Ankylosing spondylitis (AS) is a type of rheumatic disease characterized by chronic inflammation and pathological osteogenesis in the entheses. Previously, we demonstrated that enhanced osteogenic differentiation of MSC from AS patients (AS-MSC) resulted in pathological osteogenesis, and that during the enhanced osteogenic differentiation course, AS-MSC induced TNF-α-mediated local inflammation. However, whether TNF-α in turn affects AS-MSC remains unknown. Herein, we further demonstrate that a high-concentration TNF-α treatment triggers enhanced directional migration of AS-MSC in vitro and in vivo, which enforces AS pathogenesis. Mechanistically, TNF-α leads to increased expression of ELMO1 in AS-MSC, which is mediated by a METTL14 dependent m⁶A modification inELMO13′UTR. Higher ELMO1 expression of AS-MSC is found in vivo in AS patients, and inhibiting ELMO1 in SKG mice produces therapeutic effects in this spondyloarthritis model. This study may provide insight into not only the pathogenesis but also clinical therapy for AS.

Ankylosing spondylitis (AS) is a chronic inflammatory disease possessing a morbid serum microenvironment with enhanced oxidative stress. Long-term exposure to an oxidative environment usually results in cellular senescence alone with cellular dysfunction. Mesenchymal stem cells (MSCs) are a kind of stem cell possessing strong capabilities for immunoregulation, and senescent MSCs may increase inflammation and participate in AS pathogenesis. The objective of this study was to explore whether and how the oxidative serum environment of AS induces MSC senescence. Here, we found that AS serum facilitated senescence of MSCs in vitro, and articular tissues from AS patients exhibited higher expression levels of the cell cycle arrest-related proteins p53, p21 and p16. Importantly, the levels of advanced oxidative protein products (AOPPs), markers of oxidative stress, were increased in AS serum and positively correlated with the extent of MSC senescence induced by AS serum. Furthermore, MSCs cultured with AS serum showed decreased mitochondrial membrane potential and ATP production together with a reduced oxygen consumption rate. Finally, we discovered that AS serum-induced mitochondrial dysfunction resulted in elevated reactive oxygen species (ROS) in MSCs, and ROS inhibition successfully rescued MSCs from senescence. In conclusion, our data demonstrated that the oxidative serum environment of AS facilitated MSC senescence through inducing mitochondrial dysfunction and excessive ROS production. These results may help elucidate the pathogenesis of AS and provide potential targets for AS treatment.

Interleukin-6 (IL-6) with IL-6 receptor (IL-6R) play an important role in the tissue regeneration in vivo, especially bone metabolism. Bone marrow -derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells, which are main origin of osteoblasts. However, the roles of IL-6 and IL-6R in the osteogenic differentiation of BM-MSCs are still unclear. The expression of IL-6 and IL-6R was detected in BM-MSCs during osteogenic differentiation. The activation of the STAT3 pathway was assessed and its role in the osteogenic differentiation of BM-MSCs was determined using the specific inhibitor AG490. Exogenous IL-6/soluble IL-6R or antibodies against IL-6/IL-6R were used to confirm the mechanism by which the IL-6/IL-6R complex promotes the osteogenic differentiation. The levels of IL-6 and IL-6R, especially the level of membranous IL-6R but not that of soluble IL-6R, increased during osteogenic differentiation in BM-MSCs. The levels of IL-6 and IL-6R were positively correlated with the osteogenic potential of BM-MSCs. The STAT3 signaling pathway was activated during the osteogenic differentiation of BM-MSCs. AG490 markedly inhibited the activation of the STAT3 pathway and, subsequently, the osteogenic differentiation potential of BM-MSCs. Additionally, exogenous IL-6 and soluble IL-6R accelerated the osteogenic differentiation of BM-MSCs. In contrast, antibodies against IL-6 or IL-6R suppressed the osteogenic differentiation of BM-MSCs. Moreover, IL-6 and IL-6R were found to stimulate each other’s expression in BM-MSCs. IL-6 and IL-6R levels increase during the osteogenic differentiation of BM-MSCs. These two molecules form a complex to activate the downstream STAT3 signaling pathway, which promotes osteogenic differentiation in BM-MSCs via an autocrine/paracrine feedback loop.

Objective: To study the osteogenic differentiation capacity of bone marrow–derived mesenchymal stem cells (BM‐MSCs) from patients with ankylosing spondylitis (AS) and to investigate the mechanisms of abnormal osteogenic differentiation of BM‐MSCs in AS.Methods: BM‐MSCs from healthy donors (HD‐MSCs) and patients with AS (AS‐MSCs) were cultured in osteogenic differentiation medium for 0–21 days, after which their osteogenic differentiation capacity was determined using alizarin red S and alkaline phosphatase assays. Gene expression levels of osteoblastic markers and related cytokines were detected by high‐throughput quantitative reverse transcription–polymerase chain reaction. Enzyme‐linked immunosorbent assay was performed to detect protein levels of bone morphogenetic protein 2 (BMP‐2) and Noggin in the cell culture supernatant. The activation of Smad1/5/8 and MAPK signaling pathways was measured by Western blotting. The balance between BMP‐2 and Noggin expression was regulated using lentiviruses encoding short hairpin RNA and exogenous Noggin, respectively, which enabled evaluation of how this balance affected osteogenic differentiation of AS‐MSCs.Results: AS‐MSCs outperformed HD‐MSCs in osteogenic differentiation capacity. During osteogenic differentiation, AS‐MSCs secreted more BMP‐2 but less Noggin, accompanied by an overactivation of Smad1/5/8 and ERK‐1/2. When the Noggin concentration was increased or BMP‐2 expression was inhibited, the abnormal osteogenic differentiation of AS‐MSCs was rectified. In addition, the balance between BMP‐2 and Noggin secretion was restored.Conclusion: The results of this study demonstrate that an imbalance between BMP‐2 and Noggin secretion induces abnormal osteogenic differentiation of AS‐MSCs. These findings reveal a mechanism of pathologic osteogenesis in AS and provide a new perspective on inhibiting pathologic osteogenesis by regulating the balance between BMP‐2 and Noggin.

Background: The zinc transporters Zrt- and Irt-related protein (ZIP/SLC39) are overexpressed in human tumors and correlate with poor prognosis; however, their contributions to carcinogenesis and chemoresistance in osteosarcoma (OS) remain unclear.Methods: We collected 64 OS patient tissues with (n = 12) or without (n = 52) chemotherapy. The expression levels of ZIP10 were measured by immunohistochemistry and applied to prognostic analysis. ZIP10 was knocked down or overexpressed in OS cell lines to explore its effect on proliferation and chemoresistance. RNA sequencing, quantitative real-time PCR, and western blotting analysis were performed to explore ZIP10-regulated downstream target genes. A xenograft mouse model was established to evaluate the mechanisms by which ZIP10 modulates chemoresistance in OS cells.Results: The expression of ZIP10 was significantly induced by chemotherapy and highly associated with the clinical outcomes of OS. Knockdown of ZIP10 suppressed OS cell proliferation and chemoresistance. In addition, ZIP10 promoted Zn content-induced cAMP-response element binding protein (CREB) phosphorylation and activation, which are required for integrin α10 (ITGA10) transcription and ITGA10-mediated PI3K/AKT pathway activation. Importantly, ITGA10 stimulated PI3K/AKT signaling but not the classical FAK or SRC pathway. Moreover, overexpression of ZIP10 promoted ITGA10 expression and conferred chemoresistance. Treatment with the CREB inhibitor 666–15 or the PI3K/AKT inhibitor GSK690693 impaired tumor chemoresistance in ZIP10-overexpressing cells. Finally, a xenograft mouse model established by subcutaneous injection of 143B cells confirmed that ZIP10 mediates chemotherapy resistance in OS cells via the ZIP10-ITGA10-PI3K/AKT axis.Conclusions: We demonstrate that ZIP10 drives OS proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway, which might serve as a target for OS treatment.

Mesenchymal stem cells (MSCs) are a common kind of multipotent cell in vivo, but their heterogeneity limits their further applications. To identify MSC subpopulations and clarify their relationships, we performed cell mapping of bone-marrow-derived MSCs through single-cell RNA (scRNA) sequencing. In our study, three main subpopulations, namely, the stemness subpopulation, functional subpopulation, and proliferative subpopulation, were identified using marker genes and further bioinformatic analyses. Developmental trajectory analysis showed that the stemness subpopulation was the root and then became either the functional subpopulation or the proliferative subpopulation. The functional subpopulation showed stronger immunoregulatory and osteogenic differentiation abilities but lower proliferation and adipogenic differentiation. MSCs at different passages or isolated from different donors exhibited distinct cell mapping profiles, which accounted for their corresponding different functions. This study provides new insight into the biological features and clinical use of MSCs at the single-cell level, which may contribute to expanding their application in the clinic.