OP0259 Zcchc5, a ltr retrotransposon-derived neofunctionalized gene, is essential for the transcriptional activity of sox9 and the expression of col2a1 in chondrocytes and is downregulated in oa cartilage
Authors: T.M. Haqqi, N.M. Khan
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Retrotransposon-derived DNA sequences occupy approximately 40% of the mammalian genome, compared with only 1.5% of protein coding genes, and are a source of variation in the genome. LTR-derived gene <i>ZCCHC5</i> (<i>Mart3</i>) is a member of <i>gag</i>-related retrotransposon family that has lost the ability to retrotranspose. <i>ZCCHC5</i> gene encode a protein of approximately 53 Kd and contains a nucleic acid binding domain (CX<sub>2</sub>CX<sub>4</sub>HX<sub>4</sub>C), a <i>gag</i>-like region within the intact ORF and a homeobox associated leucine zipper motif indicating that this gene may have acquired new function(s) in the cell. Expression and function of <i>ZCCHC5</i> in degenerative joint diseases such as OA or other diseases has not been explored. <h3>Objectives</h3> The aim of this study was to investigate whether <i>ZCCHC5</i> is expressed in OA cartilage and chondrocytes and whether it is involved in the regulation of catabolic and/or anabolic factors in chondrocytes and its modulation under pathological conditions. <h3>Methods</h3> Chondrocytes were derived by the enzymatic digestion of human OA and normal C57BL6 mouse cartilage. Total RNA was prepared using Trizol and was made DNA-free using on-column digestion. mRNA expression was quantified using TaqMan assays. Protein expression was determined by immunohistochemistry (IHC) and Western blotting (WB) with validated antibodies. siRNA mediated depletion or plasmid mediated overexpression of ZCCHC5 gene was used to study its role in chondrocyte function under pathological conditions. Luciferase reporter vectors were used to study promoter activity in human chondrocytes. <h3>Results</h3> TaqMan, WB and IHC analyses revealed that the expression of ZCCHC5 was very low in the damaged OA cartilage compared to the levels detected in normal cartilage and the unaffected cartilage of the same patient. Stimulation of human and mouse chondrocytes with IL-1β significantly decreased the expression of ZCCHC5 (p&lt;0.05) which correlated with the inhibition of COL2A1 expression and upregulation of MMP-13. Overexpression of ZCCHC5 upregulated the COL2A1 mRNA and protein levels which were not suppressed by stimulation with IL-1β. Importantly, in these cells expression of MMP-13 was low and remained suppressed under pathological conditions. In contrast siRNA mediated depletion of ZCCHC5 expression in chondrocytes abrogated the protective effect on COL2A1 expression and increased the level of MMP-13 mRNA and protein upon treatment with IL-1b. Additionally, depletion of ZCCHC5 eliminated the SOX9 activity and COL2A1 promoter activation in human chondrocytes but this was reversed by reintroduction of ZCCHC5 expression indicating that ZCCHC5 plays an important role in SOX 9 activity and COL2A1 transcription. <h3>Conclusions</h3> Our data for the first time demonstrate that a LTR retrotransposon-derived neogene ZCCHC5 plays a key role in maintaining the differentiated functions of adult articular chondrocytes. Its repression in OA may contribute to cartilage deterioration by blocking chondrocyte anabolic functions and by enhancing expression of catabolic factors. Taken together, our data revealed a previously unidentified role of ZCCHC5 in a disease pathogenesis and uncovered a potential therapeutic approach to limit/reverse cartilage damage in OA. <h3>Acknowledgements</h3> Supported by NIH grants RO1 AT-007373, RO1 AR-067056 and funds from the Northeast Ohio Medical University <h3>Disclosure of Interest</h3> None declared