Background/Purpose: Interleukin-6 (IL-6) promotes synovial hyperplasia by inducing mediators of adhesion, migration and leukocyte infiltration in rheumatoid arthritis (RA). The extracellular enzymes sulfatase-2 (Sulf-2) and sulfatase-1 (Sulf-1) cleave specific sulfate esters from heparan sulfate proteoglycans (HSPGs) which may affect the receptor/ligand binding and signaling of chemokines, cytokines, and growth factors.  However, the potential role of sulfatases in IL-6 signaling remain unexplored. The present study evaluated the effect of the sulfatases on IL-6 signaling and induction of effector molecules in human RA synovial fibroblasts (RASFs).

Methods: Human RASFs were serum-starved overnight followed by treatment with 100 ng/ml each of IL-6 and IL-6 receptor (IL-6/IL-6R) for different time periods. Conditioned media and whole cell lysates were collected to determine the expression of adhesion (cadherin-11, ICAM-1) and migration (podoplanin) molecules using qRT-PCR and Western blotting. IL-6/IL-6R-induced MCP-1 release in conditioned media was measured by ELISA. Effect of sulfatases on IL-6/IL-6R stimulation was evaluated by small inhibitory RNA (siRNA) knockdown of Sulf-1 and Sulf-2. RASFs treated with siRNA were stimulated with IL-6/IL-6R for 24 hr for protein studies, or for 30 min to determine signaling mechanisms.

Results: Compared to IL-1β (10 ng/ml) or TNF-α (20 ng/ml), IL-6/IL-6R selectively induced the expression of Sulf-2 by 1.5-fold, without marked change in Sulf-1 expression in human RASFs (n=3). In a time-dependent study (n=3; 0-48 hr), IL-6/IL-6R caused a significant increase in the expression of cadherin-11 (2-fold; p< 0.01), podoplanin (2.5-fold; p< 0.05) and ICAM-1 (3.5-fold; p< 0.0001), and the production of MCP-1 in the conditioned media (2.6-fold; p< 0.05). Additionally, IL-6/IL-6R stimulation caused a ~4-fold increase in expression of syndecan-2, a known HSPG target of Sulf-2, in human RASFs (p< 0.01; n=3). Interestingly, densitometric analysis of Western blots showed that knockdown of Sulf-2, but not Sulf-1, preferentially reduced IL-6/IL-6R-induced cadherin-11, podoplanin, and ICAM-1 by 35%, 25%, and 30%, respectively (p< 0.05; n=3). However, siRNA targeted for both Sulf-2 and Sulf-1 proved effective in reducing IL-6/IL-6R-induced MCP-1 production by 35% and 56%, respectively (p< 0.01; n=3). Evaluation of signaling events showed that inhibition of Sulf-2, but not Sulf-1, suppressed IL-6/IL-6R-induced phosphorylation of JAK-1 (Tyr1022) by ~50% while maintaining the expression of SOCS3, a negative regulator of JAK/STAT3 signaling.

Conclusion: Our study identified a novel role of Sulf-2 in facilitating IL-6-induced signaling in RASFs and suggests potential therapeutic value of targeting Sulf-2-dependent pathways in treatment approaches for RA.  

ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-novel-role-for-extracellular-sulfatase-2-in-mediating-il-6-induced-adhesion-and-migration-molecules-in-human-rheumatoid-arthritis-synovial-fibroblasts/