Boards, Advisory Committees, Professional Organizations

  • Member, Japanese Orthopaedic Association (2012 - Present)
  • Member, Japan College of Rheumatology (2017 - Present)
  • Member, Japanese Society of Cartilage Metabolism (2017 - Present)
  • Member, Orthopaedic Research Society (2019 - Present)
  • Member, Japanese Society of Inflammation and Regeneration (2021 - Present)

Professional Education

  • Master of Medicine, Kyushu University (2021)
  • Doctor of Medicine, Kyushu University (2012)
  • Doctor of Philosophy, Kyushu University (2021)
  • Doctor of Medicine, Kyushu University (2012)
  • Ph.D., Graduated school of Kyushu University, School of Medicine (Fukuoka, Japan) (2021)
  • Board Certification, Rheumatologist of the Japanese Orthopaedic Association (2020)
  • Board Certification, Japanese Orthopaedic Association (2019)
  • M.D., Kyushu University, School of Medicine (Fukuoka, Japan) (2012)

Stanford Advisors

Current Research and Scholarly Interests

Rheumatoid Arthritis, Cartilage Metabolism

All Publications

  • Glycolytic reprogramming in macrophages and MSCs during inflammation. Frontiers in immunology Li, X., Shen, H., Zhang, M., Teissier, V., Huang, E. E., Gao, Q., Tsubosaka, M., Toya, M., Kushioka, J., Maduka, C. V., Contag, C. H., Chow, S. K., Zhang, N., Goodman, S. B. 2023; 14: 1199751


    Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs).We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE.Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines.This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

    View details for DOI 10.3389/fimmu.2023.1199751

    View details for PubMedID 37675119

    View details for PubMedCentralID PMC10477714

  • CCL2 promotes osteogenesis by facilitating macrophage migration during acute inflammation. Frontiers in cell and developmental biology Toya, M., Zhang, N., Tsubosaka, M., Kushioka, J., Gao, Q., Li, X., Chow, S. K., Goodman, S. B. 2023; 11: 1213641


    Novel minimally invasive strategies are needed to obtain robust bone healing in complex fractures and bone defects in the elderly population. Local cell therapy is one potential option for future treatment. Mesenchymal stromal cells (MSCs) are not only involved in osteogenesis but also help direct the recruitment of macrophages during bone regeneration via MSC-macrophage crosstalk. The C-C motif chemokine ligand 2 (CCL2) is an inflammatory chemokine that is associated with the migration of macrophages and MSCs during inflammation. This study investigated the use of CCL2 as a therapeutic target for local cell therapy. MSCs and macrophages were isolated from 10 to 12 week-old BALB/c male mice. Genetically modified CCL2 over-expressing MSCs were produced using murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture. Cell migration assays were also performed. MSCs transfected with murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector showed higher levels of CCL2 secretion compared to unaltered MSCs (p < 0.05). Genetic manipulation did not affect cell proliferation. CCL2 did not affect the osteogenic ability of MSCs alone. However, acute (1 day) but not sustained (7 days) stimulation with CCL2 increased the alizarin red-positive area when MSCs were co-cultured with macrophages (p < 0.001). Both recombinant CCL2 (p < 0.05) and CCL2 released from MSCs (p < 0.05) facilitated macrophage migration. We demonstrated that acute CCL2 stimulation promoted subsequent osteogenesis in co-culture of MSCs and macrophages. Acute CCL2 stimulation potentially facilitates osteogenesis during the acute inflammatory phase of bone healing by directing local macrophage migration, fostering macrophage-MSC crosstalk, and subsequently, by activating or licensing of MSCs by macrophage pro-inflammatory cytokines. The combination of CCL2, MSCs, and macrophages could be a potential strategy for local cell therapy in compromised bone healing.

    View details for DOI 10.3389/fcell.2023.1213641

    View details for PubMedID 37457301

    View details for PubMedCentralID PMC10348816

  • Bone regeneration in inflammation with aging and cell-based immunomodulatory therapy. Inflammation and regeneration Kushioka, J., Chow, S. K., Toya, M., Tsubosaka, M., Shen, H., Gao, Q., Li, X., Zhang, N., Goodman, S. B. 2023; 43 (1): 29


    Aging of the global population increases the incidence of osteoporosis and associated fragility fractures, significantly impacting patient quality of life and healthcare costs. The acute inflammatory reaction is essential to initiate healing after injury. However, aging is associated with "inflammaging", referring to the presence of systemic low-level chronic inflammation. Chronic inflammation impairs the initiation of bone regeneration in elderly patients. This review examines current knowledge of the bone regeneration process and potential immunomodulatory therapies to facilitate bone healing in inflammaging.Aged macrophages show increased sensitivity and responsiveness to inflammatory signals. While M1 macrophages are activated during the acute inflammatory response, proper resolution of the inflammatory phase involves repolarizing pro-inflammatory M1 macrophages to an anti-inflammatory M2 phenotype associated with tissue regeneration. In aging, persistent chronic inflammation resulting from the failure of M1 to M2 repolarization leads to increased osteoclast activation and decreased osteoblast formation, thus increasing bone resorption and decreasing bone formation during healing.Inflammaging can impair the ability of stem cells to support bone regeneration and contributes to the decline in bone mass and strength that occurs with aging. Therefore, modulating inflammaging is a promising approach for improving bone health in the aging population. Mesenchymal stem cells (MSCs) possess immunomodulatory properties that may benefit bone regeneration in inflammation. Preconditioning MSCs with pro-inflammatory cytokines affects MSCs' secretory profile and osteogenic ability. MSCs cultured under hypoxic conditions show increased proliferation rates and secretion of growth factors. Resolution of inflammation via local delivery of anti-inflammatory cytokines is also a potential therapy for bone regeneration in inflammaging. Scaffolds containing anti-inflammatory cytokines, unaltered MSCs, and genetically modified MSCs can also have therapeutic potential. MSC exosomes can increase the migration of MSCs to the fracture site and enhance osteogenic differentiation and angiogenesis.In conclusion, inflammaging can impair the proper initiation of bone regeneration in the elderly. Modulating inflammaging is a promising approach for improving compromised bone healing in the aging population.

    View details for DOI 10.1186/s41232-023-00279-1

    View details for PubMedID 37231450

    View details for PubMedCentralID 2880220

  • Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model. Stem cell research & therapy Teissier, V., Gao, Q., Shen, H., Li, J., Li, X., Huang, E. E., Kushioka, J., Toya, M., Tsubosaka, M., Hirata, H., Alizadeh, H. V., Maduka, C. V., Contag, C. H., Yang, Y. P., Zhang, N., Goodman, S. B. 2023; 14 (1): 99


    Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway.We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs.Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days.This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

    View details for DOI 10.1186/s13287-023-03260-4

    View details for PubMedID 37085909

    View details for PubMedCentralID PMC10122387

  • The efficiency of genetically modified mesenchymal stromal cells combined with a functionally graded scaffold for bone regeneration in corticosteroid-induced osteonecrosis of the femoral head in rabbits. Journal of biomedical materials research. Part A Tsubosaka, M., Maruyama, M., Lui, E., Moeinzadeh, S., Huang, E. E., Kushioka, J., Hirata, H., Jain, C., Storaci, H. W., Chan, C., Toya, M., Gao, Q., Teissier, V., Shen, H., Li, X., Zhang, N., Matsumoto, T., Kuroda, R., Goodman, S. B., Yang, Y. P. 2023


    Core decompression (CD) with mesenchymal stromal cells (MSCs) is an effective therapy for early-stage osteonecrosis of the femoral head (ONFH). Preconditioning of MSCs, using inflammatory mediators, is widely used in immunology and various cell therapies. We developed a three-dimensional printed functionally graded scaffold (FGS), made of beta-TCP and PCL, for cell delivery at a specific location. The present study examined the efficacy of CD treatments with genetically modified (GM) MSCs over-expressing PDGF-BB (PDGF-MSCs) or GM MSCs co-over-expressing IL-4 and PDGF-BB and preconditioned for three days of exposure to lipopolysaccharide and tumor necrosis factor-alpha (IL-4-PDGF-pMSCs) using the FGS for treating steroid-induced ONFH in rabbits. We compared CD without cell-therapy, with IL-4-PDGF-pMSCs alone, and with FGS loaded with PDGF-MSCs or IL-4-PDGF-pMSCs. For the area inside the CD, the bone volume in the CD alone was higher than in both FGS groups. The IL-4-PDGF-pMSCs alone and FGS+PDGF-MSCs reduced the occurrence of empty lacunae and improved osteoclastogenesis. There was no significant difference in angiogenesis among the four groups. The combined effect of GM MSCs or pMSCs and the FGS was not superior to the effect of each alone. To establish an important adjunctive therapy for CD for early ONFH in the future, it is necessary and essential to develop an FGS that delivers biologics appropriately and provides structural and mechanical support.

    View details for DOI 10.1002/jbm.a.37495

    View details for PubMedID 36606330

  • Therapeutic effects of MSCs, genetically modified MSCs, and NFkB-inhibitor on chronic inflammatory osteolysis in aged mice. Journal of orthopaedic research : official publication of the Orthopaedic Research Society Kushioka, J., Toya, M., Shen, H., Hirata, H., Zhang, N., Huang, E., Tsubosaka, M., Gao, Q., Teissier, V., Li, X., Utsunomiya, T., Goodman, S. B. 2022


    The number of total joint replacements is increasing, especially in elderly patients, and so too are implant-related complications such as prosthesis loosening. Wear particles from the prosthesis induce a chronic inflammatory reaction and subsequent osteolysis, leading to the need for revision surgery. This study investigated the therapeutic effect of NF-kB decoy oligodeoxynucleotides (ODN), mesenchymal stem cells (MSCs), and genetically-modified NF-kB sensing interleukin-4 over-secreting MSCs (IL4-MSCs) on chronic inflammation in aged mice. The model was generated by continuous infusion of contaminated polyethylene particles into the intramedullary space of the distal femur of aged mice (15-17-month-old) for six weeks. Local delivery of ODN showed increased bone mineral density (BMD), decreased osteoclast-like cells, increased alkaline phosphatase (ALP)-positive area, and increased M2/M1 macrophage ratio. Local injection of MSCs and IL4-MSCs significantly decreased osteoclast-like cells and increased the M2/M1 ratio, with a greater trend for IL4-MSCs than MSCs. MSCs significantly increased ALP-positive area and BMD values compared to the control. The IL4-MSCs demonstrated higher values for both ALP-positive area and BMD. These findings demonstrated the therapeutic effects of ODN, MSCs, and IL4-MSCs on chronic inflammatory osteolysis in aged mice. The two MSC-based therapies were more effective than ODN in increasing the M2/M1 macrophage ratio, reducing bone resorption, and increasing bone formation. Specifically, MSCs were more effective in increasing bone formation, and IL4-MSCs were more effective in mitigating inflammation. This study suggests potential therapeutic strategies for treating wear particle-associated inflammatory osteolysis after arthroplasty in the elderly. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/jor.25434

    View details for PubMedID 36031590

  • Ageing attenuates bone healing by mesenchymal stem cellsin a microribbon hydrogelwith a murine long bone critical-size defect model. Immunity & ageing : I & A Hirata, H., Zhang, N., Ueno, M., Barati, D., Kushioka, J., Shen, H., Tsubosaka, M., Toya, M., Lin, T., Huang, E., Yao, Z., Wu, J. Y., Zwingenberger, S., Yang, F., Goodman, S. B. 2022; 19 (1): 14


    BACKGROUND: Despite the high incidence of fractures and pseudoarthrosis in the aged population, a potential role for the use of mesenchymal stem cells (MSCs) in the treatment of bone defects in elderly patients has not been elucidated. Inflammation and the innate immune system, including macrophages, play crucial roles in the differentiation and activation of MSCs. We have developed lentivirus-transduced interleukin 4 (IL4) over-expressing MSCs (IL4-MSCs) to polarize macrophages to an M2 phenotype to promote bone healing in an established young murine critical size bone defect model. In the current study, we explore the potential of IL4-MSCs in aged mice.METHODS: A 2mm femoral diaphyseal bone defect was created and fixed with an external fixation device in 15- to 17-month-old male and female BALB/c mice. Microribbon (RB) scaffolds (Sc) with or without encapsulation of MSCs were implanted in the defect sites. Accordingly, the mice were divided into three treatment groups: Sc-only, Sc+MSCs, and Sc+IL4-MSCs. Mice were euthanized six weeks after the surgery; subsequently, MicroCT (CT), histochemical and immunohistochemical analyses were performed.RESULTS: CT analysis revealed that bone formation was markedly enhanced in the IL4-MSC group. Compared with the Sc-only, the amount of new bone increased in the Sc+MSCs and Sc+IL4-MSC groups. However, no bridging of bone was observed in all groups. H&E staining showed fibrous tissue within the defect in all groups. Alkaline phosphatase (ALP) staining was increased in the Sc+IL4-MSC group. The Sc+IL4-MSCs group showed a decrease in the number of M1 macrophages and an increase in the number of M2 macrophages, with a significant increase in the M2/M1 ratio.DISCUSSION: IL4 promotes macrophage polarization to an M2 phenotype, facilitating osteogenesis and vasculogenesis. The addition of IL4-MSCs in the RB scaffold polarized macrophages to an M2 phenotype and increased bone formation; however, complete bone bridging was not observed in any specimens. These results suggest that IL4-MSCs are insufficient to heal a critical size bone defect in aged mice, as opposed to younger animals. Additional therapeutic strategies are needed in this challenging clinical scenario.

    View details for DOI 10.1186/s12979-022-00272-1

    View details for PubMedID 35279175

  • Factors affecting patient satisfaction related to cost and treatment effectiveness in rheumatoid arthritis: results from the multicenter observational cohort study, FRANK Registry ARTHRITIS RESEARCH & THERAPY Fujiwara, T., Kondo, M., Yamada, H., Haraguchi, A., Fujimura, K., Sakuraba, K., Kamura, S., Fukushi, J., Miyahara, H., Inoue, Y., Tsuru, T., Shuto, T., Yoshizawa, S., Suematsu, E., Miyamura, T., Ayano, M., Mitoma, H., Arinobu, Y., Niiro, H., Ohishi, M., Hirata, A., Tokunaga, S., Takada, A., Hara, D., Tsushima, H., Akasaki, Y., Ikemura, S., Sueishi, T., Toya, M., Sakuragi, T., Tsutsui, T., Kai, K., Arisumi, S., Nakashima, Y. 2022; 24 (1): 53


    To further improve rheumatoid arthritis (RA) treatment, it is necessary to understand each RA patient's satisfaction and to identify the factors affecting their satisfaction. Despite the rise in medical costs for RA, little is known about the factors that influence patient satisfaction with the cost of treatment in RA patients.This is a multicenter observational study of Japanese RA patients from the FRANK Registry with data analyzed from March 2017 to August 2020. We collected data on demographic characteristics, clinical data, quality of life which was evaluated using the EuroQol 5-dimensional questionnaire (EQ5D), and patient satisfaction. The four categories of patient satisfaction were evaluated individually (i.e., cost, treatment efficacy, activities of daily living [ADL], and global treatment satisfaction). We analyzed the factors that affected each patient's satisfaction, such as age, sex, EQ5D, disease duration, disease activity, and treatment.This study included 2235 RA outpatients (406 males, 1829 females). In RA patients, "very satisfied" and "satisfied" were given for nearly half of each satisfaction aspect (cost 49%; efficacy 72%; ADL 58%; global treatment 66%) at the time of the initial registration. To investigate the factors influencing each satisfaction, multivariate analysis has revealed that the use of b/tsDMARDs increased satisfaction of treatment effect (odds ratio [OR] 0.66) and ADL (OR 0.78) but decreased cost satisfaction (OR 2.21). Age (50-64 years; OR 0.91; 65-74 years, 0.55: ≥ 75 years, 0.35), female (OR 0.81), and history of musculoskeletal surgery (OR 0.60) all increased cost satisfaction. Patients with lower disease activity and higher EQ5D scores had higher levels of satisfaction in all areas.In this study, patient satisfaction in terms of cost, treatment effect, ADL, and overall treatment was generally higher, but some patients were dissatisfied. The cost of satisfaction increased with age and a history of musculoskeletal surgery, while it decreased with a lower EQ5D score and the use of b/tsDMARDs.

    View details for DOI 10.1186/s13075-022-02746-5

    View details for Web of Science ID 000759580000003

    View details for PubMedID 35193646

    View details for PubMedCentralID PMC8862466

  • C10orf10/DEPP activates mitochondrial autophagy and maintains chondrocyte viability in the pathogenesis of osteoarthritis FASEB JOURNAL Kuwahara, M., Akasaki, Y., Kurakazu, I., Sueishi, T., Toya, M., Uchida, T., Tsutsui, T., Hirose, R., Tsushima, H., Teramura, T., Nakashima, Y. 2022; 36 (2): e22145


    Osteoarthritis (OA), the most prevalent joint disease, is characterized by the progressive loss of articular cartilage. Autophagy, a lysosomal degradation pathway, maintains cellular homeostasis, and autophagic dysfunction in chondrocytes is a hallmark of OA pathogenesis. However, the cause of autophagic dysfunction in OA chondrocytes remains incompletely understood. Recent studies have reported that decidual protein induced by progesterone (C10orf10/DEPP) positively regulates autophagic functions. In this study, we found that DEPP was involved in mitochondrial autophagic functions of chondrocytes, as well as in OA pathogenesis. DEPP expression decreased in human OA chondrocytes in the absence or presence of pro-inflammatory cytokines, and was induced by starvation, hydrogen peroxide (H2 O2 ), and hypoxia (cobalt chloride). For functional studies, DEPP knockdown decreased autophagic flux induced by H2 O2 , whereas DEPP overexpression increased autophagic flux and maintained cell viability following H2 O2 treatment. DEPP was downregulated by knockdown of forkhead box class O (FOXO) transcription factors and modulated the autophagic function regulated by FOXO3. In an OA mouse model by destabilization of the medial meniscus, DEPP-knockout mice exacerbated the progression of cartilage degradation with TUNEL-positive cells, and chondrocytes isolated from knockout mice were decreased autophagic flux and increased cell death following H2 O2 treatment. Subcellular fractionation analysis revealed that mitochondria-located DEPP activated mitochondrial autophagy via BCL2 interacting protein 3. Taken together, our data demonstrate that DEPP is a major stress-inducible gene involved in the activation of mitochondrial autophagy in chondrocytes, and maintains chondrocyte viability during OA pathogenesis. DEPP represents a potential therapeutic target for enhancing autophagy in patients with OA.

    View details for DOI 10.1096/fj.202100896R

    View details for Web of Science ID 000747987800006

    View details for PubMedID 34997944

  • Sex differences in the therapeutic effect of unaltered versus NFkappaB sensing IL-4 over-expressing mesenchymal stromal cells in a murine model of chronic inflammatory bone loss. Frontiers in bioengineering and biotechnology Shen, H., Kushioka, J., Toya, M., Utsunomiya, T., Hirata, H., Huang, E. E., Tsubosaka, M., Gao, Q., Li, X., Teissier, V., Zhang, N., Goodman, S. B. 2022; 10: 962114


    Wear particles from joint arthroplasties induce chronic inflammation associated with prolonged upregulation of nuclear factor kappa-B (NF-kappaB) signaling in macrophages and osteoclasts, which leads to osteolysis and implant loosening. Mesenchymal stromal cell (MSC)-based therapy showed great potential for immunomodulation and mitigation of osteolysis in vivo, especially in the chronic phase of inflammation. We previously generated genetically modified MSCs that secrete the anti-inflammatory cytokine interleukin 4 (IL-4) in response to NF-kappaB activation (NFkappaB-IL-4 MSCs). However, whether the impact of sexual difference in the internal environment can alter the therapeutic effects of IL-4 over-secreting MSCs that simultaneously mitigate prolonged inflammation and enhance bone formation remains unknown. This study investigated the therapeutic effects of unaltered MSCs versus NFkappaB-IL-4 MSCs in mitigating chronic inflammation and enhancing bone formation in male and female mice. The murine model was established by continuous infusion of polyethylene particles contaminated with lipopolysaccharide (cPE) into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation. Unaltered MSCs or NFkappaB-IL-4 MSCs were infused into the femoral intramedullary cavity in sex-matched groups beginning 3 weeks after primary surgery. Femurs were harvested at 6 weeks, and bone marrow density was measured with micro-computational tomography. Numbers of osteoclast-like cells, osteoblasts, and macrophages were evaluated with histochemical and immunofluorescence staining. cPE infusion resulted in severe bone loss at the surgery site, increased tartrate-resistant acid phosphatase positive osteoclasts and M1 pro-inflammatory macrophages, and decreased alkaline phosphatase expression. MSC-based therapy effectively decreased local bone loss and polarized M1 macrophages into an M2 anti-inflammatory phenotype. In females, unaltered MSCs demonstrated a larger impact in enhancing the osteogenesis, but they demonstrated similar anti-inflammatory effects compared to NFkappaB-IL-4 MSCs. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments in a sexually dimorphic manner, which could be an efficacious therapeutic strategy for treatment of periprosthetic osteolysis in both genders.

    View details for DOI 10.3389/fbioe.2022.962114

    View details for PubMedID 36046680

  • TGFβ1 signaling protects chondrocytes against oxidative stress via FOXO1-autophagy axis. Osteoarthritis and cartilage Kurakazu, I., Akasaki, Y., Tsushima, H., Sueishi, T., Toya, M., Kuwahara, M., Uchida, T., Lotz, M. K., Nakashima, Y. 2021; 29 (11): 1600-1613


    The forkhead box O1 (FOXO1) transcription factor is a key regulator of autophagy. In chondrocytes, reduced FOXO1 expression with aging causes osteoarthritis due to dysfunction of autophagy, but the mechanisms underlying regulation of FOXO1 expression and the reduction in expression with aging remain unclear. We investigated the mechanism by which transforming growth factor β1 (TGFβ1) signaling regulates the FOXO1-autophagy axis.Expression of FOXO1 was measured in chondrocytes after TGFβ1 treatment. Immunohistochemistry was performed to estimate the levels of activin receptor-like kinase 5 (ALK5) and FOXO1 in the knee joints of young, middle-aged and old mice. The effects of the ALK5 inhibitor and SMAD3 or SMAD2 knockdown on FOXO1 expression were evaluated. The role of TGFβ1 in autophagy after hydrogen peroxide (H2O2) treatment was analyzed. The protective effect of TGFβ1 against H2O2 treatment was assessed by cell viability assay and TUNEL assay.TGFβ1 promoted the expression of FOXO1 mRNA and protein. Both ALK5 and FOXO1 expression decreased with aging. ALK5 inhibition and SMAD3 knockdown suppressed induction of FOXO1 expression by TGFβ1, whereas SMAD2 knockdown increased it. TGFβ1 promoted the expression of microtubule-associated proteins 1A/1B light chain 3B (LC3)-I protein via the SMAD3-FOXO1 pathway. Furthermore, under H2O2 treatment, TGFβ1 promoted expression of LC3-II. TGFβ1 pretreatment suppressed cell death of chondrocytes following H2O2 treatment, but this protective effect was abolished by FOXO1 knockdown.TGFβ1 protects chondrocytes against oxidative stress via the FOXO1-autophagy axis, and a reduction in ALK5 expression might cause reduced FOXO1 expression with aging.

    View details for DOI 10.1016/j.joca.2021.07.015

    View details for PubMedID 34419603

  • G protein-coupled receptor kinase 5 deletion suppresses synovial inflammation in a murine model of collagen antibody-induced arthritis SCIENTIFIC REPORTS Toya, M., Akasaki, Y., Sueishi, T., Kurakazu, I., Kuwahara, M., Uchida, T., Tsutsui, T., Tsushima, H., Yamada, H., Lotz, M. K., Nakashima, Y. 2021; 11 (1): 10481


    G protein-coupled receptor kinase 5 (GRK5) regulates inflammatory responses via the nuclear factor-kappa B (NF-κB) pathway. This study investigated the functional involvement of GRK5 in the pathogenesis of inflammatory arthritis. Immunohistochemically, rheumatoid arthritis (RA) synovium had a significantly higher proportion of GRK5-positive cells in the synovial lining layer than healthy control synovium. Gene expression and NF-κB activation in lipopolysaccharide-stimulated human SW982 synovial cells were significantly suppressed by silencing of the GRK5 gene. Similarly, GRK5 kinase activity inhibition in human primary RA synovial cells attenuated gene expressions of inflammatory factors. In a murine model of collagen antibody-induced arthritis, arthritis scores and serum IL6 production of GRK5 knockout (GRK5-/-) mice were significantly lower than those of wild-type mice. Histologically, the degree of synovitis and cartilage degeneration in GRK5-/- mice was significantly lower than in wild-type mice. In in vitro analyses using activated murine macrophages and fibroblast-like synoviocytes, gene expression of inflammatory factors and p65 nuclear translocation were significantly lower in GRK5-/- mice compared to wild-type mice. In conclusion, our results suggested that GRK5 is deeply involved in the pathogenesis of inflammatory arthritis, therefore, GRK5 inhibition could be a potential therapeutic target for types of inflammatory arthritis such as RA.

    View details for DOI 10.1038/s41598-021-90020-0

    View details for Web of Science ID 000658858500013

    View details for PubMedID 34006987

    View details for PubMedCentralID PMC8131379

  • Dedifferentiated low-grade central osteosarcoma with extensive cystic change initially treated as a simple bone cyst PATHOLOGY RESEARCH AND PRACTICE Toya, M., Yamada, Y., Yokoyama, R., Taguchi, K., Nabeshima, K., Isayama, T., Oda, Y. 2020; 216 (4): 152832


    Low-grade central osteosarcoma (LG-COS) is an uncommon variant of osteosarcoma (OS) that sometimes progresses to high-grade OS post-recurrence. We herein present a case of dedifferentiated LG-COS with extensive cystic change arising in the right iliac bone of a 26-year-old man. The LG-COS was initially diagnosed and managed as a simple bone cyst. The lesion recurred thrice, and high-grade OS was diagnosed during the third recurrence. The first lesion appeared as a typical benign cystic mass on radiography. However, a huge malignant osteoblastic mass subsequently developed in the right pelvis at the third recurrence. Extended hemipelvectomy with ipsilateral hemisacral resection was performed. Histologic analysis showed tumor necrosis and irregular neoplastic tumor osteoid, while immunohistochemistry revealed that the tumor was diffusely positive for MDM2 and CDK4. The histologic diagnosis was high-grade OS dedifferentiated from a preceding cystic lesion. Our final diagnosis of the primary lesion was LG-COS with extensive cystic change.

    View details for DOI 10.1016/j.prp.2020.152832

    View details for Web of Science ID 000523598700020

    View details for PubMedID 32057514

  • GRK5 Inhibition Attenuates Cartilage Degradation via Decreased NF-kappa B Signaling ARTHRITIS & RHEUMATOLOGY Sueishi, T., Akasaki, Y., Goto, N., Kurakazu, I., Toya, M., Kuwahara, M., Uchida, T., Hayashida, M., Tsushima, H., Bekki, H., Lotz, M. K., Nakashima, Y. 2020; 72 (4): 620-631


    NF-κB-dependent signaling is an important modulator in osteoarthritis (OA), and G protein-coupled receptor kinase 5 (GRK5) regulates the NF-κB pathway. This study was undertaken to investigate the functional involvement of GRK5 in OA pathogenesis.GRK5 expression in normal and OA human knee joints was analyzed immunohistochemically. Gain- or loss-of-function experiments were performed using human and mouse chondrocytes. OA was induced in GRK5-knockout mice by destabilization of the medial meniscus, and histologic examination was performed. OA was also induced in wild-type mice, which were then treated with an intraarticular injection of amlexanox, a selective GRK5 inhibitor, every 5 days for 8 weeks.GRK5 protein expression was increased in human OA cartilage. In vitro, expression levels of OA-related factors and NF-κB transcriptional activation were down-regulated by suppression of the GRK5 gene in human OA chondrocytes (3.49-fold decrease in IL6 [P < 0.01], 2.43-fold decrease in MMP13 [P < 0.01], and 2.66-fold decrease in ADAMTS4 [P < 0.01]). Conversely, GRK5 overexpression significantly increased the expression of OA-related catabolic mediators and NF-κB transcriptional activation. On Western blot analysis, GRK5 deletion reduced IκBα phosphorylation (up to 4.4-fold decrease [P < 0.05]) and decreased p65 nuclear translocation (up to 6.4-fold decrease [P < 0.01]) in mouse chondrocytes. In vivo, both GRK5 deletion and intraarticular amlexanox protected mouse cartilage against OA.Our results suggest that GRK5 regulates cartilage degradation through a catabolic response mediated by NF-κB signaling, and is a potential target for OA treatment. Furthermore, amlexanox may be a major compound in relevant drugs.

    View details for DOI 10.1002/art.41152

    View details for Web of Science ID 000522140900015

    View details for PubMedID 31696655

  • FOXO1 transcription factor regulates chondrogenic differentiation through transforming growth factor beta 1 signaling JOURNAL OF BIOLOGICAL CHEMISTRY Kurakazu, I., Akasaki, Y., Hayashida, M., Tsushima, H., Goto, N., Sueishi, T., Toya, M., Kuwahara, M., Okazaki, K., Duffy, T., Lotz, M. K., Nakashima, Y. 2019; 294 (46): 17555-17569


    The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor β1 (TGFβ1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFβ1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFβ1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9 To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFβ1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFβ1 signaling.

    View details for DOI 10.1074/jbc.RA119.009409

    View details for Web of Science ID 000503173500031

    View details for PubMedID 31601652

    View details for PubMedCentralID PMC6873195