Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts

Cellular & Molecular Immunology volume 19, pages 1185–1195 (2022)Cite this article

Subjects

Abstract

Extracellular sulfatase-2 (Sulf-2) influences receptor–ligand binding and subsequent signaling by chemokines and growth factors, yet Sulf-2 remains unexplored in inflammatory cytokine signaling in the context of rheumatoid arthritis (RA). In the present study, we characterized Sulf-2 expression in RA and investigated its potential role in TNF-α-induced synovial inflammation using primary human RA synovial fibroblasts (RASFs). Sulf-2 expression was significantly higher in serum and synovial tissues from patients with RA and in synovium and serum from hTNFtg mice. RNA sequencing analysis of TNF-α-stimulated RASFs showed that Sulf-2 siRNA modulated ~2500 genes compared to scrambled siRNA. Ingenuity Pathway Analysis of RNA sequencing data identified Sulf-2 as a primary target in fibroblasts and macrophages in RA. Western blot, ELISA, and qRT‒PCR analyses confirmed that Sulf-2 knockdown reduced the TNF-α-induced expression of ICAM1, VCAM1, CAD11, PDPN, CCL5, CX3CL1, CXCL10, and CXCL11. Signaling studies identified the protein kinase C-delta (PKCδ) and c-Jun N-terminal kinase (JNK) pathways as key in the TNF-α-mediated induction of proteins related to cellular adhesion and invasion. Knockdown of Sulf-2 abrogated TNF-α-induced RASF proliferation. Sulf-2 knockdown with siRNA and inhibition by OKN-007 suppressed the TNF-α-induced phosphorylation of PKCδ and JNK, thereby suppressing the nuclear translocation and DNA binding activity of the transcription factors AP-1 and NF-κBp65 in human RASFs. Interestingly, Sulf-2 expression positively correlated with the expression of TNF receptor 1, and coimmunoprecipitation assays demonstrated the binding of these two proteins, suggesting they exhibit crosstalk in TNF-α signaling. This study identified a novel role of Sulf-2 in TNF-α signaling and the activation of RA synoviocytes, providing the rationale for evaluating the therapeutic targeting of Sulf-2 in preclinical models of RA.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Puimège L, Libert C, Van, Hauwermeiren F. Regulation and dysregulation of tumor necrosis factor receptor-1. Cytokine Growth Factor Rev. 2014;25:285–300.

    Article  PubMed  Google Scholar 

  2. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell. 2001;104:487–501.

    Article  CAS  PubMed  Google Scholar 

  3. Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010;233:233–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lowenthal JW, Ballard DW, Bohnlein E, Greene WC. Tumor necrosis factor alpha induces proteins that bind specifically to kappa B-like enhancer elements and regulate interleukin 2 receptor alpha-chain gene expression in primary human T lymphocytes. Proc Natl Acad Sci USA. 1989;86:2331–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Roach DR, Bean AG, Demangel C, France MP, Briscoe H, Britton WJ. TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection. J Immunol. 2002;168:4620–7.

    Article  CAS  PubMed  Google Scholar 

  6. Asif Amin M, Fox DA, Ruth JH. Synovial cellular and molecular markers in rheumatoid arthritis. Semin Immunopathol. 2017;39:385–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annu Rev Immunol. 2001;19:163–96.

    Article  CAS  PubMed  Google Scholar 

  8. Gerriets V, Bansal P, Goyal A, Khaddour K. Tumor necrosis factor inhibitors, in StatPearls. Treasure Island (FL): StatPearls; 2021.

  9. El Masri R, Seffouh A, Lortat-Jacob H, Vives RR. The “in and out” of glucosamine 6- O-sulfation: the 6th sense of heparan sulfate. Glycoconj J. 2017;34:285–98.

    Article  CAS  PubMed  Google Scholar 

  10. Rosen SD, Lemjabbar-Alaoui H. Sulf-2: an extracellular modulator of cell signaling and a cancer target candidate. Expert Opin Ther Targets. 2010;14:935–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Uchimura K, Morimoto-Tomita M, Bistrup A, Li J, Lyon M, Gallagher J, et al. HSulf- 2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparinbound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1. BMC Biochem. 2006;7:2.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Zheng X, Gai X, Han S, Moser CD, Hu C, Shire AM, et al. The human sulfatase 2inhibitor 2,4-disulfonylphenyl-tert-butylnitrone (OKN-007) has an antitumor effect in hepatocellular carcinoma mediated via suppression of TGFB1/SMAD2 and Hedgehog/GLI1 signaling. Genes Chromosomes Cancer. 2013;52:225–36.

    Article  CAS  PubMed  Google Scholar 

  13. Towner RA, Hocker J, Smith N, Saunders D, Battiste J, Hanas J. OKN-007 Alters Protein Expression Profiles in High-Grade Gliomas: Mass Spectral Analysis of Blood Sera. Brain Sci. 2022;12:100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. El Masri R, Crétinon Y, Gout E, Vivès RR. HS and inflammation: a potential playground for the Sulfs? Front Immunol. 2020;11:570.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Otsuki S, Taniguchi N, Grogan SP, D'Lima D, Kinoshita M, Lotz M. Expression of novel extracellular sulfatases Sulf-1 and Sulf-2 in normal and osteoarthritic articular cartilage. Arthritis Res Ther. 2008;10:R61.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Ratzka A, Kalus I, Moser M, Dierks T, Mundlos S, Vortkamp A. Redundant function of the heparan sulfate 6-O-endosulfatases Sulf1 and Sulf2 during skeletal development. Dev Dyn. 2008;237:339–53.

    Article  CAS  PubMed  Google Scholar 

  17. Zaman G, Staines KA, Farquharson C, Newton PT, Dudhia J, Chenu C, et al. Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing. Histochem Cell Biol. 2016;145:67–79.

    Article  CAS  PubMed  Google Scholar 

  18. Bell RD, Wu EK, Rudmann CA, Forney M, Kaiser CRW, Wood RW, et al. Selective sexual dimorphisms in musculoskeletal and cardiopulmonary pathologic manifestations and mortality incidence in the tumor necrosis factor-transgenic mouse model of rheumatoid arthritis. Arthritis Rheumatol. 2019;71:1512–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li P, Schwarz EM. The TNF-alpha transgenic mouse model of inflammatory arthritis. Springer Semin Immunopathol. 2003;25:19–33.

    Article  PubMed  Google Scholar 

  20. Tsai C, Diaz LA Jr., Singer NG, Li LL, Kirsch AH, Mitra R, et al. Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes. Arthritis Rheum. 1996;39:125–36.

    Article  CAS  PubMed  Google Scholar 

  21. Haque M, Singh AK, Ouseph MM, Ahmed S. Regulation of synovial inflammation and tissue destruction by guanylate binding protein 5 in synovial fibroblasts from patients with rheumatoid arthritis and rats with adjuvant-induced arthritis. Arthritis Rheumatol. 2021;73:943–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. McCarthy DJ, Chen Y, Smyth GK. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012;40:4288–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11:R25.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, et al. Metascape provides a biologist-oriented resource for the analysis of systemslevel datasets. Nat Commun. 2019;10:1523.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Babicki S, Arndt D, Marcu A, Liang Y, Grant JR, Maciejewski A, et al. Heatmapper: web-enabled heat mapping for all. Nucleic Acids Res. 2016;44:W147–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Singer MS, Phillips JJ, Lemjabbar-Alaoui H, Wang YQ, Wu J, Goldman R, et al. SULF2, a heparan sulfate endosulfatase, is present in the blood of healthy individuals and increases in cirrhosis. Clin Chim Acta. 2015;440:72–8.

    Article  CAS  PubMed  Google Scholar 

  27. Ahmed S, Pakozdi A, Koch AE. Regulation of interleukin-1beta-induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3- gallate in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 2006;54:2393–401.

    Article  CAS  PubMed  Google Scholar 

  28. Redl H, Schlag G, Adolf GR, Natmessnig B, Davies J. Tumor necrosis factor (TNF)- dependent shedding of the p55 TNF receptor in a baboon model of bacteremia. Infect Immun. 1995;63:297–300.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lantz M, Malik S, Slevin ML, Olsson I. Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF-binding protein in humans. Cytokine. 1990;2:402–6.

    Article  CAS  PubMed  Google Scholar 

  30. Hawari FI, Rouhani FN, Cui X, Yu ZX, Buckley C, Kaler M, et al. Release of fulllength 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors. Proc Natl Acad Sci USA. 2004;101:1297–302.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bradley JR. TNF-mediated inflammatory disease. J Pathol. 2008;214:149–60.

    Article  CAS  PubMed  Google Scholar 

  32. Mercogliano MF, Bruni S, Mauro F, Elizalde PV, Schillaci R. Harnessing tumor necrosis factor alpha to achieve effective cancer immunotherapy. Cancers. 2021;13:564.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: at the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014;1843:2563–82.

    Article  CAS  PubMed  Google Scholar 

  34. Sabir JSM, El Omri A, Banaganapalli B, Al-Shaeri MA, Alkenani NA, Sabir MJ, et al. Dissecting the role of NF-κb protein family and its regulators in rheumatoid arthritis using weighted gene co-expression network. Front Genet. 2019;10:1163.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lanzi C, Zaffaroni N, Cassinelli G. Targeting heparan sulfate proteoglycans and their modifying enzymes to enhance anticancer chemotherapy efficacy and overcome drug resistance. Curr Med Chem. 2017;24:2860–86.

    Article  CAS  PubMed  Google Scholar 

  36. Reine TM, Kusche-Gullberg M, Feta A, Jenssen T, Kolset SO. Heparan sulfate expression is affected by inflammatory stimuli in primary human endothelial cells. Glycoconj J. 2012;29:67–76.

    Article  CAS  PubMed  Google Scholar 

  37. Sikora AS, Hellec C, Carpentier M, Martinez P, Delos M, Denys A, et al. Tumournecrosis factor-α induces heparan sulfate 6-O-endosulfatase 1 (Sulf-1) expression in fibroblasts. Int J Biochem Cell Biol. 2016;80:57–65.

    Article  CAS  PubMed  Google Scholar 

  38. Alhasan AA, Spielhofer J, Kusche-Gullberg M, Kirby JA, Ali S. Role of 6-Osulfated heparan sulfate in chronic renal fibrosis. J Biol Chem. 2014;289:20295–306.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yue X, Li X, Nguyen HT, Chin DR, Sullivan DE, Lasky JA. Transforming growth factor-beta1 induces heparan sulfate 6-O-endosulfatase 1 expression in vitro and in vivo. J Biol Chem. 2008;283:20397–407.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kim HJ, Kim HS, Hong YH. Sulfatase 1 and sulfatase 2 as novel regulators of macrophage antigen presentation and phagocytosis. Yeungnam Univ J Med. 2021;38:326–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yang Y, Ahn J, Raghunathan R, Kallakury BV, Davidson B, Kennedy ZB, et al. Expression of the extracellular sulfatase SULF2 affects survival of head and neck squamous cell carcinoma patients. Front Oncol. 2020;10:582827.

    Article  PubMed  Google Scholar 

  42. Flowers SA, Zhou X, Wu J, Wang Y, Makambi K, Kallakury BV, et al. Expression of the extracellular sulfatase SULF2 is associated with squamous cell carcinoma of the head and neck. Oncotarget. 2016;7:43177–87.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Allen CT, Ricker JL, Chen Z, Van Waes C. Role of activated nuclear factor-kappaB in the pathogenesis and therapy of squamous cell carcinoma of the head and neck. Head Neck. 2007;29:959–71.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the Cooperative Human Tissue Network and National Disease Research Interchange for providing human synovial tissue specimens.

Funding

This study was supported by the NIH/NIAMS F31 Fellowship AR-076204–01 (RJS), Rheumatology Research Foundation Graduate Student Preceptorship Award (RJS/SA), and NIH/NIAMS R01 Grant AR-072615 (SA). Research by SUH and JV was made possible through the WSU College of Pharmacy and Pharmaceutical Sciences Honors Research Program.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, WA, USA

    Ruby J. Siegel, Anil K. Singh, Paul M. Panipinto, Farheen S. Shaikh, Judy Vinh, Sang U. Han & Salahuddin Ahmed

  2. Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA

    H. Mark Kenney & Edward M. Schwarz

  3. Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY, USA

    H. Mark Kenney & Edward M. Schwarz

  4. Department of Quantitative Health Sciences and Division of Rheumatology, Mayo Clinic, Rochester, MN, USA

    Cynthia S. Crowson

  5. Department of Medicine and Public Health, University of Toledo, Toledo, OH, USA

    Sadik A. Khuder

  6. Department of Pathology and Laboratory Medicine, Phoenix Children’s Hospital, Phoenix, AZ, USA

    Basil S. Khuder

  7. Department of Medicine, Division of Rheumatology and Clinical Autoimmunity Center of Excellence, University of Michigan Medical System, Ann Arbor, MI, USA

    David A. Fox

  8. Division of Rheumatology, University of Washington School of Medicine, Seattle, WA, USA

    Salahuddin Ahmed

Authors
  1. Ruby J. Siegel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anil K. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paul M. Panipinto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Farheen S. Shaikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Judy Vinh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sang U. Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. H. Mark Kenney
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Edward M. Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Cynthia S. Crowson
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sadik A. Khuder
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Basil S. Khuder
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. David A. Fox
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Salahuddin Ahmed
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RJS, AKS, and SA designed this study. RJS, AKS, JV, SUH, and HMK performed the experiments. RJS, AKS, JV, SUH, SAK, and BSK analyzed the data. RJS wrote the manuscript with participation from SA. DAF and CSC provided human RASFs and serum samples, respectively, as well as clinical guidance and manuscript feedback. SA provided support for the study.

Corresponding author

Correspondence to Salahuddin Ahmed.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Siegel, R.J., Singh, A.K., Panipinto, P.M. et al. Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts. Cell Mol Immunol 19, 1185–1195 (2022). https://doi.org/10.1038/s41423-022-00913-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41423-022-00913-x

Keywords

This article is cited by

Search

Advanced search

Quick links