OP0105 Zcchc6 deletion downregulates il-6 and reduces the severity of experimental osteoarthritis in mice
Authors: M.Y. Ansari, M.N. Khan, I. Ahmad, T.M. Haqqi
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Osteoarthritis (OA) is the most common joint disease in elderly and is a major cause of disability. Currently no disease modifying agents exist to treat OA. ZCCHC6 (zinc-finger CCHC-domain containing protein 6) is a member of family of terminal uridylyltransferases (TUTs) and is known to carry out template independent addition of Uridine at the 3' ends of miRNAs. ZCCHC6 has been implicated in miRNA-mediated regulation of cytokine gene expression; however, the role of ZCCHC6 in the pathogenesis of OA is unknown. <h3>Objectives</h3> The aim of this study was to investigate the effect of ZCCHC6 deletion on proinflammatory cytokine, IL-6, expression in human and mouse chondrocytes and to study the role of ZCCHC6 in OA pathogenesis <i>in vivo</i> using Zcchc6-/- mice. <h3>Methods</h3> Human or mouse chondrocytes were prepared by sequential enzymatic digestion and treated with recombinant human or mouse IL-1β to mimic OA pathogenic conditions. Cytokine profiling was done using qPCR array and multiplex assay. IL-6 expression in chondrocytes was analysed by qPCR and immunoblotting/ELISA. IL-6 mRNA stability was determined by Actinomycin-D chase experiments. The 3'-uridylation of miRNAs was analysed by deep sequencing of small RNAs using Illumina MiSeq. ZCCHC6 and IL-6 expression in OA cartilage was analysed by immunohistochemistry. For <i>in vivo</i> studies, OA was induced by surgical destabilisation of medial meniscus (DMM) in the knee joints of mice. The severity of OA was assessed by safranin O/fast green staining followed by OARSI scoring. <h3>Results</h3> ZCCHC6 expression was upregulated in OA cartilage. Cytokine expression profiling in ZCCHC6 depleted human and Zcchc6-/- mouse chondrocytes revealed IL-6 as a major target of ZCCHC6. Depletion of ZCCHC6 expression in human chondrocytes resulted in decreased expression of IL-6 at mRNA and protein levels. ZCCHC6 depletion suppressed IL-1β induced expression of IL-6. Overexpression of ZCCHC6 in human chondrocytes resulted in increased expression of IL-6 in the presence or absence of IL-1β. Chondrocytes from Zcchc6-/- mice also showed decreased levels of IL-6 expression compared to Zcchc6+/+. Furthermore, overexpression of mouse Zcchc6 in Zcchc6-/- chondrocytes rescued IL-6 expression. IL-6 mRNA half-life was significantly reduced in Zcchc6-/- mouse chondrocytes and ZCCHC6 depleted human chondrocytes. Deep sequencing of small RNAs in ZCCHC6 depleted human chondrocytes showed reduced 3'-uridylation of IL-6 targeting miRNAs miR-26a/26b. Human chondrocytes transfected with miR-26a/26b mimic suppressed IL-6 expression, however, miR-26b mimic with additional 'UU' failed to suppress IL-6 expression. Zcchc6-/- mice expressed low levels of MMP13 and showed lesser matrix degradation in the joints with DMM surgery. Zcchc6-/- mice developed less severe OA as determined by safranin O/fast green staining followed by OARSI scoring. Synovitis was also decreased in the Zcchc6-/- mice DMM joints in comparison to Zcchc6+/+DMM joints. <h3>Conclusions</h3> Our data demonstrate that ZCCHC6 is upregulated in OA cartilage and regulates IL-6 expression via miR-26b 3'-uridylation.These data identify a previously unknown function of ZCCHC6 in OA pathogenesis and identifies a potential therapeutic target for the management of OA. <h3>Acknowledgements</h3> This project is supported by NIH grant (RO1-AT007373, RO1-AT-005520, RO1-AR-067056) and funds from Northeast Ohio Medical University. <h3>Disclosure of Interest</h3> None declared