Kensuke Koyama

A Subcytotoxic Dose of Subtilase Cytotoxin Prevents Lipopolysaccharide-Induced Inflammatory Responses, Depending on its Capacity to Induce the Unfolded Protein Response

Subtilase cytotoxin (SubAB) is the prototype of a newly identified family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. SubAB specifically cleaves the essential endoplasmic reticulum (ER) chaperone BiP (GRP78), resulting in the activation of ER stress-induced unfolded protein response (UPR). We have recently shown that the UPR following ER stress can suppress cellular responses to inflammatory stimuli during the later phase, in association with inhibition of NF-κB activation. These findings prompted us to hypothesize that SubAB, as a selective UPR inducer, might have beneficial effects on inflammation-associated pathology via a UPR-dependent inhibition of NF-κB activation. The pretreatment of a mouse macrophage cell line, RAW264.7, with a subcytotoxic dose of SubAB-triggered UPR and inhibited LPS-induced MCP-1 and TNF-α production associated with inhibition of NF-κB activation. SubAA272B, a SubAB active site mutant that cannot induce UPR, did not show such effects. In addition, pretreatment with a sublethal dose of SubAB, but not SubAA272B, protected the mice from LPS-induced endotoxic lethality associated with reduced serum MCP-1 and TNF-α levels and also prevented the development of experimental arthritis induced by LPS in mice. Collectively, although SubAB has been identified originally as a toxin associated with the pathogenesis of hemolytic uremic syndrome, the unique ability of SubAB to selectively induce the UPR may have the potential to prevent LPS-associated inflammatory pathology under subcytotoxic conditions.

TNF-α–induced NF-κB signaling reverses age-related declines in VEGF induction and angiogenic activity in intervertebral disc tissues

We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF‐α induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF‐α–induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF‐α–induced VEGF using organ disc cultures with wild type, TNF receptor 1‐null (TNF‐RInull), or TNF receptor 2‐null (TNF‐RIInull) mice. VEGF induction was inhibited when we used TNF‐RInull‐derived disc tissues. NF‐κB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF‐α induced VEGF expression in disc cells primarily through the NF‐κB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF‐α stimulation. TNF‐α treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF‐κB inhibitors or anti‐VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF‐κB pathway, both of which are induced by TNF‐α. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patients age.

A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: Implications for herniated discs

OBJECTIVE To determine whether thymic stromal lymphopoietin (TSLP) plays a role in the resorption of herniated disc tissue. METHODS The expression of TSLP messenger RNA (mRNA) and protein in mouse intervertebral disc cells was assessed by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analysis. The ability of mouse intervertebral disc cells to respond to TSLP stimulation was examined by Western blot analysis, ELISA, and protein array analysis. Intracellular signaling pathways involved in TSLP signaling in mouse intervertebral disc cells were investigated using several chemical inhibitors. The role of TSLP in macrophage migration into the intervertebral disc was assessed by in vitro migration assay. Finally, TSLP expression in clinical specimens derived from patients with a herniated disc was examined by immunohistochemistry. RESULTS Mouse intervertebral disc cells expressed TSLP mRNA and protein upon stimulation with NF-kappaB-activating ligands such as tumor necrosis factor alpha. In addition, the mouse intervertebral disc cells expressed the TSLP receptor and produced monocyte chemotactic protein 1 (MCP-1; CCL2) and macrophage colony-stimulating factor in response to TSLP stimulation. Both anulus fibrosus and nucleus pulposus intervertebral disc cells expressed MCP-1 upon TSLP stimulation, which was mediated via the phosphatidylinositol 3-kinase/Akt pathway. Consistently, the supernatants of TSLP-activated intervertebral disc cultures had the capacity to induce macrophage migration in an MCP-1-dependent manner. Finally, TSLP and MCP-1 were coexpressed in human herniated disc specimens in which macrophage infiltration into the tissue was observed. CONCLUSION TSLP induced by NF-kappaB-activating ligands in intervertebral discs may contribute to the recruitment of macrophages to the intervertebral disc by stimulating MCP-1 production and may be involved in the resorption of herniated disc tissue.

Mechanism of signal transduction in tumor necrosis factor-like weak inducer of apoptosis-induced matrix degradation by MMP-3 upregulation in disc tissues.

Study Design. Molecular biologic and immuno-histologic analyses using in vitro murine intervertebral disc tissue culture. Objective. To investigate the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in matrix metalloproteinase 3 (MMP-3) pathway induction, and the effect of TWEAK to induce other cytokines or angiogenesis factors in disc tissues. Summary of Background Data. We previously demonstrated that TWEAK and its receptor Fn14 were expressed in murine disc tissues. TWEAK induced MMP-3 upregulation and aggrecan downregulation in disc tissues. Methods. Enzyme-Linked ImmunoSorbent Assay (ELISA), western blot, and immuno-histologic analyses were used to assess the role of TWEAK-induced MMP-3, using murine disc tissue culture. Results. TWEAK induced disc cells to generate MMP-3 as did TNF-&agr; and IL-1&bgr;. MMP-3 activity was detectable in murine disc cells. MMP-3 induction was markedly inhibited with a c-Jun N-terminal kinase (JNK) inhibitor. Phosphorylation of JNK was also confirmed. Introduction of TWEAK resulted in the degradation of disc matrix in organ disc culture, whereas proteoglycan degradation was markedly abrogated in the presence of an MMP-3 specific inhibitor or a JNK inhibitor. In addition, TWEAK also induced monocyte chemotactic protein (MCP)-1 via the NF-&kgr;B pathway, as phosphorylation of NF-&kgr;B was confirmed by western blotting. Conclusion. TWEAK plays an important role in MMP-3 induction in murine disc cells via JNK that results in degradation of disc matrix. TWEAK also induces MCP-1, which belongs to the chemokine family that recruits inflammatory cells via the NF-&kgr;B pathway.

Endogenous TGF-β activity limits TSLP expression in the intervertebral disc tissue by suppressing NF-κB activation.

Thymic stromal lymphopoietin (TSLP), an IL‐7‐like cytokine, is highly expressed in herniated disc (HD) tissue and may act as a key molecule for the initiation of macrophage recruitment into the tissue and natural resorption of HD. However, it remains unclear how TSLP expression is regulated in the intervertebral discs. This study showed that expression of TSLP and phosphorylated NF‐κB in HD tissue samples was inversely correlated with expression of phosphorylated Smad2/3 (an indicator of active TGF‐β signaling) and vice versa in posterior lumbar spinal fusion samples. The pharmacological blockades of endogenous TGF‐β activity induced TSLP expression in mouse intervertebral disc tissue culture, which was inhibited by NF‐κB inhibitors. Additionally, phosphorylation of Smad2/3 was constitutively detected in mouse intervertebral disc tissue in the steady states. Collectively, these results suggest that endogenous TGF‐β activity limits TSLP expression in intervertebral disc tissue in the steady states by suppressing NF‐κB activation. The findings reveal a regulatory mechanism how TSLP expression is induced in the intervertebral disc tissue and suggest a novel role of TGF‐β in maintaining the homeostasis of intervertebral disc tissue.

Thrombin induced by the extrinsic pathway and PAR-1 regulated inflammation at the site of fracture repair

Thrombin (coagulation factor IIa) is a serine protease encoded by the F2 gene. Pro-thrombin (coagulation factor II) is cut to generate thrombin in the coagulation cascade that results in a reduction of blood loss. Procoagulant states that lead to activation of thrombin are common in bone fracture sites. However, its physiological roles and relationship with osteoblasts in bone fractures are largely unknown. We herein report various effects of thrombin on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed proteinase-activated receptor 1 (PAR1), also known as the coagulation factor II receptor. They also produced monocyte chemoattractant protein (MCP-1), tissue factor (TF), MCSF and IL-6 upon thrombin stimulation through the PI3K-Akt and MEK-Erk1/2 pathways. Furthermore, MCP-1 obtained from thrombin-stimulated MC3T3-E1 cells induced migration by macrophage RAW264 cells. All these effects of thrombin on MC3T3-E1 cells were abolished by the selective non-peptide thrombin receptor inhibitor SCH79797. We also found that thrombin, PAR-1, MCP-1, TF as well as phosphorylated AKT and p42/44 were significantly expressed at the fracture site of mouse femoral bone. Collectively, thrombin/PAR-1 interaction regulated MCP-1, TF, MCSF and IL-6 production by MC3T3-E1 cells. Furthermore, MCP-1 induced RAW264 cell migration. Thrombin may thus be a novel cytokine that regulates several aspects of osteoblast function and fracture healing.

Development of a quick serum IL-6 measuring system in rheumatoid arthritis

Objective Interleukin‐6 (IL‐6) plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). Both fulfillment of remission criteria and assessment of other methods of evaluation of RA are important for preventing joint damage progression. Measurement of serum IL‐6 concentrations has been reported to be useful for monitoring RA disease activity. However, it takes at least 4–5 h to measure serum IL‐6 concentrations using traditional methods, which limits its utility during routine assessment in daily clinical practice settings. We established a novel method that enables measurement of serum IL‐6 within 24 min and requires a very small blood volume. We investigated the accuracy and efficacy of this system in RA patients. Methods One hundred fifty blood samples collected from 76 patients were measured using the two systems. We first developed the prototype of the Human IL‐6 RAYFAST. Then, we examined the correlation between the prototype RAYFAST and chemiluminescent enzyme immunoassay (CLEIA) methods. Finally, we compared IL‐6 concentrations and clinical parameters using both systems. Results The correlation between RAYFAST (x) and CLEIA (y) for IL‐6 was y = 0.895x − 5.94, r = 0.941 (p < 0.0001). Serum IL‐6 concentrations in RAYFAST correlated with DAS28‐CRP (r = 0.372, p < 0.05) and DAS28‐ESR (r = 0.397, p < 0.01). Serum IL‐6 concentrations in CLEIA correlated with DAS28‐CRP (r = 0.313, p < 0.001) and DAS28‐ESR (r = 0.353, p < 0.001). Conclusion This new cytokine quick measure system is as accurate as CLEIA methods. Serum IL‐6 concentrations can be measured in 24 min using the prototype RAYFAST. It might be usable in the daily clinical practice setting, thereby contributing to improved RA management.

Positive association between serum thymic stromal lymphopoietin and anti-citrullinated peptide antibodies in patients with rheumatoid arthritis

Thymic stromal lymphopoietin (TSLP) has been suggested recently to play an important role in the pathophysiology of rheumatoid arthritis (RA). However, there is little information on serum TSLP concentrations in RA and its clinical significance. The present study investigated whether serum TSLP concentrations were affected in patients with RA. Using an enzyme‐linked immunosorbent assay (ELISA), we measured TSLP concentrations in the serum obtained from 100 patients with RA, 60 patients with osteoarthritis (OA) and 34 healthy volunteers. We also investigated the correlation between serum TSLP concentrations and clinical parameters of disease activity in RA [disease activity score using 28 joint counts (DAS28)‐C‐reactive protein (CRP), DAS28‐erythrocyte sedimentation rate (ESR), Clinical Disease Activity Index (CDAI]), patients/‐physicians Visual Analogue Scale (VAS), swollen joints count, tender joints count, CRP, ESR and matrix metalloproteinase‐3 (MMP‐3) concentrations]. In addition, we investigated the correlation between serum TSLP concentrations and anti‐citrullinated peptide antibody (ACPA) and serum tumour necrosis factor (TNF)‐α. Serum TSLP levels in patients with RA were significantly higher than those in patients with OA and in healthy volunteers. Interestingly, serum TSLP concentrations were correlated significantly with ACPA titres, but not with other clinical parameters. There was a significant increase in serum TSLP concentrations in patients with RA, which was correlated positively with serum ACPA titres. These findings suggest that in patients with RA, TSLP may play a role in ACPA production by B cells.

Risk factors for clinically relevant loosening of percutaneous pedicle screws

Introduction (1) To evaluate the influence of pedicle screw loosening on clinical outcomes; (2) to clarify the association between the pull-out length and screw loosening 1 year after surgery; and (3) to determine radiographically which screw parameters predominantly influence the pull-out resistance of screws. Methods We analyzed 32 consecutive patients who underwent minimally invasive lumbar or thoracic spinal stabilization by intraoperative three-dimensional computed tomography (CT)-guided navigation without anterior reconstruction and were followed up for 1 year. The screw pull-out length was measured on axial CT images obtained both immediately after screw insertion and postoperatively. Loosening of screws and clinical outcomes were evaluated radiographically, clinically, and by CT 1 year after surgery. Results There were no significant differences in the mean age, sex, bone mineral density, mean stabilized length, and smoking habits of patients with (+) or without (−) loosening. The Oswestry Disability Index and the lumbar visual analog scale 1 year after surgery were significantly higher in patients with loosening (+) than in those without (−). The overall pedicle screw pull-out rate was 16.2% (47/290) of screws and the overall screw loosening rate was 15.2% (44/290) of screws. Screws with loosening (+) had significantly lower (axial) trajectory angles and higher screw pull-out lengths than those without (−). Approximately 82% of loosened screws had been pulled out during rod connection. Conclusions A lower axial trajectory and an increased screw pull-out length after rod reduction are crucial risk factors for screw loosening.

Platelet-Derived TGF-β Induces Tissue Factor Expression via the Smad3 Pathway in Osteosarcoma Cells: TGF-β INDUCES TF EXPRESSION VIA SMAD3 PATHWAY IN OSTEOSARCOMA CELLS

Over the last three decades, the prognosis of osteosarcoma has remained unchanged; the prognosis for patients with lung metastasis is still poor, and the development of new treatments is urgently required. We previously showed that aggressive osteosarcoma cells express more tissue factor (TF) and demonstrate enhanced extrinsic pathway capacity. Furthermore, tumor growth can be suppressed with the anticoagulant low molecular weight heparin. However, the molecular mechanisms underlying TF regulation are still unclear. Here, we report that transforming growth factor‐β (TGF‐β) upregulates TF, which can occur via activated platelets. TF was found to be expressed on osteosarcoma cell surfaces, which mediated the production of Xa and thrombin. TF induction by TGF‐β was observed in several osteosarcoma cells, and especially in MG 63 cells. Both TF expression by TGF‐β and extrinsic pathway activity through TF were rapidly increased. This reaction was inhibited by a TGF‐β type I receptor inhibitor and TGF‐β neutralizing antibody. Although TGF‐β was found to phosphorylate both Smad2 and Smad3, their roles were markedly disparate. Surprisingly, Smad2 knockdown resulted in no inhibitory effect, whereas Smad3 knockdown completely suppressed TGF‐β–induced TF expression. Next, data suggested that platelets were the source of TGF‐β. We confirmed that thrombin‐activated platelets and osteosarcoma cells could release TGF‐β, and that platelet‐derived TGF‐β could induce TF expression. These processes were also inhibited by a TGF‐β type I receptor inhibitor and Smad3 knockdown. Moreover, CD42b, TF, TGF‐β, Smad2/3, and p‐Smad2/3 were also detected in a biopsy sample from an osteosarcoma patient. Collectively, these finding suggested that the interaction between osteosarcoma cells and platelets, via thrombin and TGF‐β, results in a continuous cycle, and that anti‐platelet or anti‐TGF‐β therapy could be a promising tool for disease treatment.