Fucosyltransferase-1 Mediated Fucosylation of TGF-βR1 Is Critical to TGF-β Signaling in Scleroderma and in Bleomycin-Induced Fibrosis

W. Alexander Stinson¹, Pei-Suen Tsou^1,2, Yuxuan Du³, Huadong Cui¹, Ellen Cealey³, Nicholas Lepore⁴, Ray A. Ohara¹, Gautam Edhayan¹, Sarah Arwani¹, Rachel Morgan¹, Dinesh Khanna^1,2, David A. Fox¹ and M. Asif Amin⁵, ¹Division of Rheumatology, University of Michigan Medical Center, Ann Arbor, MI, ²University of Michigan Scleroderma Program, Ann Arbor, MI, ³Rheumatology, Division of Rheumatology, University of Michigan Medical Center, Ann Arbor, MI, ⁴University of Michigan, Division of Rheumatology, University of Michigan Medical Center, Ann Arbor, MI, ⁵Internal Medicine, Division of Rheumatology, University of Michigan Medical Center, Ann Arbor, MI

Meeting: 2016 ACR/ARHP Annual Meeting

Date of first publication: September 28, 2016

Keywords: Animal models, fibrosis, glycoproteins, scleroderma and transforming growth factor

Session Information

Date: Monday, November 14, 2016

Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's – Pathogenesis, Animal Models and Genetics - Poster II

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose: Systemic sclerosis (SSc) is a connective tissue disease characterized by systemic fibrosis. The dysregulation of transforming growth factor-β (TGF-β) signaling causes proliferation of myofibroblasts and results in the uncontrolled release of extracellular matrix. We have published that fucosyltransferase-1 (Fut1), an α-1,2 fucosyltransferase, plays an important role in rheumatoid arthritis synovial fibroblast proliferation. In this study, we examine the role of Fut1 in TGF-β receptor (TGF-βR) fucosylation, downstream signaling pathways, and target genes involved in scleroderma pathogenesis.

Methods: qPCR and ELISA were performed to assess the levels of Fut1 in SSc dermal fibroblasts and patient sera, respectively. To determine fucosylation of TGF-βR1, TGF-βR1 was immunoprecipitated from wild type (WT) dermal fibroblasts and immunoblotted with ulex europaeus agglutinin 1, which detects α-1,2 fucosylated protein. To confirm TGF-bR1 fucosylation, Fut1 was knocked down in SSc dermal fibroblasts using Fut1 shRNA. We evaluated Fut1 involvement in TGF-β signaling, myofibroblast differentiation, and TGF-b-associated target genes in WT and Fut1^-/-dermal fibroblasts via immunofluorescence (IF), qPCR, and Western blotting. An in vivo wound healing model was performed with Fut1^-/- and WT mice and wound healing was assessed over 8 days. To elucidate the role of Fut1 in an animal model of scleroderma, bleomycin was injected intradermally into Fut1^-/- and WT mice for 9 days. Mice were euthanized and skin harvested for Masson’s trichrome staining and hydroxyproline assay, a quantitative measure of collagen.

Results: Fut1 mRNA and protein were significantly elevated in SSc compared to NL dermal fibroblasts and patient sera, respectively. We found via immunoprecipitation that TGF-βR1 is fucosylated by Fut1. We found less TGF-bR1 in SSc dermal fibroblasts when transduced with Fut1 shRNA, confirming the role of Fut1 in TGF-bR1 fucosylation. TGF-β-induced phosphorylation of Smad2, Erk1/2, and Jnk was markedly reduced in Fut1^-/-dermal fibroblasts compared to WT, indicating that Fut1 is essential to TGF-βR1-mediated signaling pathways. Impaired transcription of myofibroblast differentiation inducing genes, Twist2 and Snail, was observed in Fut1^-/-dermal fibroblasts. We found a significant decrease in α-smooth muscle actin in Fut1^-/-compared to WT dermal fibroblasts as determined by IF and Western blotting, indicating a critical role for Fut1 in myofibroblast differentiation. In the wound repair model, Fut1^-/-mouse wounds healed more slowly than WT mouse wounds. Fut1^-/- mice developed significantly less bleomycin-induced skin fibrosis and collagen deposition compared to WT mice as determined by Masson’s trichrome staining and hydroxyproline assay, respectively.

Conclusion: Fut1 expression is elevated in SSc dermal fibroblasts and patient sera. Fucosylation of TGF-βR1 is critical to myofibroblast differentiation and TGF-β signaling. In addition, Fut1^-/-mice exhibit delayed wound repair and diminished bleomycin-induced skin fibrosis, indicating an essential role for Fut1 in SSc pathology. Fut1 may be a novel therapeutic target for SSc.

Disclosure: W. A. Stinson, None; P. S. Tsou, None; Y. Du, None; H. Cui, None; E. Cealey, None; N. Lepore, None; R. A. Ohara, None; G. Edhayan, None; S. Arwani, None; R. Morgan, None; D. Khanna, Bristol-Myers Squibb, 2,Pfizer Inc, 2,Roche Pharmaceuticals, 5,Sanofi-Aventis Pharmaceutical, 5,BAYER, 5,CYTORI, 5,EMD Serono, 5,Roche Pharmaceuticals, 2,Actelion Pharmaceuticals US, 5; D. A. Fox, None; M. A. Amin, None.

To cite this abstract in AMA style:

Stinson WA, Tsou PS, Du Y, Cui H, Cealey E, Lepore N, Ohara RA, Edhayan G, Arwani S, Morgan R, Khanna D, Fox DA, Amin MA. Fucosyltransferase-1 Mediated Fucosylation of TGF-βR1 Is Critical to TGF-β Signaling in Scleroderma and in Bleomycin-Induced Fibrosis [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/fucosyltransferase-1-mediated-fucosylation-of-tgf-%ce%b2r1-is-critical-to-tgf-%ce%b2-signaling-in-scleroderma-and-in-bleomycin-induced-fibrosis/. Accessed .

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