Background/Purpose: TNF-α drives RA synovial fibroblast (RASF)-mediated hyperplasia and joint tissue destruction. Extracellular sulfatase-2 (Sulf-2) influences receptor/ligand binding and subsequent signaling of chemokines, cytokines, and growth factors, however, the potential role of Sulf-2 in TNF-α signaling has remained unexplored. Our study compared Sulf-2 expression in synovial tissues and serum from hTNFtg mice and human RA patients vs. non-diseased controls and determined effects of Sulf-2 on TNF-α signaling and inflammation in human RASFs.

Methods: Sulf-2 expression was measured in human non-diseased vs. RA synovial tissues (NLSTs vs. RASTs) by qRT-PCR and Western blotting. Sulf-2 expression in wild-type C57BL/6 vs. hTNFtg mouse ankle sections was compared by fluorescence IHC. Soluble Sulf-2 in mouse and human serum was measured by ELISA. Global effects of Sulf-2 gene knockdown by siRNA on TNF-α-induced gene expression were studied using an RNA sequencing (RNA-seq) array. Differentially expressed genes (DEGs) were analyzed using edgeR package of R software. Selected gene targets were validated by qRT-PCR and Western blotting/ELISA. TNF-α signaling pathways affected by Sulf-2 knockdown were analyzed.

Results: Sulf-2 mRNA was 3-fold higher (n=6 each; p< 0.001) and protein 5-fold higher (n=5 each; p< 0.01) in RAST vs. NLST. Soluble Sulf-2 in human serum was ~2-fold higher in RA (n=48) over non-diseased (NL, n=50; p< 0.001). Sulf-2 expression was elevated in the inflamed synovium of hTNFtg mice over wild-type (n=3 each). Soluble Sulf-2 was elevated ~20% in hTNFtg male mice (n=3, n.s.) and 2-fold in females (n=3, p< 0.05) over wild-type. In the RNA-seq array of TNF-α-activated RASFs, Sulf-2 siRNA modulated ~2,500 DEG vs. NC siRNA (FDR< 0.05). Gene ontology showed Sulf-2 significantly altered expression of genes in the canonical pathway role of macrophages, fibroblasts and endothelial cells in RA. qRT-PCR confirmed that Sulf-2 knockdown reduced TNF-α-induced ICAM1 (-58%), VCAM1 (-50%), CAD11 (-45%), PDPN (-34%), CCL5 (-55%), CX3CL1 (-59%), CXCL10 (-65%), CXCL11 (-63%) compared to NC siRNA (n=4-6, p< 0.01). Signaling studies identified PKC-δ as a key intermediate in TNF-α induced adhesion proteins. Sulf-2 knockdown suppressed TNF-α-induced p-PKC-δ (Thr⁵⁰⁵)(-52%) and p-JNK(Thr¹⁸³/Tyr¹⁸⁵)(-36%)(n=6; p< 0.05) and nuclear translocation of inflammatory transcription factors including NFkB p65 and c-Rel.

Conclusion: Our results provide novel evidence of the role of Sulf-2 in TNF-α activated signaling pathways in human RASFs. Further studies may decipher the therapeutic value of targeting Sulf-2 in the treatment of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/extracellular-sulfatase-2-mediates-tnf-%ce%b1-inflammatory-signaling-in-human-rheumatoid-arthritis-synovial-fibroblasts/