Dysfunction of mRNA or RNA-binding proteins (RBPs) causes cellular aging and age-related degenerative diseases; however, information regarding the mechanism through which RBP-mediated posttranscriptional regulation affects cellular aging and related disease processes is limited. In this study, PUM1 was found to be associated with the self-renewal capacity and aging process of human mesenchymal stem cells (MSC). PUM1 interacted with the 3'-untranslated region of Toll-like receptor 4 (TLR4) to suppress TLR4 mRNA translation and regulate the activity of nuclear factor-κB (NF-κB), a master regulator of the aging process in MSCs. PUM1 overexpression protected MSCs against H 2 O 2 -induced cellular senescence by suppressing TLR4-mediated NF-κB activity. TLR4mediated NF-κB activation is a key regulator in osteoarthritis (OA) pathogenesis. PUM1 overexpression enhanced the chondrogenic potential of MSCs even under the influence of inflammation-inducing factors, such as lipopolysaccharide (LPS) or interleukin-1β (IL-1β), whereas the chondrogenic potential was reduced following the PUM1 knockdown-mediated TLR4 activation. PUM1 levels decreased under inflammatory conditions in vitro and during OA progression in human and mouse disease models. PUM1 knockdown in human chondrocytes promoted chondrogenic phenotype loss, whereas PUM1 overexpression protected the cells from inflammation-mediated disruption of the chondrogenic phenotype. Gene therapy using a lentiviral vector encoding mouse PUM1 showed promise in preserving articular cartilage integrity in OA mouse models. In conclusion, PUM1 is a novel suppressor of MSC aging, and the PUM1-TLR4 regulatory axis represents a potential therapeutic target for OA.

Background: Chondrocyte hypertrophy has been implicated in endochondral ossification and osteoarthritis (OA). In OA, hypertrophic chondrocytes contribute to the destruction and focal calcification of the joint cartilage. Although studies in this field have remarkably developed the modulation of joint inflammation using gene therapy and regeneration of damaged articular cartilage using cell therapy, studies that can modulate or prevent hypertrophic changes in articular chondrocytes are still lacking. Methods: In vitro hypertrophic differentiation and inflammation assays were conducted using human normal chondrocyte cell lines, TC28a2 cells. Human cartilage tissues and primary articular chondrocytes were obtained from OA patients undergoing total knee arthroplasty. Long non-coding RNAs (lncRNAs), LINC02035 and LOC100130207, were selected through RNA-sequencing analysis using RNAs extracted from TC28a2 cells cultured in hypertrophic medium. The regulatory mechanism was evaluated using western blotting, real-time quantitative polymerase chain reaction, osteocalcin reporter assay, RNA-immunoprecipitation (RNA-IP), RNA-in situ hybridization, and IP. Results: LncRNAs are crucial regulators of various biological processes. In this study, we identified two important lncRNAs, LINC02035 and LOC100130207, which play important roles in hypertrophic changes in normal chondrocytes, through RNA sequencing. Interestingly, the expression level of RUNX2, a master regulator of chondrocyte hypertrophy, was regulated at the post-translational level during hypertrophic differentiation of the normal human chondrocyte cell line, TC28a2. RNAimmunoprecipitation proved the potential interaction between RUNX2 protein and both lncRNAs. Knockdown (KD) of LINC02035 or LOC100130207 promoted ubiquitin-mediated proteasomal degradation of RUNX2 and prevented hypertrophic differentiation of normal chondrocyte cell lines, whereas overexpression of both lncRNAs stabilized RUNX2 protein and generated hypertrophic changes. Furthermore, the KD of the two lncRNAs mitigated the destruction of important cartilage matrix proteins, COL2A1 and ACAN, by hypertrophic differentiation or inflammatory conditions. We also confirmed that the phenotypic changes raised by the two lncRNAs could be rescued by modulating RUNX2 expression. In addition, the KD of these two lncRNAs suppressed hypertrophic changes during chondrogenic differentiation of mesenchymal stem cells. Conclusion: Therefore, this study suggests that LINC02035 and LOC100130207 contribute to hypertrophic changes in normal chondrocytes by regulating RUNX2, suggesting that these two novel lncRNAs could be potential therapeutic targets for delaying or preventing OA development, especially for preventing chondrocyte hypertrophy.

Background: Despite several studies on the effect of adeno-associated virus (AAV)-based therapeutics on osteoarthritis (OA), information on the transduction efficiency and applicable profiles of different AAV serotypes to chondrocytes in hard cartilage tissue is still limited. Moreover, the recent discovery of additional AAV serotypes makes it necessary to screen for more suitable AAV serotypes for specific tissues. Here, we compared the transduction efficiencies of 14 conventional AAV serotypes in human chondrocytes, mouse OA models, and human cartilage explants obtained from OA patients. Methods: To compare the transduction efficiency of individual AAV serotypes, green fluorescent protein (GFP) expression was detected by fluorescence microscopy or western blotting. Likewise, to compare the transduction efficiencies of individual AAV serotypes in cartilage tissues, GFP expression was determined using fluorescence microscopy or immunohistochemistry, and GFP-positive cells were counted. Results: Only AAV2, 5, 6, and 6.2 exhibited substantial transduction efficiencies in both normal and OA chondrocytes. All AAV serotypes except AAV6 and rh43 could effectively transduce human bone marrow mesenchymal stem cells. In human and mouse OA cartilage tissues, AAV2, AAV5, AAV6.2, AAV8, and AAV rh39 showed excellent tissue specificity based on transduction efficiency. These results indicate the differences in transduction efficiencies of AAV serotypes between cellular and tissue models. Conclusions: Our findings indicate that AAV2 and AAV6.2 may be the best choices for AAV-mediated gene delivery into intra-articular cartilage tissue. These AAV vectors hold the potential to be of use in clinical applications to prevent OA progression if appropriate therapeutic genes are inserted into the vector.

No abstract

Purpose Our previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells. In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model. Materials and Methods An in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1β (IL-1β, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA. Results miRNA-495 was upregulated in IL-1β-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1β in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1β. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1β-treated TC28a2 cells. Conclusion We demonstrated that inhibition of miRNA-495 alleviates IL-1β-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.