的结合位点，TGF-β1处理后纤连蛋白掺入增加可能是系膜硬化的重要发病机制。此外，纤连蛋白电荷的改变可能会增加血清免疫球蛋白在系膜中的定位。
