Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage. Collagen type II (COL2A1) was previously considered to be only a structural component of the cartilage matrix, but recently, it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy. However, the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear. In our study, a Col2a1 p.Gly1170Ser mutant mouse model was constructed, and Col2a1 loss was demonstrated in homozygotes. Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein (BMP)-SMAD1 pathway. Upon interacting with COL2A1, integrin β1 (ITGB1), the major receptor for COL2A1, competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import. COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and, through ERK1/2-SMAD1 interaction, it further repressed SMAD1 activation, thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy. Moreover, COL2A1 expression was downregulated, while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage. Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.

Osteoarthritis (OA) is characterized by cartilage destruction, chronic inflammation, and local pain. Evidence showed that retinoic acid receptor-related orphan receptor-α (RORα) is crucial in cartilage development and OA pathogenesis. Here, we investigated the role and molecular mechanism of RORα, an important member of the nuclear receptor family, in regulating the development of OA pathologic features. Investigation into clinical cartilage specimens showed that RORα expression level is positively correlated with the severity of OA and cartilage damage. In an in vivo OA model induced by anterior crucial ligament transaction, intra-articular injection of si-Rora adenovirus reversed the cartilage damage. The expression of cartilage matrix components type II collagen and aggrecan were elevated upon RORα blockade. RNA-seq data suggested that the IL-6/STAT3 pathway is significantly downregulated, manifesting the reduced expression level of both IL-6 and phosphorylated STAT3. RORα exerted its effect on IL-6/STAT3 signaling in two different ways, including interaction with STAT3 and IL-6 promoter. Taken together, our findings indicated the pivotal role of the RORα/IL-6/STAT3 axis in OA progression and confirmed that RORα blockade improved the matrix catabolism in OA chondrocytes. These results may provide a potential treatment target in OA therapy.

Accumulation of reactive oxygen species (ROS), which can be induced by inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This process can contribute to the imbalance of bone remodeling, which ultimately leads to osteoporosis. Therefore, reducing the ROS generation during osteogenesis of BMSCs may be an effective way to reverse the impairment of osteogenesis. Melatonin (MLT) has been reported to act as an antioxidant during cell proliferation and differentiation, but its antioxidant effect and mechanism of action during osteogenesis of MSCs in the inflammatory microenvironment, especially in the presence of TNF-α, remain unknown and need further study. In our study, we demonstrate that melatonin can counteract the generation of ROS and the inhibitory osteogenesis of BMSCs induced by TNF-α, by upregulating the expression of antioxidases and downregulating the expression of oxidases. Meanwhile, MLT can inhibit the phosphorylation of p65 protein and block the degradation of IκBα protein, thus decreasing the activity of the NF-κB pathway. This study confirmed that melatonin can inhibit the generation of ROS during osteogenic differentiation of BMSCs and reverse the inhibition of osteogenic differentiation of BMSCs in vitro, suggesting that melatonin can antagonize TNF-α-induced ROS generation and promote the great effect of osteogenic differentiation of BMSCs. Accordingly, these findings provide more evidence that melatonin can be used as a candidate drug for the treatment of osteoporosis.

Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro culture results in loss of MSC stemness. The inflammation that occurs at stem cell transplant sites (such as that resulting from TNF-α) is a contributing factor for stem cell treatment failure. Currently, there is little evidence regarding the protective role of melatonin with regard to the negative effects of TNF-α on the stemness of MSCs. In this study, we report a melatonin-based method to reduce the inflammatory effects on the stemness of bone marrow mesenchymal stem cells (BMMSCs). The results of colony formation assays, Alizarin red staining, western blotting, and reverse transcription-polymerase chain reactions suggest that melatonin can reverse the inflammatory damage caused by TNF-α treatment in the third, seventh, and tenth generations of primary BMMSCs (vs. control and the TNF-α-treated group). Meanwhile, a detailed analysis of the molecular mechanisms showed that the melatonin receptor and YAP signaling pathway are closely related to the role that melatonin plays in negative inflammatory effects against BMMSCs. In addition, in vivo experiments showed that melatonin could reverse the damage caused by TNF-α on bone regeneration by BMMSCs in nude mice. Overall, our results suggest that melatonin can reverse the loss of stemness caused by inflammatory factor TNF-α in BMMSCs. Our results also provide a practical strategy for the application of BMMSCs in tissue engineering and cell therapy.

Aims: Intervertebral disc degeneration (IDD) is closely related to low back pain, which is a prevalent age-related problem worldwide; however, the mechanism underlying IDD is unknown. Glutamine, a free amino acid prevalent in plasma, is recognized for its anti-inflammatory and antioxidant properties in various diseases, and the current study aims to clarify the effect and mechanism of glutamine in IDD. Results: A synergistic interplay was observed between pyroptosis and ferroptosis within degenerated human disc specimens. Glutamine significantly mitigated IDD in both ex vivo and in vivo experimental models. Moreover, glutamine protected nucleus pulposus (NP) cells after tert-butyl hydroperoxide (TBHP)-induced pyroptosis, ferroptosis, and extracellular matrix (ECM) degradation in vitro . Glutamine protected NP cells from TBHP-induced ferroptosis by promoting the nuclear factor erythroid 2-related factor 2 (Nrf2) accumulation by inhibiting its ubiquitin–proteasome degradation and inhibiting lipid oxidation. Innovation and Conclusions: A direct correlation is evident in the progression of IDD between the processes of pyroptosis and ferroptosis. Glutamine suppressed oxidative stress-induced cellular processes, including pyroptosis, ferroptosis, and ECM degradation through deubiquitinating Nrf2 and inhibiting lipid oxidation in NP cells. Glutamine is a promising novel therapeutic target for the management of IDD.