Hideo Morioka

IL-1β and TNFα-initiated IL-6–STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes irreversible joint damage and significant disability. However, the fundamental mechanisms underlying how inflammation and joint destruction in RA develop and are sustained chronically remain largely unknown. Here, we show that signal transducer and activator of transcription 3 (STAT3) is the key mediator of both chronic inflammation and joint destruction in RA. We found that inflammatory cytokines highly expressed in RA patients, such as IL-1β, tumor necrosis factor alpha and IL-6, activated STAT3 either directly or indirectly and in turn induced expression of IL-6 family cytokines, further activating STAT3 in murine osteoblastic and fibroblastic cells. STAT3 activation also induced expression of receptor activator of nuclear factor kappa B ligand (RANKL), a cytokine essential for osteoclastogenesis, and STAT3 deficiency or pharmacological inhibition promoted significant reduction in expression of both IL-6 family cytokines and RANKL in vitro. STAT3 inhibition was also effective in treating an RA model, collagen-induced arthritis, in vivo through significant reduction in expression of IL-6 family cytokines and RANKL, inhibiting both inflammation and joint destruction. Leukemia inhibitory factor expression and STAT3 activation by IL-1β were mainly promoted by IL-6 but still induced in IL-6-deficient cells. Thus, our data provide new insight into RA pathogenesis and provide evidence that inflammatory cytokines trigger a cytokine amplification loop via IL-6-STAT3 that promotes sustained inflammation and joint destruction.

The Blimp1–Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis

Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte–induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets ‘osteoclastic’ molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1–Bcl6–osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.

Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization

The mobilization of hematopoietic stem cells does not require osteoclasts, which may even have an inhibitory effect.

C-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis

The development of cancer is due to the growth and proliferation of transformed normal cells. Recent evidence suggests that the nature of oncogenic stress and the state of the cell of origin critically affect both tumorigenic activity and tumor histological type. However, this mechanistic relationship in mesenchymal tumors is currently largely unexplored. To clarify these issues, we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (−/−) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARγ in tripotent MSC-like cells and overexpressing PPARγ in bipotent cells affected cell proliferation and tumorigenic activity. Our findings indicate that differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS.

Osteoclast stimulatory transmembrane protein and dendritic cell–specific transmembrane protein cooperatively modulate cell–cell fusion to form osteoclasts and foreign body giant cells

Cell–cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC‐STAMP), and analyzed its function. OC‐STAMP–deficient mice exhibited a complete lack of cell–cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage‐lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC‐STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC‐STAMP–overexpressing transgenic mice with OC‐STAMP–deficient mice restored inhibited osteoclast and FBGC cell–cell fusion seen in OC‐STAMP–deficient mice. Thus, fusogenic mechanisms in macrophage‐lineage cells are regulated via OC‐STAMP and DC‐STAMP.

The IRE1α-XBP1 pathway is essential for osteoblast differentiation through promoting transcription of Osterix.

During skeletal development, osteoblasts produce large amounts of extracellular matrix proteins and must therefore increase their secretory machinery to handle the deposition. The accumulation of unfolded protein in the endoplasmic reticulum induces an adoptive mechanism called the unfolded protein response (UPR). We show that one of the most crucial UPR mediators, inositol‐requiring protein 1α (IRE1α), and its target transcription factor X‐box binding protein 1 (XBP1), are essential for bone morphogenic protein 2‐induced osteoblast differentiation. Furthermore, we identify Osterix (Osx, a transcription factor that is indispensible for bone formation) as a target gene of XBP1. The promoter region of the Osx gene encodes two potential binding motifs for XBP1, and we show that XBP1 binds to these regions. Thus, the IRE1α–XBP1 pathway is involved in osteoblast differentiation through promoting Osx transcription.

Trabectedin monotherapy after standard chemotherapy versus best supportive care in patients with advanced, translocation-related sarcoma: a randomised, open-label, phase 2 study.

BACKGROUND Trabectedin binds to the minor groove of DNA and blocks DNA repair machinery. Preclinical data have shown that trabectedin also modulates the transcription of the oncogenic fusion proteins of translocation-related sarcomas. We aimed to assess the efficacy and safety of trabectedin as second-line therapy or later for patients with advanced translocation-related sarcoma. METHODS We did a multicentre randomised open-label study in Japan. Eligible patients had pathological diagnosis of translocation-related sarcoma, were aged 19 years or older, were unresponsive or intolerant to standard chemotherapy regimens, no more than four previous chemotherapy regimens, Eastern Cooperative Oncology Group performance status 0 or 1, adequate bone marrow reserve, renal and liver functions, and had measurable lesions. Patients were randomly assigned (1:1) by the minimisation method to receive either trabectedin (1·2 mg/m(2) given via a central venous line over 24 h on day 1 of a 21 day treatment cycle) or best supportive care, which was adjusted centrally by pathological subtype. Investigators, patients, and the sponsor were unmasked to the treatment assignment. Progression-free survival and objective responses were assessed by a masked central radiology imaging review. Efficacy was assessed by masked central radiology imaging review. The primary endpoint was progression-free survival for the full analysis set population. Follow-up is ongoing for the patients under study treatment. The study is registered with Japan Pharmaceutical Information Center, number JapicCTI-121850. FINDINGS Between July 11, 2012, and Jan 20, 2014, 76 patients were enrolled and allocated to receive either trabectedin (n=39) or best supportive care (n=37). After central review to confirm pathological subtypes, 73 patients (37 in the trabectedin group and 36 in the best supportive care group) were included in the primary efficacy analysis. Median progression-free survival of the trabectedin group was 5·6 months (95% CI 4·1-7·5) and the best supportive care group was 0·9 months (0·7-1·0). The hazard ratio (HR) for progression-free survival of trabectedin versus best supportive care was 0·07 (90% CI 0·03-0·14 and 95% CI 0·03-0·16) by a Cox proportional hazards model (p<0·0001). The most common drug-related adverse events for patients treated with trabectedin were nausea (32 [89%] of 36), decreased appetite (21 [58%]), decreased neutrophil count (30 [83%]), increased alanine aminotransferase (24 [67%]), and decreased white blood cell count (20 [56%]). INTERPRETATION Trabectedin significantly reduced the risk of disease progression and death in patients with advanced translocation-related sarcoma after standard chemotherapy such as doxorubicin, and should be considered as a new therapeutic treatment option for this patient population. FUNDING Taiho Pharmaceutical Co., Ltd.

Clear cell sarcoma of tendons and aponeuroses: a study of 75 patients.

Clear cell sarcoma (CCS) of tendons and aponeuroses (malignant melanoma of soft parts) is a rare melanocytic soft tissue sarcoma. The objective of this study was to determine the clinical features, prognostic factors, and optimal treatment policy for patients with this rare disease.

Reduced Argininosuccinate Synthetase Is a Predictive Biomarker for the Development of Pulmonary Metastasis in Patients with Osteosarcoma

Pulmonary metastasis is the most significant prognostic determinant for osteosarcoma, but methods for its prediction and treatment have not been established. Using oligonucleotide microarrays, we compared the global gene expression of biopsy samples between seven osteosarcoma patients who developed pulmonary metastasis within 4 years after neoadjuvant chemotherapy and curative resection, and 12 patients who did not relapse. We identified argininosuccinate synthetase (ASS) as a gene differentially expressed with the highest statistical significance (Welchs t test, P = 2.2 × 10−5). Immunohistochemical analysis of an independent cohort of 62 osteosarcoma cases confirmed that reduced expression of ASS protein was significantly correlated with the development of pulmonary metastasis after surgery (log-rank test, P < 0.05). Cox regression analysis revealed that ASS was the sole significant predictive factor (P = 0.039; hazard ratio, 0.319; 95% confidence interval, 0.108-0.945). ASS is one of the enzymes required for the production of a nonessential amino acid, arginine. We showed that osteosarcoma cells lacking ASS expression were auxotrophic for arginine and underwent G0-G1 arrest in arginine-free medium, suggesting that an arginine deprivation therapy could be effective in patients with osteosarcoma. Recently, phase I and II clinical trials in patients with melanoma and hepatocellular carcinoma have shown the safety and efficacy of plasma arginine depletion by stabilized arginine deiminase. Our data indicate that in patients with osteosarcoma, reduced expression of ASS is not only a novel predictive biomarker for the development of metastasis, but also a potential target for pharmacologic intervention. Mol Cancer Ther; 9(3); 535–44

Repair of full-thickness articular cartilage defects using injectable type II collagen gel embedded with cultured chondrocytes in a rabbit model

BackgroundRecently, tissue-engineered chondrocyte transplantation has been tried to treat full-thickness cartilage defects. We developed an injectable type II collagen gel scaffold by chemically reacting type II collagen with polyethylene glycol crosslinker. This type II collagen was prepared from the nasal septa of cattle. In the present study, chondrocytes embedded in type II collagen gel were injected into rabbit full-thickness cartilage defects without a periosteal graft, and the feasibility for clinical application of the gel was evaluated.MethodsChondrocytes were isolated from 1-kg New Zealand white rabbits. A full-thickness articular cartilage defect (5 mm diameter, 4 mm depth) was created on the patellar groove of the femur of 16 male 3-kg New Zealand white rabbits. A type II collagen solution of mixed chondrocytes at a density of 1 × 107 cells/ml was injected and transplanted into the defect in the right knee. The controls were the defect only in the left knee. At 4, 8, 12, and 24 weeks after operation, four cases from each group were evaluated macroscopically and histologically.ResultsAfter injection into the cartilage defect, the gel bonded to the adjacent cartilage and bone within several minutes. Macroscopic examination revealed that the surface of the transplanted area was smooth and exhibited similar coloration and good integration with the surrounding cartilage at 12 and 24 weeks after transplantation. Histological examination at 8 weeks revealed favorable hyaline cartilage regeneration with good chondrocyte morphology. At 12 and 24 weeks, reparative cartilage remained rich in type II collagen. According to O’Driscoll histological scores, significant differences between the transplanted and control groups were apparent at 12 and 24 weeks. Immunohistochemical staining indicated sufficient type II collagen synthesis in regenerated cartilage 8 weeks after transplantation, and it was maintained until 24 weeks.ConclusionsThese results indicate that type II collagen gel is suitable for injection into cartilage defects without any covering of a graft and offers a useful scaffold during chondrocyte transplantation.