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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

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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.

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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.

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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

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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.

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Correspondence to Salahuddin Ahmed.

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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

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