The Effect of Transforming Growth Factor β 1 on Mesangial Cell Fibronectin Synthesis: Increased Incorporation into the Extracellular Matrix and Reduced p I but No Effect on Alternative Splicing

Abstract

Fibronectin is a multidomain glycoprotein which accumulates in mesangial proliferative glomerulonephritis (MPGN). Recent evidence has implicated transforming growth factor β (TGF-β) in the pathogenesis of experimental MPGN. We have, therefore, examined the influence of TGF-β 1 on mesangial cell fibronectin synthesis. Considering, first, levels of mRNA, TGF-β 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-β 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-β 1. TGF-β 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-β 1 increases incorporation of fibronectin into the ECM. Because fibronectin possesses binding sites for other ECM proteins, greater incorporation of fibronectin following TGF-β 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.