FIGURE 6.

Klotho binds to TGFβR2 and inhibits TGF-β1 binding to the receptor. A, direct protein-protein interaction between Klotho and TGFβR2. Klotho protein was incubated with recombinant receptor ectodomain proteins with a human Fc tag and pulled down with protein A beads. The beads-bound protein was detected by immunoblot analysis using anti-Klotho (upper panel) or anti-human IgG (Fc, lower panel) antibodies. TGFβR2; Type-II TGF-β receptor, FGFR1b; fibroblast growth factor receptor 1α (IIIb), FGFR1c; fibroblast growth factor receptor 1α (IIIc), TGFβR1; Type-I TGF-β receptor, LRP-6; LDL receptor-related protein 6, EGFR; epidermal growth factor receptor, PDGFR; platelet-derived growth factor receptor-α. Klotho was reported to bind to FGFR1c but not to FGFR1b (6, 39), which served as positive and negative controls, respectively. B, Klotho inhibits TGF-β1 binding to TGFβR2 in vitro. TGF-β1 binding assays were performed using recombinant TGFβR2 protein on protein A beads in the presence of Klotho protein. Data indicate means of duplicated measurement and a fitting curve (R² = 0.776). C, Klotho inhibits TGF-β1 binding to renal epithelial cells. TGF-β1 binding assay was performed in NRK52E cells in the presence of Klotho protein. Data indicate means ± S.E. (n = 4 for each dose). p = 0.017 by one-way ANOVA.