Abstract
Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.
Highlights
Osteoarthritis (OA) is a most common degenerative joint disorder and is characterized by degradation of articular cartilage, thickening of subchondral bone, and synovial inflammation[1,2]
Previous reports showed that HS6ST2 expression was significantly reduced in the cartilage of OA patients and the change of HS6ST2 could alter the expression of cartilage-related genes[24], suggesting a pivotal role of HS6ST2 during chondrocyte differentiation and progression of OA
In OA-damaged cartilage tissues, HS6ST2 was reduced at RNA and protein levels, while MMP13 mRNA was enhanced, which are consistent with published reports about overexpression of MMP13 in OA-damaged cartilage[41] and our previous study[24]
Summary
There are relationships among the expression levels of HS6ST2, MMP13, and miR-23b-3p in damaged cartilage from OA patients. The protein expression of HS6ST2 was downregulated after the administration of mimic miR-23b-3p, whereas transfection with the anti-miR-23b3p sequences could upregulate the protein expression of HS6ST2 (Fig. 2c, lower panel). In C28/I2 cells, upregulation of MMP13 from ectopic miR23b-3p or si-HS6ST2 was shown under TNF-α stimulation (Supplementary Figure S4). To confirm the matrix degradation caused by mimic miR-23b-3p via the target relationship between miR-23b3p and HS6ST2 mRNA, the plasmid for ectopic expression of HS6ST2 with FLAG tag was constructed. MMP13 protein level decreased and matrix staining increased under overexpression of HS6ST2 treated with TNF-α (Fig. 4c, d). Under TNF-α stimulation, the matrix staining was significantly reduced with ectopic miR-23b-3p, but the degradation was rescued by HS6ST2 overexpression (Fig. 4f), suggesting that miR-23b-3p participates in matrix degradation via regulating HS6ST2 expression. Findings provided evidence that HS6ST2 mediated by miR- 23b-3p might regulate cartilage degradation depending on the activity of p38 MAPK
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