Background/Purpose:

Interleukin-1β (IL-1β) is the major cytokine involved in cartilage catabolism in osteoarthritis(OA) and induces the expression of pro-inflammatory cytokine IL-6. Members of cytoplasmic RNA nucleotidyl transferases superfamily catalyze the addition of nucleotides to the 3’end of mRNAs. However, the expression or role of RNA nucleotidyl transferases in regulating cytokine expression in OA is unknown. The aim of this study was to investigate whether RNA nucleotidyl transferase ZCCHC6 is expressed in OA cartilage and whether it is involved in the regulation of IL-6 expression in OA chondrocytes.

Methods:

Chondrocytes were derived by enzymatic digestion of human cartilage obtained from OA patients (n=14) undergoing knee joint replacement. Chondrocytes were stimulated with IL-1β (5ng/ml) or treated with Actinomycin D (5 µg/ml) or NF-κB inhibitor SC514 (75 µM). Total RNA from grounded cartilage and from chondrocytes was purified using Qiagen RNeasy kit (Qiagen). Reverse transcription was performed using the Quantitect Reverse Transcription kit and the ZCCHC6 or IL-6 mRNA was quantified using TaqMan assays. SiRNA-mediated depletion of ZCCHC6 in human chondrocytes was used to study the effect on inflammatory cytokine expression using a cytokine array (Ray Biotech). Protein expression of IL-6 was studied using Western immunoblotting and by ELISA in culture supernatants. Data was analyzed using Origin 6.1 software package and p<0.05 was considered significant.

Results:

Our results showed differential expression of nucleotidyl transferase ZCCHC6 in the damaged cartilage compared to unaffected cartilage. Higher expression of IL-6 (6.0-fold ± 1.44) in damaged cartilage compared to smooth cartilage (n=3; p<0.05) from OA patients was also observed. We further demonstrate that IL-1β stimulation resulted in significant increase in the expression of ZCCHC6 (11.3-fold ± 1.6) and the mRNA of the inflammatory cytokine IL-6 (4956-fold ± 40.6) in human chondrocytes (n=4; p<0.05). Similar increase in the protein expression of both ZCCHC6 and IL-6 was also observed. Depletion of ZCCHC6 significantly decreased the expression of IL-6 mRNA (~77-95%) and protein in IL-1β-stimulated human chondrocytes. Importantly, IL-6 mRNAs in IL-1β-stimulated human chondrocytes treated with ZCCHC6 siRNA had shorter poly-A tails (n=3; p<0.05). Cell supernatants from control or ZCCHC6 siRNA treated chondrocytes stimulated with IL-1β were analyzed using a cytokine array. A subset of cytokines including IL-6 was substantially decreased by loss of ZCCHC6. Our results also showed that the IL-1β-induced activation of NF-ΚB has no role in the regulation of ZCCHC6 expression in OA chondrocytes (n=3; p<0.05). Additionally, OA chondrocytes transfected with ZCCHC6 siRNA also showed a decrease (~99%) in constitutive IL-6 mRNA expression (n=3; p<0.05).

Conclusion: Taken together our results demonstrate that ZCCHC6 is highly expressed in damaged human cartilage from OA patients. Furthermore, ZCCHC6 modulates IL-6 expression in human chondrocytes at the post-transcriptional level by influencing cytokine mRNA stability. These results identify ZCCHC6 as a possible therapeutic target for the treatment of OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/zinc-finger-protein-zcchc6-is-highly-expressed-in-osteoarthritic-cartilage-and-regulate-the-expression-of-interleukin-6-in-human-chondrocytes/