Altered dynamics of transforming growth factor β (TGF-β) receptors in scleroderma fibroblasts

Objectives To investigate the difference in the dynamics of transforming growth factor beta (TGF-beta) receptors between normal and scleroderma fibroblasts. Methods The cell surface expression levels of TGF-beta receptors were determined by biotinylation and immunoprecipitation assay. The dynamics of TGF-beta receptors on the cell surface was determined by the reversible biotinylation assay. The subcellular localisation of TGF-beta receptors was determined by immunoprecipitation using antibodies against clathrin and caveolin. Results Although the total expression levels of TGF-beta receptors were elevated in scleroderma fibroblasts compared with normal fibroblasts, there was no significant difference in the cell surface expression levels of TGF-beta receptors between these two groups. However, the internalisation rate of TGF-beta receptors was higher in scleroderma fibroblasts compared with normal fibroblasts. Furthermore, caveolin constitutively made a complex with TGF-beta receptors, while the interaction of clathrin with TGF-beta receptors was marginal in scleroderma fibroblasts. Conclusions The dynamics of TGF-beta receptors on the cell surface is accelerated in scleroderma fibroblasts. Considering that the activation state of TGF-beta signalling is regulated by a balance between the clathrin-dependent internalisation and the lipid raft-caveolar internalisation, the accumulation of TGF-beta receptors in caveolin-positive vesicles may result in the deceleration of caveolin-dependent internalisation and subsequently lead to the relative acceleration of clathrin-dependent internalisation.