Skip to main content

Advertisement

Log in

Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids

  • Original Paper
  • Published:
Journal of Molecular Histology Aims and scope Submit manuscript

Abstract

Keloid, a benign skin disorder, forms during wound healing in genetically susceptible individuals. To better control keloid and understand the molecular mechanisms, this study screened gene hypermethylations of GEO database microarray data on keloids and identified the hypermethylation of the secreted frizzled related protein-1 (SFRP1) promoter. Subsequently, hypermethylation and mRNA and protein levels were assessed in 57 cases of keloid vs. normal skin tissues. Fibroblasts from tissues were isolated for the assessment of gene regulation in vitro. The methods used were bioinformatic analysis, lentiviral infection carrying SFRP1 cDNA, qRT-PCR, western blot, immunohistochemistry, luciferase reporter assay, methylation-specific PCR and methylated DNA immunoprecipitation-qPCR, ELISA, and/or 5-Aza-2′-deoxycytidine treatment. The data revealed that the SFRP1 promoter was hypermethylated in keloid tissues, compared with that in normal skin tissues. The SFRP1 promoter methylation contributed to the downregulation of SFRP1 mRNA and protein in keloid tissues and keloid fibroblasts. The 5-Aza treatment significantly upregulated SFRP1 mRNA and protein level in keloid fibroblasts. Furthermore, the knockdown of DNMT1 expression, and not the expression of DNMT3a or DMNT3b, was responsible for the hypermethylation of the SFRP1 promoter and upregulation of SFRP1 mRNA and protein in keloid fibroblasts. In addition, the infection of lentivirus carrying SFRP1 cDNA significantly inhibited the signaling activity of Wnt/β-catenin and the mRNA and protein expression of β-catenin and α-SMA in keloid fibroblasts. In summary, the lost SFRP1 expression-induced Wnt/β-catenin signaling due to the hypermethylation of the SFRP1 promoter could associate with keloid development, suggesting that SFRP1 might be a therapeutic target for keloid treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

SFRP1:

Secreted frizzled-related protein 1

ORF:

Open reading frame

MSP:

Methylation-specific PCR

MeDIP-qPCR:

Methylated DNA immunoprecipitation-qPCR

5-Aza:

5-Aza-2′-deoxycytidine

ELISA:

Enzyme-linked immunosorbent assay

α-SMA:

Alpha smooth muscle actin

KF:

Keloid fibroblast

NFFB:

Normal skin fibroblast

References

  • Andrews JP, Marttala J, Macarak E, Rosenbloom J, Uitto J (2016) Keloids: the paradigm of skin fibrosis—Pathomechanisms and treatment. Matrix Biol 51:37–46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Caldwell GM, Jones C, Gensberg K, Jan S, Hardy RG, Byrd P, Chughtai S, Wallis Y, Matthews GM, Morton DG (2004) The Wnt antagonist sFRP1 in colorectal tumorigenesis. Cancer Res 64:883–888

    Article  CAS  PubMed  Google Scholar 

  • Cheng W, Qi Y, Tian L, Wang B, Huang W, Chen Y (2017) Dicer promotes tumorigenesis by translocating to nucleus to promote SFRP1 promoter methylation in cholangiocarcinoma cells. Cell Death Dis 8:e2628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chim CS, Pang R, Fung TK, Choi CL, Liang R (2007) Epigenetic dysregulation of Wnt signaling pathway in multiple myeloma. Leukemia 21:2527–2536

    Article  CAS  PubMed  Google Scholar 

  • Chiu HY, Tsai TF (2011) Images in clinical medicine. Keloidal morphea. N Engl J Med 364:e28

    Article  CAS  PubMed  Google Scholar 

  • Chua AW, Ma D, Gan SU, Fu Z, Han HC, Song C, Sabapathy K, Phan TT (2011) The role of R-spondin2 in keratinocyte proliferation and epidermal thickening in keloid scarring. J Invest Dermatol 131:644–654

    Article  CAS  PubMed  Google Scholar 

  • Dees C, Schlottmann I, Funke R, Distler A, Palumbo-Zerr K, Zerr P, Lin NY, Beyer C, Distler O, Schett G, Distler JH (2014) The Wnt antagonists DKK1 and SFRP1 are downregulated by promoter hypermethylation in systemic sclerosis. Ann Rheum Dis 73:1232–1239

    Article  CAS  PubMed  Google Scholar 

  • Delic S, Lottmann N, Stelzl A, Liesenberg F, Wolter M, Götze S, Zapatka M, Shiio Y, Sabel MC, Felsberg J, Reifenberger G, Riemenschneider MJ (2014) MiR-328 promotes glioma cell invasion via SFRP1-dependent Wnt-signaling activation. Neuro-oncology 16:179–190

    Article  CAS  PubMed  Google Scholar 

  • Fu HD, Wang BK, Wan ZQ, Lin H, Chang ML, Han GL (2016) Wnt5a mediated canonical Wnt signaling pathway activation in orthodontic tooth movement: possible role in the tension force-induced bone formation. J Mol Histol 47:455–466

    Article  CAS  PubMed  Google Scholar 

  • Gauglitz GG, Korting HC, Pavicic T, Ruzicka T, Jeschke MG (2011) Hypertrophic scarring and keloids: pathomechanisms and current and emerging treatment strategies. Mol Med 17:113–125

    Article  CAS  PubMed  Google Scholar 

  • Glass DA 2nd (2017) Current understanding of the genetic causes of keloid formation. J Investig Dermatol Symp Proc 18:S50–S53

    Article  PubMed  Google Scholar 

  • Hagele T, Nyanda H, Patel N, Russell N, Cohen G, Nelson C (2017) Advanced management of severe keloids. Skinmed 15:365–370

    PubMed  Google Scholar 

  • Hamburg-Shields E, DiNuoscio GJ, Mullin NK, Lafyatis R, Atit RP (2015) Sustained β-catenin activity in dermal fibroblasts promotes fibrosis by up-regulating expression of extracellular matrix protein-coding genes. J Pathol 235:686–697

    Article  CAS  PubMed  Google Scholar 

  • Han W, Liu J (2017) Epigenetic silencing of the Wnt antagonist APCDD1 by promoter DNA hyper-methylation contributes to osteosarcoma cell invasion and metastasis. Biochem Biophys Res Commun 491:91–97

    Article  CAS  PubMed  Google Scholar 

  • Igota S, Tosa M, Murakami M, Egawa S, Shimizu H, Hyakusoku H, Ghazizadeh M (2013) Identification and characterization of Wnt signaling pathway in keloid pathogenesis. Int J Med Sci 10:344–354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ishii T, Murakami J, Notohara K, Cullings HM, Sasamoto H, Kambara T, Shirakawa Y, Naomoto Y, Ouchida M, Shimizu K, Tanaka N, Jass JR, Matsubara N (2007) Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa. Gut 56:13–19

    Article  CAS  PubMed  Google Scholar 

  • Jones LR, Young W, Divine G, Datta I, Chen KM, Ozog D, Worsham MJ (2015) Genome-wide scan for methylation profiles in keloids. Dis Markers 2015:943176

    Article  PubMed  PubMed Central  Google Scholar 

  • Kele J, Andersson ER, Villaescusa JC, Cajanek L, Parish CL, Bonilla S, Toledo EM, Bryja V, Rubin JS, Shimono A, Arenas E (2012) SFRP1 and SFRP2 dose-dependently regulate midbrain dopamine neuron development in vivo and in embryonic stem cells. Stem Cells 30:865–875

    Article  CAS  PubMed  Google Scholar 

  • Kim J, Kim S (2014) In silico identification of SFRP1 as a hypermethylated gene in colorectal cancers. Genomics Inform 12:171–180

    Article  PubMed  PubMed Central  Google Scholar 

  • Kloten V, Becker B, Winner K, Schrauder MG, Fasching PA, Anzeneder T, Veeck J, Hartmann A, Knüchel R, Dahl E (2013) Promoter hypermethylation of the tumor-suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening. Breast Cancer Res 15:R4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lagathu C, Christodoulides C, Tan CY, Virtue S, Laudes M, Campbell M, Ishikawa K, Ortega F, Tinahones FJ, Fernández-Real JM, Orešič M, Sethi JK, Vidal-Puig A (2010) Secreted frizzled-related protein 1 regulates adipose tissue expansion and is dysregulated in severe obesity. Int J Obes 34:1695–1705

    Article  CAS  Google Scholar 

  • Lee WJ, Park JH, Shin JU, Noh H, Lew DH, Yang WI, Yun CO, Lee KH, Lee JH (2015) Endothelial-to-mesenchymal transition induced by Wnt 3a in keloid pathogenesis. Wound Repair Regen 23:435–442

    Article  PubMed  Google Scholar 

  • Li Y, Zhang J, Zhang W, Liu Y, Li Y, Wang K, Zhang Y, Yang C, Li X, Shi J, Su L, Hu D (2017) MicroRNA-192 regulates hypertrophic scar fibrosis by targeting SIP1. J Mol Histol 48:357–366

    Article  CAS  PubMed  Google Scholar 

  • Lien WH, Fuchs E (2014) Wnt some lose some: transcriptional governance of stem cells by Wnt/β-catenin signaling. Genes Dev 28:1517–1532

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mun JH, Kim YM, Kim BS, Kim JH, Kim MB, Ko HC (2014) Simvastatin inhibits transforming growth factor-β1-induced expression of type I collagen, CTGF, and α-SMA in keloid fibroblasts. Wound Repair Regen 22:125–133

    Article  PubMed  Google Scholar 

  • Ogunbiyi A (2016) Acne keloidalis nuchae: prevalence, impact, and management challenges. Clin Cosmet Investig Dermatol 9:483–489

    Article  PubMed  PubMed Central  Google Scholar 

  • Otterson GA, Khleif SN, Chen W, Coxon AB, Kaye FJ (1995) CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2′deoxycytidine. Oncogene 11:1211–1216

    CAS  PubMed  Google Scholar 

  • Polakis P (2000) Wnt signaling and cancer. Genes Dev 14:1837–1851

    CAS  PubMed  Google Scholar 

  • Rodriguez J, Esteve P, Weinl C, Ruiz JM, Fermin Y, Trousse F, Dwivedy A, Holt C, Bovolenta P (2005) SFRP1 regulates the growth of retinal ganglion cell axons through the Fz2 receptor. Nat Neurosci 8:1301–1309

    Article  CAS  PubMed  Google Scholar 

  • Roh MR, Kumar R, Rajadurai A, Njauw C, Ryoo UH, Chung KY, Tsao H (2017) Beta-catenin causes fibrotic changes in the extracellular matrix via upregulation of collagen I transcription. Br J Dermatol 177:312–315

    Article  CAS  PubMed  Google Scholar 

  • Sato M (2006) Upregulation of the Wnt/beta-catenin pathway induced by transforming growth factor-beta in hypertrophic scars and keloids. Acta Derm Venereol 86:300–307

    Article  CAS  PubMed  Google Scholar 

  • Serizawa RR, Ralfkiaer U, Steven K, Lam GW, Schmiedel S, Schüz J, Hansen AB, Horn T, Guldberg P (2011) Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events. Int J Cancer 129:78–87

    Article  CAS  PubMed  Google Scholar 

  • Sideek MA, Teia A, Kopecki Z, Cowin AJ, Gibson MA (2016) Co-localization of LTBP-2 with FGF-2 in fibrotic human keloid and hypertrophic scar. J Mol Histol 47:35–45

    Article  CAS  PubMed  Google Scholar 

  • Sun XJ, Wang Q, Guo B, Liu XY, Wang B (2017) Identification of skin-related lncRNAs as potential biomarkers that involved in Wnt pathways in keloids. Oncotarget 8:34236–34244

    PubMed  PubMed Central  Google Scholar 

  • Svensson A, Norrby M, Libelius R, Tågerud S (2008) Secreted frizzled related protein 1 (Sfrp1) and Wnt signaling in innervated and denervated skeletal muscle. J Mol Histol 39:329–337

    Article  CAS  PubMed  Google Scholar 

  • Valenzuela Salas I, Fernández Miralbell A (2015) Images in clinical medicine. Incisional keloid. N Engl J Med 372:1453

    Article  PubMed  Google Scholar 

  • Vallée A, Lecarpentier Y, Guillevin R, Vallée JN (2017) Interactions between TGF-β1, canonical WNT/β-catenin pathway and PPAR γ in radiation-induced fibrosis. Oncotarget 8:90579–90604

    Article  PubMed  PubMed Central  Google Scholar 

  • Veeck J, Niederacher D, An H, Klopocki E, Wiesmann F, Betz B, Galm O, Camara O, Dürst M, Kristiansen G, Huszka C, Knüchel R, Dahl E (2006) Aberrant methylation of the Wnt antagonist SFRP1 in breast cancer is associated with unfavourable prognosis. Oncogene 25:3479–3488

    Article  CAS  PubMed  Google Scholar 

  • Xie Q, Chen L, Shan X, Shan X, Tang J, Zhou F, Chen Q, Quan H, Nie D, Zhang W, Huang AL, Tang N (2014) Epigenetic silencing of SFRP1 and SFRP5 by hepatitis B virus X protein enhances hepatoma cell tumorigenicity through Wnt signaling pathway. Int J Cancer 135:635–646

    Article  CAS  PubMed  Google Scholar 

  • Yang L, Zheng Z, Zhou Q, Bai X, Fan L, Yang C, Su L, Hu D (2017) miR-155 promotes cutaneous wound healing through enhanced keratinocytes migration by MMP-2. J Mol Histol 48:147–155

    Article  CAS  PubMed  Google Scholar 

  • Yang L, Liu L, Ying H, Yu Y, Zhang D, Deng H, Zhang H, Chai J (2018) Acute downregulation of miR-155 leads to a reduced collagen synthesis through attenuating macrophages inflammatory factor secretion by targeting SHIP1. J Mol Histol. https://doi.org/10.1007/s10735-018-9756-5 (Epub ahead of print)

    Google Scholar 

  • Yasuniwa Y, Izumi H, Wang KY, Shimajiri S, Sasaguri Y, Kawai K, Kasai H, Shimada T, Miyake K, Kashiwagi E, Hirano G, Kidani A, Akiyama M, Han B, Wu Y, Ieiri I, Higuchi S, Kohno K (2010) Circadian disruption accelerates tumor growth and angio/stromagenesis through a Wnt signaling pathway. PLoS ONE 5:e15330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu D, Shang Y, Yuan J, Ding S, Luo S, Hao L (2016) Wnt/β-catenin signaling exacerbates keloid cell proliferation by regulating telomerase. Cell Physiol Biochem 39:2001–2013

    Article  CAS  PubMed  Google Scholar 

  • Yuan YP, Huang K, Xu YM, Chen XC, Li HH, Cai BZ, Liu Y, Zhang H, Li Y, Lin CM (2016) Canonical and non-canonical Wnt signaling control the regeneration of amputated rodent vibrissae follicles. J Mol Histol 47:1–8

    Article  CAS  PubMed  Google Scholar 

  • Zhao B, Zhang Y, Han S, Zhang W, Zhou Q, Guan H, Liu J, Shi J, Su L, Hu D (2017) Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation. J Mol Histol 48:121–132

    Article  CAS  PubMed  Google Scholar 

  • Zhou M, Guo S, Yuan L, Zhang Y, Zhang M, Chen H, Lu M, Yang J, Ma J (2017) Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae. J Mol Histol 48:389–401

    Article  CAS  PubMed  Google Scholar 

  • Zhu HY, Bai WD, Li C, Zheng Z, Guan H, Liu JQ, Yang XK, Han SC, Gao JX, Wang HT, Hu DH (2016) Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-β2 by targeting ZNF217 via miR-200c in keloid fibroblasts. Sci Rep 6:24728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by National Natural Science Foundation of China (Nos. 81201470, 81772071, 81372069), Natural Science Foundation of Shaanxi Province (Nos. 2017JQ8031, 2014KTCL03-09) and China Postdoctoral Science Foundation (No. 2014M562600).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Zhao Zheng or Dahai Hu.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interest in this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, J., Zhu, H., Wang, H. et al. Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids. J Mol Hist 49, 185–193 (2018). https://doi.org/10.1007/s10735-018-9758-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10735-018-9758-3

Keywords

Navigation