Increased Cthrc1 Activates Normal Fibroblasts and Suppresses Keloid Fibroblasts by Inhibiting TGF-β/Smad Signal Pathway and Modulating YAP Subcellular Location

Abstract

Keloid may induce severe impairment of life quality for the patients, although keloid is a cutaneous benign tumor. Collagen triple helix repeat containing protein 1 (Cthrc1) was identified as a novel gene that was originally found in adventitial fibroblasts after arterial injury. To address the role of Cthrc1 in keloid, the expression level of Cthrc1 was assessed in normal skin and keloid tissue, as well as in normal fibroblasts (NFs) and keloid fibroblasts (KFs) by using quantitative PCR, Western blotting and immunohistochemical analysis. The results showed that Cthrc1 was increased in keloid tissue and KFs as compared with normal skin and NFs. Meanwhile, CCK8 and Transwell assays found the cellular proliferation and migration of KFs were increased as compared with NFs. Further, to verify the function of Cthrc1 in NFs and KFs, we increased Cthrc1 expression by transfecting lentivirus (LV) vectors LV-Cthrc1. The cellular proliferation and migration, collagen synthesis and the influence on TGF-β and YAP signaling were tested. The cellular proliferation and migration were increased in NFs-Cthrc1 as compared with NFs-control. Meanwhile, YAP expression and nuclear-location was increased in NFs-Cthrc1. On the contrary, when Cthrc1 was overexpressed in KFs, the cellular migration was suppressed and YAP expression was reduced and transferred to cytoplasm in KFs-Cthrc1 as compared with KFs-control. But the expression level of collagen I was decreased and pSmad2/3 nucleus transfer was suppressed in both NFs-Cthrc1 and KFs-Cthrc1 by using Western blotting and immunofluorescence. Increased Cthrc1 activated NFs by promoting YAP nucleus translocation, whereas suppressed KFs by inhibiting YAP nucleus translocation. Enhanced Cthrc1 decreased collagen I in both NFs and KFs by inhibiting TGF-β/Smad pathway. In conclusion, Cthrc1 may play a role in the pathogenesis of keloid by inhibiting collagen synthesis and fibroblasts migration via suppressing TGF-β/Smad pathway and YAP nucleus translocation.

Keywords

keloid / Collagen triple helix repeat containing protein 1 / TGF-β / YAP

References

[1] ArnoAI, GauglitzGG, BarretJP, et al.. Up-to-date approach to manage keloids and hypertrophic scars: A useful guide. Burns, 2014, 40(7): 1255-1266

[2] LiuF, LagaresD, ChoiKM, et al.. Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis. Am J Physiol Lung Cell Mol Physiol, 2014, 308(4): L344-L357

[3] PyagayP, HeroultM, WangQ, et al.. Collagen triple helix repeat containing 1, a novel secreted protein in injured and diseased arteries, inhibits collagen expression and promotes cell migration. Circ Res, 2005, 96(2): 261-268

[4] DurmusT, LeClairRJ, ParkKS, et al.. Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1). Gene Expr Patterns, 2006, 6(8): 935-940

[5] LeClairR, LindnerV. The role of collagen triple helix repeat containing 1 in injured arteries, collagen expression, and transforming growth factor beta signaling. Trends Cardiovasc Med, 2007, 17(6): 202-205

[6] BianZ, MiaoQ, ZhongW, et al.. Treatment of cholestatic fibrosis by altering gene expression of Cthrc1: Implications for autoimmune and nonautoimmune liver disease. J Autoimmun, 2015, 63: 76-87

[7] UnahabhokhaT, SucontphuntA, NimmannitU, et al.. Molecular signalings in keloid disease and current therapeutic approaches from natural based compounds. Pharm Biol, 2015, 53(3): 457-463

[8] LeClairRJ, DurmusT, WangQ, et al.. Cthrc1 is a novel inhibitor of transforming growth factor-beta signaling and neointimal lesion formation. Circ Res, 2007, 100(6): 826-833

[9] LiJ, CaoJ, LiM, et al.. Collagen triple helix repeat containing-1 inhibits transforming growth factor-β1-induced collagen type I expression in keloid. Br J Dermatol., 2011, 164(5): 1030-1036

[10] LeeMJ, ByunMR, Furutani-SeikiM, et al.. YAP and TAZ Regulate Skin Wound Healing. J Invest Dermatol, 2014, 134(2): 518-525

[11] ZhaoB, LiL, LuQ, et al.. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev, 2011, 25(1): 51-63

[12] BarryER, MorikawaT, ButlerBL, et al.. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature, 2013, 493(7430): 106-110

[13] LiuM, ZouW, HuangM, et al.. Isolation and cultivation of bovine corneal stromal fibroblasts by two-step collagenase digestion method. Zhongguo Zuzhi Gongcheng Yanjiu (Chinese)., 2012, 16(7): 1201-1205

[14] KeZ, HeW, LaiY, et al.. Overexpression of collagen triple helix repeat containing 1 (CTHRC1) is associated with tumour aggressiveness and poor prognosis in human non-small cell lung cancer. Oncotarget, 2014, 5(19): 9410-9424

[15] TanF, LiuF, LiuH, et al.. CTHRC1 is associated with peritoneal carcinomatosis in colorectal cancer: a new predictor for prognosis. Med Oncol, 2013, 30(1): 473

[16] HouM, ChengZ, ShenH, et al.. High expression of CTHRC1 promotes EMT of epithelial ovarian cancer (EOC) and is associated with poor prognosis. Oncotarget, 2015, 6(34): 35813-35829

[17] WangL, XiangYN, ZhangYH, et al.. Collagen triple helix repeat containing-1 in the differential diagnosis of dermatofibrosarcoma protuberans and dermatofibrom. Br J Dermatol, 2011, 164(1): 135-140

[18] IpW, Wellman-LabadieO, TangL, et al.. Collagen triple helix repeat containing 1 promotes melanoma cell adhesion and survival. J Cutan Med Surg, 2011, 15(2): 103-110

[19] KharaishviliG, MagdalenaC, KaterinaB, et al.. Collagen triple helix repeat containing 1 protein, periostin and versican in primary and metastatic breast cancer: an immunohistochemical study. J Clin Pathol, 2011, 64(11): 977-982

[20] GurtnerGC, WernerS, BarrandonY, et al.. Wound repair and regeneration. Nature, 2008, 453(7193): 314-321

[21] LiuX, HeH, YinJQ. Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis. Liver Int, 2006, 26(1): 8-22

[22] MehalWZ, IredaleJ, FriedmanSL. Scraping fibrosis: expressway to the core of fibrosis. Nat Med, 2011, 17(5): 552-553

[23] DentonCP, MerkelPA, FurstDE, et al.. Recombinant human anti-transforming growth factor beta1 antibody therapy in systemic sclerosis: a multicenter, randomized, placebo-controlled phase I/II trial of CAT-192. Arthritis Rheum, 2007, 56(1): 323-333

[24] WangL, XiangYN, ZhangYH, et al.. CTHRC1 is upregulated by promoter demethylation and transforming growth factor-beta1 and may be associated with metastasis in human gastric cancer. Cancer Sci, 2012, 103(7): 1327-1333

[25] PiersmaB, BankRA, BoersemaM. Signaling in fibrosis: TGF-beta, WNT, and YAP/TAZ converge. Front Med (Lausanne), 2015, 2: 59

[26] VarelasX, WranaJL. Coordinating developmental signaling: novel roles for the Hippo pathway. Trends Cell Biol, 2012, 22(2): 88-96

[27] PreisserF, GiehlK, RehmM, et al.. Inhibitors of oxygen sensing prolyl hydroxylases regulate nuclear localization of the transcription factors Smad2 and YAP/TAZ involved in CTGF synthesis. Biochim Biophys Acta, 2016, 1863(8): 2027-2036

[28] GrannasK, ArngardenL, LonnP, et al.. Crosstalk between Hippo and TGFβ: subcellular localization of YAP/TAZ/Smad complexes. J Mol Biol, 2015, 427(21): 3407-3415