Dysregulation of TGF‐β Signaling: A Key Role for Fucosyltransferase‐1 in Scleroderma and Bleomycin‐Induced Fibrosis

Abstract

BackgroundSystemic sclerosis (SSc) is a connective tissue disease characterized by systemic fibrosis. The dysregulation of transforming growth factor‐β (TGF‐β) causes proliferation of myofibroblasts and results in the uncontrolled release of extracellular matrix. We have published that fucosyltransferase‐1 (Fut1) plays an important role in rheumatoid arthritis synovial tissue fibroblast proliferation and in the expression of adhesion molecules and cytokines, but its contribution to SSc pathogenesis has not yet been examined.MethodsQuantitative polymerase chain reaction was performed to assess the levels of Fut1 in SSc dermal fibroblasts. We then examined the expression of Fut1 in SSc patient sera and on skin biopsies. TGF‐β receptor (TGF‐βR) and TGF‐βR mediated signaling indermediates were examined in wild type (wt) and Fut1−/− dermal fibroblasts via Western blotting and immunofluorescence. To confirm the contribution of Fut1 in SSc, Fut1 was knocked down in SSc and normal (NL) human dermal fibroblasts using Fut1 shRNA. We evaluated the role of Fut1 in skin fibroblast migration and proliferation by performing an in vitro scratch wound assay. An in vivo wound healing model was performed with Fut1−/− and wt mice. To elucidate the role of Fut1 in an animal model of scleroderma, Fut1−/− and wt mice were injected with bleomycin intradermally. Mice were euthanized and fibrotic skin harvested for Masson's trichrome staining and immunohistochemistry.ResultsFut1 mRNA and protein was significantly elevated in SSc compared to NL dermal fibroblasts and patient sera, respectively. Fut1 was markedly higher on SSc compared to NL skin biopsies. Fut1 shRNA transduced SSc and NL fibroblasts had diminished TGF‐βR1 and TGF‐βR2 expression compared to controls, indicating a critical role for Fut1 in scleroderma. In addition, TGF‐β‐induced p‐Smad3, p‐Jnk, and p‐Erk were decreased in Fut1−/−dermal fibroblasts compared to wt. TGF‐β stimulated Fut1−/− dermal fibroblasts showed decreased alpha‐smooth muscle actin via immunofluorescence and Western blot, suggesting the contribution of Fut1 in myofibroblast differentiation. Fut1−/− dermal fibroblasts exhibited decreased cell proliferation and growth in vitro. In the wound repair model, wound healing was delayed in Fut1−/− mice compared to wt mice. As wound repair shares many of the fibrogenic features of SSc, Fut1 may play a role in SSc fibrosis. Fut1−/− mice developed significantly less bleomycin‐induced skin fibrosis compared to wt mice as determined by Masson's trichrome staining, demonstrating the role of Fut1 in SSc pathology.ConclusionFut1 expression is elevated in SSc dermal fibroblasts, patient sera and on skin biopsies. Fut1−/− dermal fibroblasts exhibit impaired myofibroblast differentiation, migration and proliferation in vitro in part due to impaired TGF‐β signaling. In addition, Fut1−/− mice exhibit delayed wound repair and highly diminished bleomycin‐induced skin fibrosis, indicating an essential role for Fut1 in SSc pathology. Fut1 may be a potential novel therapeutic target to treat SSc.