OSTF1 knockdown mitigates IL-1ß-induced chondrocyte injury via inhibiting the NF-κB signaling pathway.

Osteoarthritis (OA) is an age-related joint disease characterized by progressive heterogeneous changes in articular cartilage and subchondral bone. Osteoclast stimulating factor 1 (OSTF1) is a small intracellular protein involved in bone formation and bone resorption. However, to our best knowledge, its role in OA is still unclear. In this study, an OA rat model was established by anterior cruciate ligament transection (ALCT). OSTF1 was increased in the cartilage tissues of OA patients and OA rats. Next, the role of OSTF1 in interleukin-1ß (IL-1ß)-induced chondrocyte apoptosis, inflammation and extracellular matrix degradation was explored through loss of function assays. Strikingly, OSTF1 knockdown relieved IL-1ß-induced chondrocyte apoptosis, with decreased cleaved caspase-3 and cleaved PARP levels. Besides, OSTF1 knockdown restrained IL-1ß-induced inflammation and degradation of extracellular matrix of chondrocytes. Subsequently, the molecular mechanism of OSTF1 was explored. Transcriptomic analysis revealed the potential gene network map regulated by OSTF1 knockdown. Some differentially expressed genes (DEGs) were involved in regulating the NF-κB signaling pathway. Furthermore, our results demonstrated that OSTF1 knockdown inhibited IL-1ß-activated the NF-κB signaling pathway. Ultimately, we analyzed the potential gene network map regulated by OSTF1 and its downstream NF-κB. Bioinformatics analysis showed that 18 DEGs in OSTF1-silenced chondrocytes overlapped with the NF-κB downstream targets. Collectively, our findings indicate that OSTF1 knockdown mitigates IL-1ß-induced chondrocyte injury via inhibiting the NF-κB signaling pathway.

CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway.

BACKGROUND: Osteoarthritis (OA), the most common joint disease, is linked with chondrocyte apoptosis and extracellular matrix (ECM) degradation. Charged multivesicular body protein 5 (CHMP5), a member of the multivesicular body, has been reported to serve as an anti-apoptotic protein to participate in leukemia development. However, the effects of CHMP5 on apoptosis and ECM degradation in OA remain unclear. METHODS: In this study, quantitative proteomics was performed to analyze differential proteins between normal and OA patient articular cartilages. The OA mouse model was constructed by the destabilization of the medial meniscus (DMM). In vitro, interleukin-1 beta (IL-1ß) was used to induce OA in human chondrocytes. CHMP5 overexpression and silencing vectors were created using an adenovirus system. The effects of CHMP5 on IL-1ß-induced chondrocyte apoptosis were investigated by CCK-8, flow cytometry, and western blot. The effects on ECM degradation were examined by western blot and immunofluorescence. The potential mechanism was explored by western blot and Co-IP assays. RESULTS: Downregulated CHMP5 was identified by proteomics in OA patient cartilages, which was verified in human and mouse articular cartilages. CHMP5 overexpression repressed cell apoptosis and ECM degradation in OA chondrocytes. However, silencing CHMP5 exacerbated OA chondrocyte apoptosis and ECM degradation. Furthermore, we found that the protective effect of CHMP5 against OA was involved in nuclear factor kappa B (NF-κB) signaling pathway. CONCLUSIONS: This study demonstrated that CHMP5 repressed IL-1ß-induced chondrocyte apoptosis and ECM degradation and blocked NF-κB activation. It was shown that CHMP5 might be a novel potential therapeutic target for OA in the future.

MSRB2 Ameliorates H2O2-induced Chondrocyte Oxidative Stress and Suppresses Apoptosis in Osteoarthritis.

BACKGROUND: Chondrocyte oxidative stress and apoptosis are critical factors contributing to the pathogenesis of osteoarthritis (OA). Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial protein that protects cells from oxidative stress and is involved in apoptosis. This study aimed to investigated the expression of MSRB2 in articular cartilage tissues and elucidated its effect on H2O2-stimulated chondrocytes. METHODS: Human chondrocytes were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12. MSRB2 overexpression in chondrocytes was achieved by transfecting with an MSRB2 overexpression plasmid. Western blot, quantitative RT-PCR, Immunofluorescence staining, and TUNEL assay were employed in this study. RESULTS: MSRB2 expression was found to be reduced in OA patients. Furthermore, overexpression of MSRB2 in H2O2-induced chondrocytes mitigated apoptosis and enhanced cell viability. Elevated MSRB2 expression diminished chondrocyte ROS contents, decreased cytochrome C (Cyc) in the cytoplasm, and regulated mitochondrial membrane potential to maintain mitochondrial homeostasis. Interestingly, knockdown of charged multivesicular body protein 5 (CHMP5) led to a decreased inMSRB2 expression in chondrocytes. Additionally, protein levels of CHMP5 and MSRB2 were reduced in H2O2-stimulated chondrocytes, and silencing CHMP5 reduced MSRB2 expression. Knockdown of CHMP5 increased cleaved caspase-3 expression in H2O2-induced chondrocytes and elevated TUNEL-positive chondrocytes. CONCLUSION: MSRB2 decreased in OA, and overexpression of MSRB2 alleviated oxidative stress and apoptosis of chondrocyte.

miR-29a promotes osteoblast proliferation by downregulating DKK-1 expression and activating Wnt/ß-catenin signaling pathway.

BACKGROUND: MicroRNA (miRNA) is a kind of non-coding small RNA with a negative regulating function. Some miRNAs play a role in regulating the differentiation and function of osteoblasts, chondrocytes and osteoclasts. OBJECTIVES: In this study, we analyzed the role of miR-29a and dickkopf-1 (DKK-1) in osteoblast differentiation. MATERIAL AND METHODS: Specimens were collected from the surgical resection of pathological ankylosing spondylitis (AS) tissue and some normal tissues. The expression of miR-29a, DKK-1 and ß-catenin in normal and AS tissues were detected with real-time polymerase chain reaction (RT-PCR) and western blotting. Cell proliferation was detected with a Cell Counting Kit-8, cell migration and invasion were determined using a Transwell system and cell apoptosis was analyzed with flow cytometry. The luciferase reporter gene plasmid pGL3-DKK-1 and a point-mutation of the luciferase reporter gene plasmid mut-pGL3-DKK-1 were constructed. RESULTS: It was found that miR-29a could promote the proliferation of hFOB1.19 cells, while DKK-1 inhibited their proliferation. Also, miR-29a was able to inhibit the apoptosis of hFOB1.19 cells, while DKK-1 was able to promote the apoptosis of hFOB1.19 cells. When it comes to the invasion and migration of hFOB1.19 cells, miR-29a was found to promote it, while DKK-1 did not. CONCLUSIONS: These findings will lead to a better understanding of the proliferation and differentiation of osteoblasts and will provide new insights for the treatment of this disease.

Knockdown of EIF3C promotes human U-2OS cells apoptosis through increased CASP3/7 and Chk1/2 by upregulating SAPK/JNK.

BACKGROUND: As a component of the EIF3 complex, EIF3C is essential for several steps in protein synthesis initiation. Recently, it has been addressed that EIF3C is overexpressed in several human cancers and plays a pivotal role in cell proliferation and tumorigenesis. MATERIALS AND METHODS: Immunohistochemistry, quantitative real-time PCR (qPCR), and Western blotting assays were employed to determine the expression of EIF3C in osteosarcoma (OsC) tissues obtained from 60 patients. The levels of EIF3C mRNA and protein were assessed by qPCR and Western blotting, respectively. The effect of EIF3C knockdown on OsC cell proliferation was detected by MTT and colony formation assays, respectively. Cell apoptosis induced by EIF3C silencing was analyzed by flow cytometric analysis. PathScan stress and apoptosis signaling antibody array kit was used to analyze the potential effects of EIF3C knockdown on OsC cells. RESULTS: The levels of EIF3C were high in OsC tissues and cell lines. In addition, EIF3C knockdown by lentivirus-mediated shRNA targeting EIF3C significantly suppressed cell proliferation and colony formation and induced apoptosis in U-2OS cells. Moreover, EIF3C knockdown led to the upregulated expression of CASP3/7, Chk1/2, and SAPK/JNK, indicating that the downregulated expression of EIF3C might be associated with pro-apoptosis of U-2OS cells. CONCLUSION: EIF3C may be a promising target for gene therapy of human OsC. However, the precise mechanisms behind the effect of EIF3C on OsC tumorigenesis require further analysis.

Wnt3a is critical for endothelial progenitor cell-mediated neural stem cell proliferation and differentiation.

The present study aimed to determine whether co-culture with bone marrow­derived endothelial progenitor cells (EPCs) affects the proliferation and differentiation of spinal cord-derived neural stem cells (NSCs), and to investigate the underlying mechanism. The proliferation and differentiation of the NSCs were evaluated by an MTT cell proliferation and cytotoxicity assay, and immunofluorescence, respectively. The number of neurospheres and the number of ß­tubulin III­positive cells were detected by microscopy. The wingless­type MMTV integration site family, member 3a (Wnt3a)/ß-catenin signaling pathway was analyzed by western blot analysis and reverse transcription­quantitative polymerase chain reaction to elucidate the possible mechanisms of EPC­mediated NSC proliferation and differentiation. The results revealed that co­culture with EPCs significantly induced NSC proliferation and differentiation. In addition, co­culture with EPCs markedly induced the expression levels of Wnt3a and ß­catenin and inhibited the phosphorylation of glycogen synthase kinase 3ß (GSK­3ß). By contrast, Wnt3a knockdown using a short hairpin RNA plasmid in the EPCs reduced EPC­mediated NSC proliferation and differentiation, accompanied by inhibition of the EPC­mediated expression of ß­catenin, and its phosphorylation and activation of GSK­3ß. Taken together, the findings of the present study demonstrated that Wnt3a was critical for EPC­mediated NSC proliferation and differentiation.