Fortilin interacts with TGF-β1 and prevents TGF-β receptor activation

Fortilin prevents the activation of the TGF-beta 1 receptor by occupying the pocket of TGF-beta 1 and competing with TGF-beta RII to bind with TGF-beta 1. This inhibits Smad3 phosphorylation and the differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-beta 1 and prevents it from activating the TGF-beta 1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-beta 1 and its isoforms. The modified ELISA assay shows that TGF-beta 1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-beta 1. The SPR analysis also reveals that fortilin and the TGF-beta receptor II (TGF beta RII) compete for TGF-beta 1. Both luciferase and secreted alkaline phosphatase reporter assays show that fortilin prevents TGF-beta 1 from activating Smad3 binding to Smad-binding element. Fortilin inhibits the phosphorylation of Smad3 in both quantitative western blot assays and ELISA. Finally, fortilin inhibits TGF beta-1-induced differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. A computer-assisted virtual docking reveals that fortilin occupies the pocket of TGF-beta 1 that is normally occupied by TGF beta RII and that TGF-beta 1 can bind either fortilin or TGF beta RII at any given time. These data support the role of extracellular fortilin as a negative regulator of the TGF-beta 1 signaling pathway.

Automated summary

Fortilin prevents the activation of the TGF-beta 1 signaling pathway by binding to TGF-beta 1 and inhibiting its interaction with the TGF-beta RII receptor. This inhibits the phosphorylation of Smad3 and the differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. These findings suggest that extracellular fortilin acts as a negative regulator of the TGF-beta 1 signaling pathway.