Ran GTPase-activating protein 1 (RanGAP1) is a known regulator of nucleocytoplasmic transport; however, its specific function within innate immunity remains undefined. Here, we show that low RanGAP1 expression correlates with poor survival outcomes in septic patients and demonstrate that RanGAP1 expression and subcellular localization are dynamically regulated in macrophages following lipopolysaccharide (LPS) stimulation. We observed that deletion of RanGAP1 in macrophages exacerbates sepsis progression by using a cecal ligation and puncture (CLP) murine model. Mechanistically, RanGAP1 deficiency significantly impairs macrophage anti-infective functions, including inflammatory cytokine production, pathogen clearance, and antigen processing. These findings highlight RanGAP1 as a central regulator of macrophage immune responses, suggesting that its activity is a critical determinant of septic outcomes.

Abstract Objective Enhanced glycolysis is a metabolic hallmark of chondrocytes in osteoarthritis (OA); however, the roles of the glycolytic rate-limiting enzyme hexokinase 2 (HK2) in cartilage remain poorly understood. Methods Pharmacological approach (3-bromopyruvate (3-BrPA) treatment) and mice model involving HK2 knockout in Col2a1-expressing chondrocytes are utilized to access the impact of HK2 blockage on cartilage ex vivo and in vivo . The in vivo effects of HK2 inhibition on OA progression were evaluated using a destabilization of the medial meniscus (DMM)-induced OA mouse model, through both intra-articular 3-BrPA administration and chondrocyte HK2 deletion. Additionally, we analyzed published single-cell RNA sequencing (scRNA-seq) datasets from human articular cartilage and integrated these with bulk RNA-seq data from HK2-deficient chondrocytes to characterize HK2 expression features across conditions. Results Both pharmacological inhibition and genetic deletion of HK2 impair cartilage formation ex vivo . Bulk RNA-seq analysis and ex vivo studies demonstrated a promoted ossification-like process due to HK2 ablation in chondrocytes. Through pseudotime analysis of published single-cell RNA sequencing (scRNA-seq) datasets from human articular cartilages, we further identified that HK2 is differentially expressed across conditions, with a feature of a relatively high expression level at terminal stages of chondrocyte differentiation in the context of OA. We next confirmed HK2 deficiency in chondrocytes significantly exacerbated OA progression but having no impact on skeletal development in mice. Conclusions HK2 plays a critical role in maintaining cartilage health, likely through the regulation of calcification, thereby highlighting the potential risks associated with targeting glycolytic enzymes as a therapeutic strategy for OA.

Type II topoisomerase α (Top2a) is crucial for maintaining the stemness of embryonic stem cells (ESCs) and is upregulated in various human cancers, yet the mechanisms underlying its cell type-specific functions remain elusive. Here, using an unbiased proteomic approach, we discovered that Trim28, along with subunits of various chromatin remodeling complexes and modifiers, interacts with Top2a on ESC chromatin. Specifically, Top2a directly interacts with Trim28 in mESCs but only weakly, if at all, in mouse embryonic fibroblasts (MEFs). Functionally, Trim28 cooperates with Top2a to maintain ESC status and enhance induced pluripotency. Genome-wide approaches revealed that Top2a and Trim28 co-activate a subset of genes encoding key metabolic enzymes involved in glycolysis in both ESCs and breast cancer cells. Mechanistically, high levels of Top2a fine-tune promoter bivalency by recruiting Trim28 to co-activated genes, while preventing its binding to co-repressed genes. Strikingly, targeting TOP2A in combination with TRIM28 in breast cancer cells leads to a superior anti-tumorigenic effect. Our findings thereby define a novel Top2a-Trim28 partnership for ESC-specific gene regulation, providing a rationale for the dual targeting of this axis in cancers with elevated TOP2A/TRIM28. A novel Top2a-Trim28 axis transcriptionally orchestrates glycolysis to control ESC stemness and tumorigenesis, supporting dual targeting in cancers with elevated TOP2A/TRIM28.

Colorectal cancer (CRC) remains largely refractory to immune-checkpoint blockade, with adenomatous polyposis coli (APC) mutations present in 80%–90% of cases. Loss of APC was previously thought to promote tumor progression mainly through deregulated Wnt/β-catenin signaling. Here, we report that APC loss leads to inhibition of CD8+ T cell infiltration and CRC immune evasion through the dephosphorylation of signal transducers and activators of transcription 1 (STAT1) by protein tyrosine phosphatase non-receptor type 13 (PTPN13), independently of β-catenin. Peptides containing the last 11 C-terminal amino acid (aa) residues of APC (APC11) bind directly to PTPN13 to block PTPN13–STAT1 interactions and facilitate STAT1 phosphorylation, interferon regulatory factor-1 (IRF1) expression, major histocompatibility complex (MHC) class I antigen presentation, and T cell intratumoral infiltration, all of which eventually inhibit tumor progression and enhance the effects of programmed cell death 1 (PD1) blockade. Thus, we have identified a previously unknown APC/PTPN13/STAT1-dependent tumor immune-suppressive mechanism. The potent tumor-suppressing effect of combining anti-PD1 antibodies with APC11 peptides provides a compelling target and rationale for future development of anti-tumor drugs for patients with CRC.

Metabolic disorders have been recognized as a major contributor to the occurrence and progression of osteoarthritis (OA). Identifying novel therapeutic agents to ameliorate the progression of OA with metabolic disorder is crucial. In this study, we demonstrate that semaglutide (SG), a glucagon-like peptide-1 receptor (GLP-1R) agonist, exhibits strong chondroprotective effects in an OA mouse model with obesity, as evidenced by reduced pathological changes, including cartilage degeneration, osteophyte formation, synovial lesion, and pain sensitivity. A randomized pilot clinical study (ChiCTR2200066291) further supports these findings. By designing a precise diet-controlled setting to rule out the effect of appetite suppression and weight loss induced by SG, we demonstrate a weight loss-independent mechanism. Through regulating the "GLP-1R-AMPK-PFKFB3" axis, the SG reprograms chondrocyte metabolism profile from glycolysis to oxidative phosphorylation under inflammatory conditions, resulting in cartilage restoration.

The anti-osteoporotic effects of parathyroid hormone (PTH) analogs for patients with severe osteoporosis decrease with age; however, the underlying mechanism(s) are poorly understood. Here we provide evidence that enhanced TNF-mediated chronic inflammation during aging may play a critical role in promoting bone loss and attenuating the anabolic effects of PTH on bone in aged individuals. Specifically, we find that abolishment of TNF signaling activation by global deletion of the Tnfr1/2 genes, which encode the TNF receptors, prevents aging-related bone mass loss and improves the anabolic effects of PTH (1-34) analog to increase bone mass and bone mineral density in aged, but not young adult, mice. These effects are achieved mainly by enhancing PTH signaling in osteoblastic cells, leading to accelerated bone formation. TNF-α inhibition of PTH signaling is mediated via TRAF2, an E3 ligase. TRAF2 promotes the non-canonical K27- and K33-linked ubiquitination of Gαs, which increases Gαs protein stability but blocks Gαs activation and downstream signaling. Of clinical significance, infliximab, a TNF-α neutralizing antibody, reduces the bone loss in aged mice and improves the skeletal response to PTH. Collectively, we demonstrate a novel mechanism whereby TNF/TNFR/TRAF2 promotes aging-related bone loss and impairs the anabolic effects of PTH on bone in aged animals by inhibiting Gαs activation, and our findings suggest that modulating the TNF/TNFR signaling pathway may improve the effectiveness of PTH treatment in senile osteoporosis patients.

Ankylosing spondylitis (AS) is an autoimmune disease that can cause severe deformities, and the immunological patterns associated with its onset and progression remain poorly understood. Here, after recruiting healthy donors and patients in different stages, we performed single-cell RNA sequencing for peripheral blood mononuclear cells to investigate the cytotaxonomic and immunological hallmarks associated with AS onset, aggravation and remission and explore the intrinsic laws causing AS lesions. The results showed that innate antibacterial defense functions were generally enhanced in most cell types at disease onset and were negatively associated with AS severity. The abundance and exogenous antigen presentation scores of the natural killer (NK) cell subset characterized as antigen-presenting cells (APC-NK) increased during disease aggravation but decreased during remission. Generally, APC-NK abundance and their presentation scores were negatively correlated with innate defense scores for multiple cell types. CD4+ effector T cell abundance and cytotoxicity, as well as the enhancement of CD4+ T cell responses by HLA-DRB1+ NK cells (similar to APC-NK), were associated with AS severity. The implantation of HLA-DRB1+ NK cells accelerated AS-like alterations in SKG modeling mice with curdlan induction; this was blocked with CD4+ T cell exhaustion. NK cell exhaustion improved the phenotypes of AS-like mice. HLA-DPB1/DPA1 in APC-NK participated in AS aggravation by mediating antigen presentation targeting CD4+ T cells. Overall, innate defense antigen presentation coupling drives AS lesions and different outcomes. Furthermore, the trade-off between innate defense and NK-dependent exogenous antigen presentation results in CD4+ T cell activation or inactivation, thereby contributing to AS aggravation or remission; this reveals that APC-NK is a crucial factor causing ankylosing deformities.

Age-related inflammation plays a pivotal role in osteoarthritis (OA) pathogenesis, but the mechanism is not fully understood. Here, we identify decreased IL-36 receptor antagonists (IL-36Ra) in epidermal keratinocytes from a premature-aged skin mice model, aged mice and patients. Decreased IL-36Ra leads to increased secretion of IL-36 agonists to serum and joints, which activates proinflammatory signaling and promotes senescence in chondrocytes and synovial fibroblasts, thereby aggravates OA progression. Deletion of IL-36Ra in keratinocytes exacerbates, whereas intra-articular inhibition of IL-36R signaling effectively attenuates OA progression in male mice. Moreover, we also generate microneedles loaded with mouse recombinant IL-36Ra protein or spesolimab, insert them directly into skin to sustainably inhibit IL-36R signaling, which both clearly attenuate OA progression in male mice. Overall, our results reveal that IL-36 agonists are age-related systemic inflammatory factors released from skin to joints and contribute to OA development, and targeting IL-36R signaling in aged skin with microneedles represents a promising disease-modifying approach. Age-related inflammation is a key mediator of the pathogenesis of osteoarthritis (OA), but the mechanism is not fully understood. Here, the authors show that IL-36 agonists are systemic inflammatory factors released from aged skin to promote senescence in joint cells and aggravate OA progression.

This study investigated the therapeutic potential of CD200Fc in a mouse model of chronic neuroinflammation induced by intraperitoneal lipopolysaccharide (LPS) injections administered every other day for four weeks. Wild-type mice (Male mice aged 8 weeks) were divided into four groups: WT + PBS, WT + LPS, LPS + PBS, and LPS+CD200Fc (n = 16 per group). CD200Fc (4 µg per mouse, prepared at 2 µg/µL) was administered via intracerebroventricular stereotaxic injection at an infusion rate of 0.2µL/min, and intraperitoneal LPS injection was performed continuously within two weeks before and after treatment. The motor function of mice was assessed one week after model establishment using open-field, rotarod, and pole tests. Neuroinflammatory markers, microglial activation, autophagy function, and neuronal damage were evaluated via immunofluorescence and Western blot. The WT + LPS group exhibited significant motor dysfunction compared to WT + PBS controls, along with elevated levels of neuroinflammation-related proteins (including IL-18, IL-1β, and NLRP3) and the microglial activation marker CD68. These changes were accompanied by autophagic dysfunction and dopaminergic neuron loss. In contrast, the LPS+CD200Fc group showed notable improvements in motor function, reduced NLRP3 inflammasome activation, attenuated neuroinflammation, and restoration of autophagy. Dopaminergic neuronal damage was also significantly alleviated. These results indicate that CD200Fc significantly ameliorates LPS-induced neuroinflammation and motor dysfunction by suppressing microglial activation and restoring autophagic function. This supports further investigation into CD200Fc as a promising candidate for treating neuroinflammatory disorders of the central nervous system (CNS).

Osteoclast-development patterns and their alterations across Ankylosing Spondylitis (AS) conditions are mysterious, making AS treatment difficult. Our study aims to clarify osteoclast-precursor (OCP) development patterns from monocytes and their variations under AS conditions. We performed single-cell transcriptomics in peripheral blood mononuclear cells (PBMCs) from healthy donors and AS patients in the early, aggravated and remission stages. After monocytic reclustering, OCP-development patterns and the alterations upon AS onset and different outcomes were revealed based on single-cell trajectory. The trajectories revealed two monocyte states with strong OCP features, and AS pathogenesis was characterized by their reduction. Ribosome synthesis was considered the essential function for the development towards OCP-featured states, and this function and its representative molecule, RPS17, showed a decreasing trend with AS onset and outcomes. Histology assessment showed that RPS17 underexpression participated in AS inflammatory osteogenesis and ankylosing destruction. Conditional knockout of RPS17 ameliorated ovariectomy-induced bone loss and enhanced osteoclastogenesis, and RPS17 overexpression improved the phenotype of AS-like mice. Importantly, local injection of RPS17-overexpressed monocytic OCPs markedly ameliorated the joint alterations of AS-like mice without promoting bone loss; this was associated with enhanced osteoclastogenesis adjacent to the articular surface and T-cell-suppressive property in monocytic OCPs. Overall, the evolution of monocytes towards OCP-lineage fate mainly depends on ribosome synthesis, and OCP-development disorder participates in AS lesions due to a reduction in RPS17-dependent ribosome synthesis. Notably, RPS17-overexpressed monocytic OCPs have translational potential in preventing and treating AS peripheral lesions.

MicroRNAs (miRNAs) are emerging as important regulators in osteoarthritis (OA) pathogenesis. In our study, a real-time PCR assay revealed that miR-483-5p was upregulated in articular cartilage from OA patients and experimental OA mice induced by destabilization of the medial meniscus compared to their controls. Overexpression of miR-483-5p by intra-articular injection of lentivirus LV3-miR-483-5p significantly enhanced the severity of experimental OA. Consequently, we synthesized antago-miR-483-5p to silence the endogenous miR-483-5p and delivered it intra-articularly, which revealed that antago-miR-483-5p delayed the progression of experimental OA. To investigate the functional mechanism of miR-483-5p in OA development, we generated doxycycline-inducible miR-483 transgenic (TG483) mice. TG483 mice exhibited significant acceleration and increased severity of OA, and age-related OA occurred with higher incidence and greater severity in TG483 mice compared with their controls. Furthermore, our results revealed miR-483-5p directly targeted to the cartilage matrix protein matrilin 3 (Matn3) and tissue inhibitor of metalloproteinase 2 (Timp2) to stimulate chondrocyte hypertrophy, extracellular matrix degradation, and cartilage angiogenesis, and it consequently initiated and accelerated the development of OA. In conclusion, our findings reveal an miRNA functional pathway important for OA development. Targeting of miR-483-5p by intra-articular injection of antago-miR-483-5p represents an approach that could prevent the onset of OA and delay its progression.

Non-shivering thermogenesis of brown adipose tissue (BAT) is tightly controlled by neural innervation. However, the underlying mechanism remains unclear. Here, we reveal that BAT regulates its own thermoadaptive innervation by crosstalk with Schwann cells (SCs). Loss of Olfm4 (encoding Olfactomedin-4), a risk gene in human obesity, causes BAT dysfunction and reduces whole-body thermogenesis, predisposing to obesity in mice. Mechanistically, BAT-derived OLFM4 traps Noggin, an endogenous inhibitor of BMPs, liberating BMP7-BMPR1B signaling to promote SC differentiation. Conversely, Olfm4 loss reduced BMP7 signaling in mature SCs, leading to MEK/ERK-dependent dedifferentiation and dysfunction, ultimately impairing both sensory and sympathetic innervation. Thermoneutrality exposure reduces Olfm4 expression in BAT, resulting in a similar phenotype. MEK/ERK inhibition, ERK1 depletion, or cold exposure reverses this SC dedifferentiation, enhancing resistance to obesity. These findings suggest that this neurotrophic BAT-SC crosstalk controls thermoadaptive BAT innervation. Reactivating OLFM4 signaling may be a promising therapeutic strategy for obesity and related metabolic diseases.

The development of human organotypic models of cartilage provides essential insights into chondrogenesis and chondrocyte hypertrophy while enabling advanced applications in drug discovery, gene editing, and tissue regeneration. Here, we present a robust and efficient protocol for differentiating human expanded pluripotent stem cells (hEPSCs) into hypertrophic chondrocytes through a sclerotome intermediate. The protocol involves initial sclerotome induction, followed by 3D chondrogenic culture and subsequent hypertrophic maturation induced by bone morphogenetic protein-4 (BMP4), thyroid hormone (T3), and β-glycerophosphate. This protocol also allows for sensitive testing of the effects of various compounds on hypertrophic differentiation during the maturation process. Furthermore, we identify an α-adrenergic receptor antagonist, phentolamine, as an inhibitor of hypertrophic differentiation. This organoid system provides a practical platform for exploring cartilage hypertrophy mechanisms and testing therapeutic strategies for cartilage regeneration. Key features • This differentiation protocol generates hypertrophic chondrocytes from hEPSCs through a sclerotome intermediate. • This protocol facilitates sensitive testing of compounds during the hypertrophic maturation stage, enabling the study of molecular mechanisms and therapeutic interventions for cartilage hypertrophy. • This protocol identifies the α-adrenergic receptor antagonist phentolamine as a modulator of hypertrophic differentiation.

Aims: Osteoarthritis (OA) is a chronic and debilitating joint disease. Despite its prevalence, especially in ageing and obese populations, effective treatments targeting the molecular mechanisms of OA are limited. This study aimed to investigate the role of carpaine (CP), a major alkaloid from the Carica papaya leaf, in inhibiting articular cartilage destruction and synovitis during OA progression, and to elucidate the underlying molecular mechanisms. Methods: CP (purity > 98%) was dissolved in dimethyl sulfoxide (DMSO). Various antibodies and reagents were sourced from Sigma-Aldrich, Abcam, and other suppliers. Peritoneal macrophages (pMACs) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and treated with CP to assess its effects on inflammatory cytokine production and nuclear factor-kappa B (NF-κB) signalling. A total of 40 ten-week-old male C57/BL6 mice underwent destabilization of the medial meniscus (DMM) surgery to induce OA. Post-surgery, mice were treated with CP (0.5 or 3 mg/kg) or vehicle via intra-articular injections for up to ten weeks. Cartilage degradation and synovitis were evaluated using Safranin O, Fast Green staining, haematoxylin and eosin (H&E) staining, immunohistochemistry, and quantitative polymerase chain reaction (PCR). Results: CP treatment significantly reduced cartilage degeneration and maintained hyaline cartilage thickness compared to the vehicle group. Indicators of cartilage degeneration, such as collagen X (Col X) and matrix metallopeptidase 13 (MMP13), were markedly decreased in the CP-treated group. CP-treated mice exhibited significantly lower synovitis scores at both five and ten weeks post-DMM surgery. CP prominently decreased the production of proinflammatory cytokines (interleukin (IL)-1β, IL-6) in M1 polarized macrophages both in vitro and in vivo. CP impeded NF-κB signalling by promoting p65 degradation through the E3 ubiquitin ligase LRSAM1. The defensive effect of CP was reversed by Lrsam1 small interfering RNA (siRNA), confirming the role of LRSAM1 in CP-mediated NF-κB inhibition. Conclusion: CP acts as a ‘physiological brake’ on NF-κB activation, thereby mitigating synovial inflammation and cartilage destruction in OA. These findings suggest that targeting synovitis via CP could be a promising therapeutic strategy for OA. Cite this article: Bone Joint Res 2025;14(4):353–364.

Mechanical overload is a critical contributor to cartilage degeneration in osteoarthritis (OA) pathogenesis. Circular RNA (circRNA) is expected to provide a long-lasting therapy for OA. However, the involvement of the circRNA-associated competitive endogenous RNA network in chondrocyte senescence induced by mechanical overloading remains unestablished. A mechanical overloading-induced chondrocyte senescence model in human primary chondrocytes is constructed, and differences in the expression of circRNAs and miRNAs were analyzed. The biological roles of circKIAA0586/miR-335-5p in chondrocyte senescence and OA progression under mechanical overloading and its downstream targets were determined using gain- and loss-of-function experiments in various biochemical assays in human chondrocytes. The in vivo effects of circKIAA0586 overexpression were also determined in destabilization of the medial meniscus (DMM) OA mice and aged spontaneous OA mice. The mechanical overloading-induced chondrocyte senescence was aggravated by miR-335-5p or circKIAA0586 knockdown. Accumulated DNA damage response was observed following mechanical overloading, which reduced after miR-335-5p inhibition or circKIAA0586 supplementation. MiR-335-5p was regulated by circKIA0586 adsorption. HELLS was prominently down-regulated following mechanical overloading treatment. Moreover, miR-335-5p bound to lymphoid-specific helicase (HELLS) mRNA during mechanical overloading was demonstrated to mediate the nonhomologous end joining (NHEJ) pathway, thereby inducing DNA damage and senescence. In addition, the senescence delaying and cartilage protective functions of circKIAA0586 and HELLS were validated in DMM OA mice and aged spontaneous OA mice. Our findings suggest that miR-335-5p, which escapes circKIAA0586 adsorption, facilitates mechanical overloading-induced chondrocyte senescence and OA progression by impairing the NHEJ pathway through HELLS inhibition. Overall, targeting circKIAA0586/miR-335-5p/HELLS signaling provides a novel therapeutic approach for OA.