Background/Purpose:

RANTES/CCL5 (RANTES) is a C-C chemokine that binds to its receptor (CCR5) and initiates inflammatory processes in rheumatoid arthritis (RA) by facilitating leukocyte infiltration. However, the role of RANTES/CCL5 beyond its chemotactic activity is not fully known. The present study was carried out to study the effect of RANTES/CCL5 on matrix metalloproteinase (MMP)-1 and MMP-13 expression using human rheumatoid arthritis synovial fibroblasts (RA-FLS).

Methods:

Human RA-FLS isolated from RA synovial tissues were serum-starved overnight followed by treatment with RANTES/CCL5 (20-100 ng/ml) for different time period. Conditioned media was collected and cells were lysed with RNA lysis buffer. Effect of RANTES/CCL5 on MMP and TIMP activation in the conditioned media from RA-FLS was evaluated using human MMPs protein array. MMP-1 and MMP-13 mRNA expression and protein synthesis was evaluated using qRT-PCR, ELISA, and Western blotting methods. Effect of RANTES/CCL5 on collagenase activity was studied using in-gel zymography. The inhibitory effect of RANTES/CCL5 antagonist, Met-RANTES (50-200 ng/ml), on RANTES/CCL5-induced MMP-1 and MMP-13 production was evaluated. RA-FLS were stimulated with RANTES/CCL5 alone or the presence of signaling inhibitors for 5-30 mins to determine signaling mechanisms involved. Statistical value of p<0.05 was considered significant.

Results:

Results from human MMP antibody array showed that RANTES/CCL5 selectively induces MMP-1, MMP-13, TIMP-1, and TIMP-2 in RA-FLS (p<0.05; n=3). The confirmatory results using ELISA and qRT-PCR analysis showed that RANTES/CCL5 (20-100 ng/ml) significantly induced MMP-1 (~3-6-fold) and MMP-13 (~3-8-fold) expression in a dose-dependent manner (p<0.05 for 100 ng/ml). Collagen zymography results showed that RANTES/CCL5 induced collagenase activity in human RA-FLS dose-dependently (p<0.05; n=3). RA-FLS pretreatment with met-RANTES significantly reduced the ability of RANTES/CCL5 to induced MMP-1 and MMP-13 expression (p<0.05; n=3). Interestingly, silencing of RANTES/CCL5 gene using siRNA approach also markedly inhibited TNF-α-induced MMP-1 and MMP-13 production. Stimulation of RA-FLS with RANTES/CCL5 selectively activated PKC-δ and JNK pathways. In addition, pretreatment of RA-FLS with selective inhibitors for PKC-δ, ERK and JNK markedly inhibited RANTES/CCL5-induced MMP-1 and MMP-13 production.

Conclusion:

RANTES regulates MMP-1 and MMP-13 expression in RA-FLS via selective activation of PKC-δ, ERK, and JNK pathways and may also contribute to tissue destruction in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rantesccl5-selectively-induces-mmp-1-and-mmp-13-production-in-rheumatoid-arthritis-synovial-fibroblasts-via-pkc-i-and-jnk-pathways/