Dna methylation regulates the expression of cxcl12 in rheumatoid arthritis synovial fibroblasts

ABSTRACT In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the

methylation status of its promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite

sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and

stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased

the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we

observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was

expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs.

Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction. Access through your institution Buy or subscribe This is a preview of subscription content,

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12 May 2025 REFERENCES * Ospelt C, Gay S . The role of resident synovial cells in destructive arthritis. _Best Pract Res Clin Rheumatol_ 2008; 22: 239–252. Article  CAS  Google Scholar  *

Karouzakis E, Gay RE, Michel BA, Gay S, Neidhart M . DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. _Arthritis Rheum_ 2009; 60: 3613–3622. Article  CAS  Google Scholar  *

Wade PA . Methyl CpG-binding proteins and transcriptional repression. _Bioessays_ 2001; 23: 1131–1137. Article  CAS  Google Scholar  * Reik W, Dean W, Walter J . Epigenetic reprogramming in

mammalian development. _Science_ 2001; 293: 1089–1093. Article  CAS  Google Scholar  * Hewagama A, Richardson B . The genetics and epigenetics of autoimmune diseases. _J Autoimmun_ 2009; 33:

3–11. Article  CAS  Google Scholar  * Esteller M . Epigenetics in cancer. _N Engl J Med_ 2008; 358: 1148–1159. Article  CAS  Google Scholar  * Petit I, Jin D, Rafii S . The SDF-1-CXCR4

signaling pathway: a molecular hub modulating neo-angiogenesis. _Trends Immunol_ 2007; 28: 299–307. Article  CAS  Google Scholar  * Kucia M, Jankowski K, Reca R, Wysoczynski M, Bandura L,

Allendorf DJ _et al_. CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion. _J Mol Histol_ 2004; 35: 233–245. Article  CAS  Google Scholar  * Burns JM, Summers BC, Wang Y, Melikian A,

Berahovich R, Miao Z _et al_. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. _J Exp Med_ 2006; 203: 2201–2213. Article  CAS

  Google Scholar  * Raggo C, Ruhl R, McAllister S, Koon H, Dezube BJ, Fruh K _et al_. Novel cellular genes essential for transformation of endothelial cells by Kaposi's

sarcoma-associated herpesvirus. _Cancer Res_ 2005; 65: 5084–5095. Article  CAS  Google Scholar  * Miao Z, Luker KE, Summers BC, Berahovich R, Bhojani MS, Rehemtulla A _et al_. CXCR7 (RDC1)

promotes breast and lung tumor growth _in vivo_ and is expressed on tumor-associated vasculature. _Proc Natl Acad Sci USA_ 2007; 104: 15735–15740. Article  CAS  Google Scholar  * De Paepe B,

Schroder JM, Martin JJ, Racz GZ, De Bleecker JL . Localization of the alpha-chemokine SDF-1 and its receptor CXCR4 in idiopathic inflammatory myopathies. _Neuromuscul Disord_ 2004; 14:

265–273. Article  Google Scholar  * Gu J, Marker-Hermann E, Baeten D, Tsai WC, Gladman D, Xiong M _et al_. A 588-gene microarray analysis of the peripheral blood mononuclear cells of

spondyloarthropathy patients. _Rheumatology (Oxford)_ 2002; 41: 759–766. Article  CAS  Google Scholar  * Krumbholz M, Theil D, Cepok S, Hemmer B, Kivisakk P, Ransohoff RM _et al_. Chemokines

in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment. _Brain_ 2006; 129 (part 1): 200–211. Article  Google Scholar  * Kryczek I,

Wei S, Keller E, Liu R, Zou W . Stroma-derived factor (SDF-1/CXCL12) and human tumor pathogenesis. _Am J Physiol Cell Physiol_ 2007; 292: C987–C995. Article  CAS  Google Scholar  * Kanbe K,

Takagishi K, Chen Q . Stimulation of matrix metalloprotease 3 release from human chondrocytes by the interaction of stromal cell-derived factor 1 and CXC chemokine receptor 4. _Arthritis

Rheum_ 2002; 46: 130–137. Article  CAS  Google Scholar  * Nanki T, Hayashida K, El-Gabalawy HS, Suson S, Shi K, Girschick HJ _et al_. Stromal cell-derived factor-1-CXC chemokine receptor 4

interactions play a central role in CD4+ T cell accumulation in rheumatoid arthritis synovium. _J Immunol_ 2000; 165: 6590–6598. Article  CAS  Google Scholar  * Burger JA, Zvaifler NJ,

Tsukada N, Firestein GS, Kipps TJ . Fibroblast-like synoviocytes support B-cell pseudoemperipolesis via a stromal cell-derived factor-1- and CD106 (VCAM-1)-dependent mechanism. _J Clin

Invest_ 2001; 107: 305–315. Article  CAS  Google Scholar  * Nanki T, Nagasaka K, Hayashida K, Saita Y, Miyasaka N . Chemokines regulate IL-6 and IL-8 production by fibroblast-like

synoviocytes from patients with rheumatoid arthritis. _J Immunol_ 2001; 167: 5381–5385. Article  CAS  Google Scholar  * Blades MC, Ingegnoli F, Wheller SK, Manzo A, Wahid S, Panayi GS _et

al_. Stromal cell-derived factor 1 (CXCL12) induces monocyte migration into human synovium transplanted onto SCID Mice. _Arthritis Rheum_ 2002; 46: 824–836. Article  CAS  Google Scholar  *

Watanabe N, Ando K, Yoshida S, Inuzuka S, Kobayashi M, Matsui N _et al_. Gene expression profile analysis of rheumatoid synovial fibroblast cultures revealing the overexpression of genes

responsible for tumor-like growth of rheumatoid synovium. _Biochem Biophys Res Commun_ 2002; 294: 1121–1129. Article  CAS  Google Scholar  * Grassi F, Cristino S, Toneguzzi S, Piacentini A,

Facchini A, Lisignoli G . CXCL12 chemokine up-regulates bone resorption and MMP-9 release by human osteoclasts: CXCL12 levels are increased in synovial and bone tissue of rheumatoid

arthritis patients. _J Cell Physiol_ 2004; 199: 244–251. Article  CAS  Google Scholar  * Garcia-Vicuna R, Gomez-Gaviro MV, Dominguez-Luis MJ, Pec MK, Gonzalez-Alvaro I, Alvaro-Gracia JM _et

al_. CC and CXC chemokine receptors mediate migration, proliferation, and matrix metalloproteinase production by fibroblast-like synoviocytes from rheumatoid arthritis patients. _Arthritis

Rheum_ 2004; 50: 3866–3877. Article  CAS  Google Scholar  * Kanbe K, Takemura T, Takeuchi K, Chen Q, Takagishi K, Inoue K . Synovectomy reduces stromal-cell-derived factor-1 (SDF-1) which is

involved in the destruction of cartilage in osteoarthritis and rheumatoid arthritis. _J Bone Joint Surg Br_ 2004; 86: 296–300. Article  CAS  Google Scholar  * Kondo Y, Issa JP . Epigenetic

changes in colorectal cancer. _Cancer Metastasis Rev_ 2004; 23: 29–39. Article  CAS  Google Scholar  * Lin CH, Hsieh SY, Sheen IS, Lee WC, Chen TC, Shyu WC _et al_. Genome-wide

hypomethylation in hepatocellular carcinogenesis. _Cancer Res_ 2001; 61: 4238–4243. CAS  PubMed  Google Scholar  * Herman JG, Baylin SB . Gene silencing in cancer in association with

promoter hypermethylation. _N Engl J Med_ 2003; 349: 2042–2054. Article  CAS  Google Scholar  * Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E _et al_. Analysis of repetitive

element DNA methylation by MethyLight. _Nucleic Acids Res_ 2005; 33: 6823–6836. Article  CAS  Google Scholar  * Roman-Gomez J, Jimenez-Velasco A, Agirre X, Cervantes F, Sanchez J, Garate L

_et al_. Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia. _Oncogene_ 2005; 24:

7213–7223. Article  CAS  Google Scholar  * Richardson B . Primer: epigenetics of autoimmunity. _Nat Clin Pract Rheumatol_ 2007; 3: 521–527. Article  CAS  Google Scholar  * Lu Q, Wu A,

Richardson BC . Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs. _J Immunol_ 2005; 174: 6212–6219.

Article  CAS  Google Scholar  * Kaplan MJ, Lu Q, Wu A, Attwood J, Richardson B . Demethylation of promoter regulatory elements contributes to perforin overexpression in CD4+ lupus T cells.

_J Immunol_ 2004; 172: 3652–3661. Article  CAS  Google Scholar  * Oelke K, Lu Q, Richardson D, Wu A, Deng C, Hanash S _et al_. Overexpression of CD70 and overstimulation of IgG synthesis by

lupus T cells and T cells treated with DNA methylation inhibitors. _Arthritis Rheum_ 2004; 50: 1850–1860. Article  CAS  Google Scholar  * Calonge E, Alonso-Lobo JM, Escandon C, Gonzalez N,

Bermejo M, Santiago B _et al_. c/EBPbeta is a major regulatory element driving transcriptional activation of the CXCL12 promoter. _J Mol Biol_ 2010; 396: 463–472. Article  CAS  Google

Scholar  * Ceradini DJ, Gurtner GC . Homing to hypoxia: HIF-1 as a mediator of progenitor cell recruitment to injured tissue. _Trends Cardiovasc Med_ 2005; 15: 57–63. Article  CAS  Google

Scholar  * Garcia-Moruja C, Alonso-Lobo JM, Rueda P, Torres C, Gonzalez N, Bermejo M _et al_. Functional characterization of SDF-1 proximal promoter. _J Mol Biol_ 2005; 348: 43–62. Article 

CAS  Google Scholar  * Wendt MK, Johanesen PA, Kang-Decker N, Binion DG, Shah V, Dwinell MB . Silencing of epithelial CXCL12 expression by DNA hypermethylation promotes colonic carcinoma

metastasis. _Oncogene_ 2006; 25: 4986–4997. Article  CAS  Google Scholar  * Wendt MK, Cooper AN, Dwinell MB . Epigenetic silencing of CXCL12 increases the metastatic potential of mammary

carcinoma cells. _Oncogene_ 2008; 27: 1461–1471. Article  CAS  Google Scholar  * Pietkiewicz PP, Lutkowska A, Lianeri M, Jagodzinski PP . Tamoxifen epigenetically modulates CXCL12 expression

in MCF-7 breast cancer cells. _Biomed Pharmacother_ 2010; 64: 54–57. Article  CAS  Google Scholar  * Bradfield PF, Amft N, Vernon-Wilson E, Exley AE, Parsonage G, Rainger GE _et al_.

Rheumatoid fibroblast-like synoviocytes overexpress the chemokine stromal cell-derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within

synovial tissue. _Arthritis Rheum_ 2003; 48: 2472–2482. Article  CAS  Google Scholar  * del Rey MJ, Izquierdo E, Caja S, Usategui A, Santiago B, Galindo M _et al_. Human inflammatory

synovial fibroblasts induce enhanced myeloid cell recruitment and angiogenesis through a hypoxia-inducible transcription factor 1alpha/vascular endothelial growth factor-mediated pathway in

immunodeficient mice. _Arthritis Rheum_ 2009; 60: 2926–2934. Article  CAS  Google Scholar  * Pablos JL, Santiago B, Galindo M, Torres C, Brehmer MT, Blanco FJ _et al_. Synoviocyte-derived

CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis. _J Immunol_ 2003; 170: 2147–2152. Article  CAS  Google Scholar  * Sato N, Matsubayashi H, Fukushima N,

Goggins M . The chemokine receptor CXCR4 is regulated by DNA methylation in pancreatic cancer. _Cancer Biol Ther_ 2005; 4: 70–76. Article  CAS  Google Scholar  * Law NM, Rosenzweig SA .

Characterization of the G-protein linked orphan receptor GPRN1/RDC1. _Biochem Biophys Res Commun_ 1994; 201: 458–465. Article  CAS  Google Scholar  * Balabanian K, Lagane B, Infantino S,

Chow KY, Harriague J, Moepps B _et al_. The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. _J Biol Chem_ 2005; 280: 35760–35766. Article  CAS

  Google Scholar  * Infantino S, Moepps B, Thelen M . Expression and regulation of the orphan receptor RDC1 and its putative ligand in human dendritic and B cells. _J Immunol_ 2006; 176:

2197–2207. Article  CAS  Google Scholar  * Zabel BA, Wang Y, Lewen S, Berahovich RD, Penfold ME, Zhang P _et al_. Elucidation of CXCR7-mediated signaling events and inhibition of

CXCR4-mediated tumor cell transendothelial migration by CXCR7 ligands. _J Immunol_ 2009; 183: 3204–3211. Article  CAS  Google Scholar  * Kalatskaya I, Berchiche YA, Gravel S, Limberg BJ,

Rosenbaum JS, Heveker N . AMD3100 is a CXCR7 ligand with allosteric agonist properties. _Mol Pharmacol_ 2009; 75: 1240–1247. Article  CAS  Google Scholar  * Rajagopal S, Kim J, Ahn S, Craig

S, Lam CM, Gerard NP _et al_. Beta-arrestin- but not G protein-mediated signaling by the ″decoy″ receptor CXCR7. _Proc Natl Acad Sci USA_ 2010; 107: 628–632. Article  Google Scholar  *

Gurevich EV, Gurevich VV . Arrestins: ubiquitous regulators of cellular signaling pathways. _Genome Biol_ 2006; 7: 236. Article  Google Scholar  * Konttinen YT, Ainola M, Valleala H, Ma J,

Ida H, Mandelin J _et al_. Analysis of 16 different matrix metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and rheumatoid arthritis. _Ann Rheum

Dis_ 1999; 58: 691–697. Article  CAS  Google Scholar  * Jungel A, Ospelt C, Lesch M, Thiel M, Sunyer T, Schorr O _et al_. Effect of the oral application of a highly selective MMP-13

inhibitor in three different animal models of rheumatoid arthritis. _Ann Rheum Dis_ 2010; 69: 898–902. Article  CAS  Google Scholar  * Rutkauskaite E, Zacharias W, Schedel J, Muller-Ladner

U, Mawrin C, Seemayer CA _et al_. Ribozymes that inhibit the production of matrix metalloproteinase 1 reduce the invasiveness of rheumatoid arthritis synovial fibroblasts. _Arthritis Rheum_

2004; 50: 1448–1456. Article  CAS  Google Scholar  * Iliopoulos D, Malizos KN, Tsezou A . Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible

molecular target for osteoarthritis therapeutic intervention. _Ann Rheum Dis_ 2007; 66: 1616–1621. Article  CAS  Google Scholar  * Roach HI, Yamada N, Cheung KS, Tilley S, Clarke NM, Oreffo

RO _et al_. Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions.

_Arthritis Rheum_ 2005; 52: 3110–3124. Article  CAS  Google Scholar  * Couillard J, Demers M, Lavoie G, St-Pierre Y . The role of DNA hypomethylation in the control of stromelysin gene

expression. _Biochem Biophys Res Commun_ 2006; 342: 1233–1239. Article  CAS  Google Scholar  * Ogata Y, Enghild JJ, Nagase H . Matrix metalloproteinase 3 (stromelysin) activates the

precursor for the human matrix metalloproteinase 9. _J Biol Chem_ 1992; 267: 3581–3584. CAS  PubMed  Google Scholar  * Goldberg GI, Strongin A, Collier IE, Genrich LT, Marmer BL .

Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the

proenzyme with stromelysin. _J Biol Chem_ 1992; 267: 4583–4591. CAS  PubMed  Google Scholar  * Benbow U, Brinckerhoff CE . The AP-1 site and MMP gene regulation: what is all the fuss about?

_Matrix Biol_ 1997; 15: 519–526. Article  CAS  Google Scholar  * Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS _et al_. The American Rheumatism Association 1987 revised

criteria for the classification of rheumatoid arthritis. _Arthritis Rheum_ 1988; 31: 315–324. Article  CAS  Google Scholar  * Ospelt C, Kurowska-Stolarska M, Neidhart M, Michel BA, Gay RE,

Laufer S _et al_. The dual inhibitor of lipoxygenase and cyclooxygenase ML3000 decreases the expression of CXCR3 ligands. _Ann Rheum Dis_ 2007; 67: 524–529. Article  Google Scholar  * Li LC,

Dahiya R . MethPrimer: designing primers for methylation PCRs. _Bioinformatics_ 2002; 18: 1427–1431. Article  CAS  Google Scholar  * Bock C, Reither S, Mikeska T, Paulsen M, Walter J,

Lengauer T . BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. _Bioinformatics_ 2005; 21: 4067–4068. Article  CAS  Google Scholar  * Distler

JH, Jungel A, Huber LC, Seemayer CA, Reich III CF, Gay RE _et al_. The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell

microparticles. _Proc Natl Acad Sci USA_ 2005; 102: 2892–2897. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Mrs Maria Comazzi and Mr Ferenc Pataky for their

excellent technical work. We also thank our collaborators Professor C Buckley and Dr A Filer for providing us with the human lung fibroblast cultures. This work was supported by the

Articulum Fellowship Program (YR), the Institute of Arthritis Research, Epilanges, Switzerland, and by the European Community's FP6 (Autocure) and FP7 funding (Masterswitch). This

publication reflects only the author's views. The European Community is not liable for any use that may be made of the information herein. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Center of Experimental Rheumatology and Zurich Center of Integrative Human Physiology (ZIHP), University Hospital Zurich, Zurich, Switzerland E Karouzakis, Y Rengel, A Jüngel, R E Gay, B A

Michel, S Gay, M Neidhart & C Ospelt * Schulthess Clinic, Zurich, Switzerland C Kolling * Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of

Amsterdam, Amsterdam, The Netherlands P P Tak & C Ospelt Authors * E Karouzakis View author publications You can also search for this author inPubMed Google Scholar * Y Rengel View

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View author publications You can also search for this author inPubMed Google Scholar * R E Gay View author publications You can also search for this author inPubMed Google Scholar * B A

Michel View author publications You can also search for this author inPubMed Google Scholar * P P Tak View author publications You can also search for this author inPubMed Google Scholar * S

Gay View author publications You can also search for this author inPubMed Google Scholar * M Neidhart View author publications You can also search for this author inPubMed Google Scholar *

C Ospelt View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to E Karouzakis. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no conflict of interest. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Karouzakis, E., Rengel, Y., Jüngel, A. _et al._ DNA methylation

regulates the expression of CXCL12 in rheumatoid arthritis synovial fibroblasts. _Genes Immun_ 12, 643–652 (2011). https://doi.org/10.1038/gene.2011.45 Download citation * Received: 20

January 2011 * Revised: 03 May 2011 * Accepted: 01 June 2011 * Published: 14 July 2011 * Issue Date: December 2011 * DOI: https://doi.org/10.1038/gene.2011.45 SHARE THIS ARTICLE Anyone you

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Springer Nature SharedIt content-sharing initiative KEYWORDS * CXCL12 * rheumatoid arthritis synovial fibroblasts * epigenetics * DNA methylation