14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA. METHODS: Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA. RESULTS: Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes. CONCLUSIONS: This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.

Progranulin promotes bone fracture healing via TNFR pathways in mice with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) significantly increases bone fragility and fracture risk. Progranulin (PGRN) promotes bone fracture healing in both physiological and type 1 diabetic conditions. The present study aimed to investigate the role of PGRN in T2DM bone fracture healing. MKR mice (with an FVB/N genetic background) were used as the T2DM model. Drill-hole and Bonnarens and Einhorn models were used to investigate the role of PGRN in T2DM fracture healing in vivo. Primary bone marrow cells were isolated for molecular and signaling studies, and reverse transcription-polymerase chain reaction, immunohistochemical staining, and western blotting were performed to assess PGRN effects in vitro. PGRN mRNA and protein expression were upregulated in the T2DM model. Local administration of recombinant PGRN effectively promoted T2DM bone fracture healing in vivo. Additionally, PGRN could induce anabolic metabolism during endochondral ossification through the TNFR2-Akt and Erk1/2 pathways. Furthermore, PGRN showed anti-inflammatory activity in the T2DM bone regeneration process. These findings suggest that local administration of exogenous PGRN may be an alternative strategy to support bone regeneration in patients with T2DM. Additionally, PGRN might hold therapeutic potential for other TNFR-related metabolic disorders.

Progranulin promotes diabetic fracture healing in mice with type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by insulin deficiency, and patients with diabetes have an increased risk of bone fracture and significantly impaired fracture healing. Proinflammatory cytokine tumor necrosis factor-alpha is significantly upregulated in diabetic fractures and is believed to underlie delayed fracture healing commonly observed in diabetes. Our previous genetic screen for the binding partners of progranulin (PGRN), a growth factor-like molecule that induces chondrogenesis, led to the identification of tumor necrosis factor receptors (TNFRs) as the PGRN-binding receptors. In this study, we employed several in vivo models to ascertain whether PGRN has therapeutic effects in diabetic fracture healing. Here, we report that deletion of PGRN significantly delayed bone fracture healing and aggravated inflammation in the fracture models of mice with T1DM. In contrast, recombinant PGRN effectively promoted diabetic fracture healing by inhibiting inflammation and enhancing chondrogenesis. In addition, both TNFR1 proinflammatory and TNFR2 anti-inflammatory signaling pathways are involved in PGRN-stimulated diabetic fracture healing. Collectively, these findings illuminate a novel understanding concerning the role of PGRN in diabetic fracture healing and may have an application in the development of novel therapeutic intervention strategies for diabetic and other types of impaired fracture healing.

Progranulin derivative Atsttrin protects against early osteoarthritis in mouse and rat models.

BACKGROUND: Atsttrin, an engineered protein composed of three tumor necrosis factor receptor (TNFR)-binding fragments of progranulin (PGRN), shows therapeutic effect in multiple murine models of inflammatory arthritis . Additionally, intra-articular delivery of PGRN protects against osteoarthritis (OA) progression. The purpose of this study is to determine whether Atsttrin also has therapeutic effects in OA and the molecular mechanisms involved. METHODS: Surgically induced and noninvasive rupture OA models were established in mouse and rat, respectively. Cartilage degradation and OA were evaluated using Safranin O staining, immunohistochemistry, and ELISA. Additionally, expressions of pain-related markers, degenerative factors, and anabolic and catabolic markers known to be involved in OA were analyzed. Furthermore, the anabolic and anti-catabolic effects and underlying mechanisms of Atsttrin were determined using in-vitro assays with primary chondrocytes. RESULTS: Herein, we found Atsttrin effectively prevented the accelerated OA phenotype associated with PGRN deficiency. Additionally, Atsttrin exhibited a preventative effect in OA by protecting articular cartilage and reducing OA-associated pain in both nonsurgically induced rat and surgically induced murine OA models. Mechanistic studies revealed that Atsttrin stimulated TNFR2-Akt-Erk1/2-dependent chondrocyte anabolism, while inhibiting TNFα/TNFR1-mediated inflammatory catabolism. CONCLUSIONS: These findings not only provide new insights into the role of PGRN and its derived engineered protein Atsttrin in cartilage homeostasis as well as OA in vivo, but may also lead to new therapeutic alternatives for OA as well as other relative degenerative joint diseases.

Interleukin-17A- or tumor necrosis factor α-mediated increase in proliferation of T cells cocultured with synovium-derived mesenchymal stem cells in rheumatoid arthritis.

INTRODUCTION: Mesenchymal stem cells (MSCs) represent promising applications in rheumatoid arthritis (RA). However, the inflammatory niche in the RA synovium could adversely affect MSC function. This study was designed to investigate biologic and immunologic properties of synovium-derived MSCs (SMSCs) in RA, with particular focus on whether cytokines can mediate increase of proliferation of T cells cocultured with SMSCs in RA. METHODS: Compared with SMSCs from eight healthy donors (HDs), SMSCs from 22 patients with RA (RAp) were evaluated. The methyl thiazolyl tetrazolium (MTT) assay was used to assess cell-population doubling and viability. Multipotentiality of SMSCs was examined by using appropriate culture conditions. Flow cytometry was used to investigate the marker phenotype of SMSCs. Immunomodulation potential of SMSCs was examined by mixed peripheral blood mononuclear cells (PBMCs) reactions, and then by PBMCs or synovial T cells with or without the addition of inflammatory cytokines (interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) after stimulation with phytohemagglutinin (PHA), respectively. RESULTS: SMSCs from RA patients (RA-SMSCs) showed normal population doubling, cell viability, multiple differentiation characteristics, and surface markers. In either mixed PBMC reactions or PBMC proliferation stimulated with PHA, RA-SMSCs showed normal immunomodulation function compared with SMSCs from healthy donors (HD-SMSCs). However, the increase in proliferation of T cells was observed when IL-17A and TNF-α were added alone or in combination. CONCLUSIONS: Our data suggest that the inflammatory niche, especially these cytokines, may increase the proliferation of T cells cocultured with SMSCs in RA.

Recombinant human erythropoietin prevents motor neuron apoptosis in a rat model of cervical sub-acute spinal cord compression.

The objective of the study was to investigate the effects of recombinant human erythropoietin (rhEPO) in a rat model of cervical sub-acute spinal cord compression. 80 Wistar rats were randomly divided into 4 groups. Rats in the sham group (Group A, n=5) underwent surgical procedures without cervical spinal cord compression; while rats in other groups were subjected to the spinal compression process. In the control group (Group B, n=25), rats received an i.v. injection of 1 mL saline at day 7 post-surgery. Rats in the low-dose group (Group C, n=25) and the high-dose group (Group D, n=25) were treated with rhEPO at 500 units/kg body-weight and 5000 units/kg, respectively, via intravenous injection at day 7 post surgery. Limb motor function was scored by Basso-Beattie-Bresnahan (BBB) standards at 3, 7, 14, 21 and 28 days post-surgery. The distribution and quantities of EPO and its receptor (EPO-R) in the compressed segment of the spinal cord were detected by immunohistochemistry. Motor neuron apoptosis in the spinal cord was evaluated using TUNEL staining and flow cytometry at the indicated time points. Finally, IL-8, TNF-α, IL-6, and IL-1ß levels in the compressed cervical spinal cord were determined by ELISA within the lesion epicenter at each time point post-surgery. The data suggest that expression of EPO-R was significantly increased following sub-acute cervical spinal cord compression; Groups C and D exhibited better BBB scores at all observed time points compared with the control group (p<0.01). Using TUNEL staining and FCM, we observed that rhEPO profoundly inhibited motor neuron apoptosis in the spinal cord at day 21 (p<0.01). Additionally, treatment with rhEPO halted the elevation of inflammatory cytokines. rhEPO administration decreased motor neuron apoptosis in the cervical spinal cord, improved motor functions and reduced the inflammatory response in a sub-acute cervical spinal cord compression model. Moreover, sustained treatment with low doses of rhEPO revealed a positive therapeutic effect.