ZCCHC6 (TUT7) deletion inhibit the expression of IL-6 and MMP-13 and reduces the severity of post-traumatic osteoarthritis in a murine model

Abstract

Purpose: Approximately 20-50% of patients diagnosed with osteoarthritis (OA) develop the disease in joints that previously sustained trauma. RNA terminal uridyltransferases (TUTs), including Zcchc6 (zinc-finger CCHC-domain containing protein 6/TUT7), have been implicated in the post-transcriptional regulation of inflammatory mediators. However, role of Zcchc6 (TUT7) in the pathogenesis of OA or post-traumatic OA has not been explored. Methods: All murine studies were approved by the IACUC and the studies with deidentified, discarded human cartilage were approved by the IRB. Zcchc6 knockout mice were generated by gene targeting (KOMP, UC-Davis). Human or murine chondrocytes were prepared by the enzymatic digestion of the OA cartilage or knee cartilage respective and treated with recombinant human or mouse IL-1β (5ng/ml, R&D Systems, #: 201-LB-025 and #:401-ML-005 respectively) for in vitro studies. IL-6 and MMP13 gene and protein expression in chondrocytes was analyzed by qPCR and immunoblotting respectively. IL-6 mRNA stability was determined by Actinomycin-D chase experiments. For in vivo studies, post-traumatic OA in the knee joints of Zcchc6 deficient male mice and wild type littermates was induced by the surgical destabilization of medial meniscus (DMM) and mice were sacrificed after eight weeks of DMM and the severity of OA was determined per OARSI guidelines for murine OA. Contra lateral knee joints with sham surgery served as controls. Expression of MMP13 (Santa Cruz Biotechnology, #sc-30071), Type-II Collagen cleavage (IBEX Pharmaceuticals, #C1,2C) and Zcchc6 (Santa Cruz Biotechnology, #sc-137947) in vivo were analyzed by immunohistochemistry. Fluorescence in situ hybridization (FISH) was performed using the RNAScope technology. RNA immunoprecipitation (RIP) was performed using validated antibodies. siRNAs were used to deplete the expression of ZCCHC6. Results: Knee cartilage chondrocytes prepared from Zcchc6 KO mice showed increased levels of IL-6 and MMP13 mRNA expression compared to WT chondrocytes in response to IL-1β stimulation. Of interest was the observation that the protein levels of IL-6 and MMP13 were significantly downregulated in IL-1β stimulated Zcchc6 KO chondrocytes lysate and culture supernatants in comparison to WT chondrocytes. FISH and RIP analysis revealed that ZCCHC6 protein binds to IL-6 mRNAs in primary human OA chondrocytes under pathological conditions. Furthermore, IL-6 mRNA half-life was reduced by 50% in Zcchc6 KO murine chondrocytes and ZCCHC6 depleted human primary chondrocytes. The expression of Zcchc6 in the knee joints of wild type mice with DMM surgery was highly upregulated in comparison to control joints. Zcchc6 KO mice expressed low levels of MMP13 and showed lesser matrix degradation in the joints with DMM surgery compared to joints of the WT mice and the control contralateral joints and developed less severe OA as determined by Safranin-O-Fast green staining followed by OARSI scoring. Synovitis was also found to be decreased in the KO mice DMM joints in comparison to WT DMM joints. Conclusions: Our data demonstrate that Zcchc6 is upregulated in DMM joints and functions as a positive regulator of IL-6 and MMP13 expression in murine chondrocytes and cartilage in vivo. Severity of the posttraumatic OA and cartilage degradation was significantly low in Zcchc6 KO mice and correlated with reduced expression of MMP-13. These results identify ZCCHC6 as a potential therapeutic target for the treatment of OA.