The Fibronectin Production Is Increased by Thrombospondin via Activation of TGF-β in Cultured Human Mesangial Cells

Abstract

Thrombospondin (TSP) is a multifunctional glycoprotein that is synthesized by a variety of cells including mesangial cells (MCs). To clarify the effect of TSP on the pathogenesis of diabetic nephropathy, we studied the effect of glucose concentrations on TSP synthesis in cultured human MCs. Thereafter, the effects of TSP on the activation of transforming growth factor beta (TGF-β) and fibronectin production were investigated in MCs. Incubating MCs with elevated glucose levels for 6 days resulted in an increase in TSP synthesis, measured by an enzyme-linked immunosorbent assay, both in culture media and cell layers. Treatment of MCs with TSP (final concentrations 1 and 5 µg/ml) for 24 h resulted in an increase (1.3- and 2.1-fold, respectively) in active TGF-β, which was determined with an enzyme-linked immunosorbent assay using TGF-β-soluble receptor type II, in the culture media without having any effect on the production of total TGF-β. Exposure of MCs to TSP caused enhancement of fibronectin production in both media and cell layers in a TSP dose-dependent manner with the maximum at a TSP concentration of 1 µg/ml. The TSP-induced increase in fibronectin production from MCs was completely prevented by concomitant treatment with 10 µg/ml anti-TGF-β neutralizing antibody. These results indicate that the TSP production is promoted by a high ambient glucose concentration in human MCs and that TSP, in turn, causes an increase in fibronectin production via activation of TGF-β.

Suppression Subtractive Hybridization Identifies High Glucose Levels as a Stimulus for Expression of Connective Tissue Growth Factor and Other Genes in Human Mesangial Cells

“…Differential expression of 15 of the 16 subcloned fragments was confirmed by Northern blot (Table I). Sequence analysis revealed induction of four genes implicated previously in the pathogenesis of diabetic nephropathy: fibronectin, caldesmon, plasminogen activator inhibitor-1 (PAI-1), and thrombospondin (15)(16)(17)(18). Of 11 other cDNA fragments, two encoded novel genes, designated IHG-1 and IHG-2 (increased in high glucose), and nine encoded known genes whose induction by high glucose had not been reported previously.…”

Blockade of TSP1-Dependent TGF-β Activity Reduces Renal Injury and Proteinuria in a Murine Model of Diabetic Nephropathy

“…49 TSP1 increases fibronectin expression by cultured renal tubular epithelial cells through TGF-␤-dependent and independent mechanisms 58 and also in glucose-treated mesangial cells through a TGF-␤-dependent mechanism. 50 Blockade of CD36 through RNA silencing in cultured renal proximal tubular cells reduced bioactive TGF-␤1 and fibronectin in the presence of profibrogenic concentrations of albumin. 59 Together, these data suggest that TSP1-mediated activation of latent TGF-␤ is a key factor in diabetic tubulointerstitial fibrosis and proteinuria.…”

“…48,49 Furthermore, TSP1 increases fibronectin expression by cultured mesangial cells in a TGF-␤-dependent manner. 50 Therefore, the effects of LSKL treatment on renal fibronectin expression were examined by immunostaining of renal sections and by immunoblotting of renal lysates. Immunoblotting of renal lysates from high-dose LSKL-treated mice showed a Ͼ66% reduction in fibronectin expression (Figure 2).…”

Nitric Oxide and cGMP-dependent Protein Kinase Regulation of Glucose-mediated Thrombospondin 1-dependent Transforming Growth Factor-β Activation in Mesangial Cells

“…TSP1, a disulfide-linked 180-kDa trimer, is a multifunctional protein that is produced by a variety of cells, including mesangial cells. In vitro and in vivo studies show that high glucose up-regulates TSP1 expression (13)(14)(15)(16)31) and that TSP1 protein is increased in the kidneys of diabetic patients (17). Recently, we and others showed that TSP1 is responsible for the activation of TGF-␤ in mesangial cells when exposed to high glucose for either short (2 days) or long term (3 weeks) treatment (18,19).…”