Attenuation by HSP90 inhibitors of EGF-elicited migration of osteoblasts: involvement of p44/p42 MAP kinase.

BACKGROUND: We have demonstrated that epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells is mediated through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK), and Akt.The molecular chaperone heat shock protein 90 (HSP90) is abundantly expressed in osteoblasts. However, the role of HSP90 in osteoblast migration remains obscure. OBJECTIVE: In this study, we investigated the effect of HSP90 inhibitors on the EGF-induced migration of MC3T3-E1 cells and the mechanism. METHODS: Clonal osteoblast-like MC3T3-E1 cells were treated with the HSP90 inhibitors geldanamycin or onalespib and then stimulated with EGF. Cell migration was evaluated using the transwell cell migration assay and wound-healing assay. The viability of MC3T3-E1 cells was analyzed using the Cell Counting Kit-8. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, Akt, and protein kinase-like endoplasmic reticulum kinase (PERK) was evaluated by western blot analysis. RESULTS: EGF-induced migration was significantly suppressed by geldanamycin and onalespib, evaluated by both transwell cell migration assay and wound-healing assay. Geldanamycin and onalespib did not significantly alter cell viability. Geldanamycin and onalespib markedly reduced the EGF-induced phosphorylation of p44/p42 MAP kinase, but not p38 MAP kinase or Akt. By contrast, geldanamycin and onalespib increased the EGF-induced phosphorylation of SAPK/JNK. PERK phosphorylation was not significantly affected by geldanamycin or onalespib. CONCLUSION: Our results strongly suggest that HSP90 inhibitors reduce the EGF-induced osteoblast migration through the p44/p42 MAP kinase.

Upregulation of TGF-ß-induced HSP27 by HSP90 inhibitors in osteoblasts.

BACKGROUND: Heat shock protein (HSP) 90 functions as a molecular chaperone and is constitutively expressed and induced in response to stress in many cell types. We have previously demonstrated that transforming growth factor-ß (TGF-ß), the most abundant cytokine in bone cells, induces the expression of HSP27 through Smad2, p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in mouse osteoblastic MC3T3-E1 cells. This study investigated the effects of HSP90 on the TGF-ß-induced HSP27 expression and the underlying mechanism in mouse osteoblastic MC3T3-E1 cells. METHODS: Clonal osteoblastic MC3T3-E1 cells were treated with the HSP90 inhibitors and then stimulated with TGF-ß. HSP27 expression and the phosphorylation of Smad2, p44/p42 MAPK, p38 MAPK, and SAPK/JNK were evaluated by western blot analysis. RESULT: HSP90 inhibitors 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) and onalespib significantly enhanced the TGF-ß-induced HSP27 expression. TGF-ß inhibitor SB431542 reduced the enhancement by 17-DMAG or onalespib of the TGF-ß-induced HSP27 expression levels. HSP90 inhibitors, geldanamycin, onalespib, and 17-DMAG did not affect the TGF-ß-stimulated phosphorylation of Smad2. Geldanamycin did not affect the TGF-ß-stimulated phosphorylation of p44/p42 MAPK or p38 MAPK but significantly enhanced the TGF-ß-stimulated phosphorylation of SAPK/JNK. Onalespib also increased the TGF-ß-stimulated phosphorylation of SAPK/JNK. Furthermore, SP600125, a specific inhibitor for SAPK/JNK, significantly suppressed onalespib or geldanamycin's enhancing effect of the TGF-ß-induced HSP27 expression levels. CONCLUSION: Our results strongly suggest that HSP90 inhibitors upregulated the TGF-ß-induced HSP27 expression and that these effects of HSP90 inhibitors were mediated through SAPK/JNK pathway in osteoblasts.

Bone parameters of elite athletes with oligomenorrhea and prevalence seeking medical attention: a cross-sectional study.

INTRODUCTION: Since the definition of secondary amenorrhea is cessation of regular menses for more than 3 months, it is likely that athletes with irregular menstrual cycles, including oligomenorrhea, do not consider the condition as serious. However, the consequences of untreated oligomenorrhea have not been investigated in elite track and field athletes. MATERIALS AND METHODS: The cohort consisted of 91 elite-level track and field athletes. Body compositions, including bone parameters and bone turnover markers (BTMs), were measured. RESULTS: Among the 91 participants, 52 were eumenorrheic and 33 were oligomenorrheic. The eumenorrheic athletes had significantly higher bone mineral density (BMD) and bone mineral content (BMC) of the lumbar spine, lower extremities, and whole body than had the oligomenorrheic athletes (p < 0.01). There were no significant differences in BTMs between the two groups, but oligomenorrheic athletes had significantly lower percent body fat. CONCLUSION: More than 40% of the elite-level female track and field athletes in this study reported menstrual disorders with oligomenorrhea as the most common. However, none sought medical attention. As compared to the eumenorrheic athletes, the oligomenorrheic athletes had lower BMC and BMD. Hence, if an athlete is oligomenorrheic, bone parameter measurements are considerably important.

HSP90 inhibitors strengthen extracellular ATP-stimulated synthesis of interleukin-6 in osteoblasts: Amplification of p38 MAP kinase.

Heat shock protein 90 (HSP90) is expressed ubiquitously in a variety of cell types including osteoblasts. HSP90 acts as a key driver of proteostasis under pathophysiological conditions. Here, we investigated the involvement of HSP90 in extracellular ATP-stimulated interleukin (IL)-6 synthesis and HSP90 downstream signalling in osteoblast-like MC3T3-E1 cells. In osteoblasts, extracellular ATP stimulates the synthesis of IL-6, a bone-remodelling agent. Geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and onalespib, three different HSP90 inhibitors, amplified the ATP-stimulated IL-6 release. Geldanamycin increased IL-6 mRNA expression elicited by ATP. ATP enhanced the triiodothyronine-induced osteocalcin release, but HSP90 inhibitors suppressed the release. Extracellular ATP induced the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), p70 S6 kinase, Akt, and myosin phosphatase-targeting subunit (MYPT), a Rho-kinase substrate. SB203580, an inhibitor of p38 MAPK, suppressed ATP-stimulated IL-6 release. Inhibitors of MEK1/2 (PD98059), JNK (SP600125), upstream kinase of p70 S6 kinase (rapamycin) and Akt (deguelin), all increased IL-6 release. Y27632, a Rho-kinase inhibitor, failed to affect the IL-6 release stimulated by ATP. Geldanamycin and 17-AAG both amplified ATP-induced p38 MAPK phosphorylation, although geldanamycin inhibited the phosphorylation of Akt induced by ATP. In addition, SB203580 significantly reduced the amplification by geldanamycin of the IL-6 release. Taken together, our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 MAPK activation in osteoblasts. SIGNIFICANCE OF THE STUDY: Heat shock protein 90 (HSP90) acts as a key driver of proteostasis under pathophysiological conditions in a variety of cell types. We have previously shown that HSP90 is expressed at high levels in osteoblast-like MC3T3-E1 cells, even in their quiescent state, consistent with HSP90 performing an important physiological function in osteoblasts. In the present study, we investigated whether HSP90 is implicated in extracellular ATP-induced interleukin (IL)-6 synthesis in osteoblast-like MC3T3-E1 cells. Our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 mitogen-activated protein kinase activation in osteoblasts.

Bone Metabolism, Bone Mineral Content, and Density in Elite Late Teen Female Sprinters.

With intensive training, bone injuries are a major concern for athletes. To assess bone condition, we often measure bone turnover markers, bone mineral content and density; however, in junior athletes, it is not easy to distinguish changes caused by bone injuries from those caused by growth, because the metabolism is increased in both cases. Moreover, although some studies have examined female endurance athletes, knowledge regarding changes in static and dynamic bone conditions in late teen athletes is limited. In this study, we measured the bone mineral content and density, as well as bone turnover markers, in 40 elite female sprinters in their late teens. Whole body mode dual-energy X-ray absorptiometry was performed to measure bone mineral content and density. Blood samples were collected to determine bone resorption and formation markers at the end of track season in 2016 and during the same period of the following year. Body weight and bone mineral content significantly increased, and tartrate-resistant acid phosphatase type 5b, bone-type alkaline phosphatase, and osteocalcin significantly decreased after a year. Furthermore, the rate of change in bone mineral content was higher in younger athletes, indicating that bone growth approaches completion in the late teen years and that bone metabolism accordingly decreases.

Status Estimation and In-Process Connection of Kanbans Using BLE Beacons and LPWA Network to Implement Intra-Traceability for the Kanban System.

IoT-based measurement systems for manufacturing have been widely implemented. As components that can be implemented at low cost, BLE beacons have been used in several systems developed in previous research. In this work, we focus on the Kanban system, which is a measure used in manufacturing strategy. The Kanban system emphasizes inventory management and is used to produce only required amounts. In the Kanban system, the Kanban cards are rotated through the factory along with the products, and when the products change to a different process route, the Kanban card is removed from the products and the products are assigned to another Kanban. For this reason, a single Kanban cannot trace products from plan to completion. In this work, we propose a system that uses a Bluetooth low energy (BLE) beacon to connect Kanbans in different routes but assigned to the same products. The proposed method estimates the beacon status of whether the Kanban is inside or outside a postbox, which can then be computed by a micro controller at low computational cost. In addition, the system connects the Kanbans using the beacons as paired connection targets. In an experiment, we confirmed that the system connected 70% of the beacons accurately. We also confirmed that the system could connect the Kanbans at a small implementation cost.

Efficacy of combination-chemotherapy with pirarubicin, ifosfamide, and etoposide for soft tissue sarcoma: a single-institution retrospective analysis.

BACKGROUND: The standard chemotherapy regimens for soft tissue sarcoma are doxorubicin-based. This retrospective study aimed to assess the efficacy and safety of pirarubicin, ifosfamide, and etoposide combination therapy for patients with this disease. METHODS: Between 2008 and 2017, 25 patients with soft tissue sarcoma were treated with pirarubicin (30 mg/m2, 2 days), ifosfamide (2 g/m2, 5 days), and etoposide (100 mg/m2, 3 days) every 3 weeks. The primary endpoint was overall response, and the secondary endpoint was adverse events of this regimen. RESULTS: Responses to this regimen according to RECIST criteria were partial response (n = 9, 36%), stable disease (n = 9, 36%) and progressive disease (n = 7, 28%). During the treatment phase, frequent grade 3 or worse adverse events were hematological toxicities including white blood cell decreases (96%), febrile neutropenia (68%), anemia (68%), and platelet count decreases (48%). No long-term adverse events were reported during the study period. CONCLUSION: This regimen was comparable to previously published doxorubicin-based combination chemotherapy in terms of response rate. Although there were no long-lasting adverse events, based on our results, severe hematological toxicity should be considered.

Resveratrol suppresses insulin-like growth factor I-induced osteoblast migration: attenuation of the p44/p42 MAP kinase pathway.

Resveratrol is a natural polyphenol with beneficial antioxidant properties. It suppresses the migration of osteoblast-like MC3T3-E1 cells induced by epidermal growth factor, via SIRT1-mediated inhibition of SAPK/JNK and Akt. Moreover, insulin-like growth factor-I (IGF-I) stimulates the migration involving the pathways of p44/p42 mitogen-activated protein (MAP) kinase and Akt. Therefore, we investigated the effects of resveratrol on IGF-I-induced cell migration. Resveratrol and SRT1720, an activator of SIRT1, suppressed IGF-I-induced migration. Inauhzin, a SIRT1 inhibitor, significantly rescued the inhibition of IGF-I-induced cell migration by resveratrol. Resveratrol inhibited IGF-I-induced phosphorylation of p44/p42 MAP kinase but not Akt. SRT1720 inhibited IGF-I-induced phosphorylation of p44/p42 MAP kinase. Furthermore, PD98059, p44/p42 MAP kinase inhibitor, alone suppressed IGF-I-induced osteoblast migration, but did not affect the suppressive effect of resveratrol when administered concomitantly. These findings strongly suggest that resveratrol suppresses IGF-I-induced osteoblast migration via SIRT1 activation at least partially by attenuating the p44/p42 MAP kinase pathway.

Local dissemination of osteosarcoma observed after massage therapy: a case report.

BACKGROUND: Limited evidence is available regarding the dissemination of tumor tissues due to compression during massage therapy, a routine procedure in patients with various symptoms in Asian countries. CASE PRESENTATION: A 12-year-old male presented at a massage clinic with pain and swelling of his left knee, which worsened the same night. Consistent with conventional osteosarcoma, radiography revealed cortical bone destruction, osteoblastic changes, and periosteal reactions. Magnetic resonance imaging revealed a tumor in the distal femur, an extraskeletal mass, and an infiltrative lesion in the intramuscular and neurovascular areas surrounding the distal femur; this was considered as hemorrhage and dissemination of the tumor tissue. 18Fluorine-labelled fluorodeoxyglucose-positron emission tomography and computed tomography revealed multiple metastases in the spine, liver, and lung. Consistent with osteosarcoma, histopathological examination revealed tumor cell proliferation with extensive pleomorphism and mitoses. Despite undergoing chemotherapy, radiation therapy, and hip disarticulation, the patient died due to multiple metastases 13 months after the initial diagnosis. CONCLUSIONS: The present case suggests association of massage therapy with the local dissemination of tumor tissues, although influence of massage therapy on metastatic lesions remains unclear. Massage therapists should be aware of the possibility for dissemination of hidden malignancies due to the procedure.

Enhancement by HSP90 inhibitor of PGD2-stimulated HSP27 induction in osteoblasts: Suppression of SAPK/JNK and p38 MAP kinase.

Heat shock protein (HSP) 90 that is ubiquitously expressed in various tissues is a major molecular chaperone. We have previously demonstrated that prostaglandin D2 (PGD2), a bone remodeling factor, elicits the expression of HSP27, a small HSP, through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of HSP90 in the PGD2-stimulated HSP27 induction and the underlying mechanism in MC3T3-E1 cells. Onalespib, an inhibitor of HSP90, significantly enhanced the PGD2-stimulated HSP27 induction. In addition, geldanamycin, another HSP90 inhibitor, potentiated the HSP27 induction. Both onalespib and geldanamycin markedly amplified the PGD2-induced phosphorylation of SAPK/JNK and p38 MAP kinase. SP600125, an inhibitor of SAPK/JNK, and SB203580, an inhibitor of p38 MAP kinase, suppressed the amplification by onalespib of the PGD2-stimulated HSP27 induction. These results strongly suggest that HSP90 plays a negative role in the HSP27 induction stimulated by PGD2 in osteoblasts, and that the inhibitory effect of HSP90 is mediated through the regulation of SAPK/JNK and p38 MAP kinase.

The Janus kinase inhibitor tofacitinib inhibits TNF-α-induced gliostatin expression in rheumatoid fibroblast-like synoviocytes.

OBJECTIVES: Gliostatin (GLS) is known to have angiogenic and arthritogenic activity, and GLS expression levels in serum from patients with rheumatoid arthritis (RA) are significantly correlated with the disease activity. Tofacitinib is a novel oral Janus kinase (JAK) inhibitor and is effective in treating RA. However, the mechanism of action of tofacitinib in fibroblast-like synoviocytes (FLSs) has not been elucidated. The purpose of this study was to investigate the modulatory effects of tofacitinib on serum GLS levels in patients with RA and GLS production in FLSs derived from patients with RA. METHODS: Six patients with RA who had failed therapy with at least one TNF inhibitor and were receiving tofacitinib therapy were included in the study. Serum samples were collected to measure CRP, MMP-3 and GLS expression. FLSs derived from patients with RA were cultured and stimulated by TNFα with or without tofacitinib. GLS expression levels were determined using reverse transcription-polymerase chain reaction (RT-PCR), EIA and immunocytochemistry, and signal transducer and activator of transcription (STAT) protein phosphorylation levels were determined by western blotting. RESULTS: Treatment with tofacitinib decreased serum GLS levels in all patients. GLS mRNA and protein expression levels were significantly increased by treatment with TNF-α alone, and these increases were suppressed by treatment with tofacitinib, which also inhibited TNF-α-induced STAT1 phosphorylation. CONCLUSIONS: JAK/STAT activation plays a pivotal role in TNF-α-mediated GLS up-regulation in RA. Suppression of GLS expression in FLSs has been suggested to be one of the mechanisms through which tofacitinib exerts its anti-inflammatory effects.

Efficacy of radio-hyperthermo-chemotherapy as salvage therapy for recurrent or residual malignant soft tissue tumors.

BACKGROUND: Recurrence after wide excision or residual tumor after an unplanned excision of a malignant soft tissue sarcoma (STS) is a complex problem, due to a higher recurrence rate and poorer survival rate compared with primary resection. Regional hyperthermia was used, with the expectation that it will enhance the anti-tumor effects of chemotherapy and radiotherapy. This study aimed to assess the efficacy of neoadjuvant concomitant radiotherapy, hyperthermia, and chemotherapy (RHC) for salvage of recurrent or residual malignant STS. METHODS: We identified 64 patients with recurrent or residual STS treated between 1994 and 2013. After excluding those with low-grade malignancy, with recurrent bone tumor in the soft tissues, with truncal STS, and who declined to participate, 23 patients (7 with recurrence and 16 with residual tumor) underwent RHC. The histologic diagnoses were undifferentiated pleomorphic sarcoma (n = 11), synovial sarcoma (n = 3), leiomyosarcoma and myxoid liposarcoma (n = 2 each), and other histologic types. As primary outcomes, the 5-year overall survival (OS), distant metastasis-free survival (D-MFS), and local control (LC) rates were evaluated by Kaplan-Meier analysis. RESULTS: The median follow-up period was 112.3 months. The 5-year OS, D-MFS, and LC were 86.4%, 77.4%, and 86.7%, respectively. In the univariate analysis, tumor depth was considered as a negative prognostic factor for OS and D-MFS, and a positive margin was also a negative prognostic factor for OS, D-MFS LC with retained on Cox proportional hazards model in OS, and D-MFS. CONCLUSION: RHC is an effective option for salvage treatment of recurrent and residual STS.

Treatment of aneurysmal bone cysts using endoscopic curettage.

BACKGROUND: Although aneurysmal bone cysts (ABCs) are benign tumours, they have the potential to be locally aggressive. Various treatment approaches, such as en bloc resection, open curettage, radiotherapy, sclerotherapy, and embolization have been proposed, but the most appropriate treatment should be selected after considering the risk of tumour recurrence and treatment complications. Endoscopic curettage (ESC) may be a less invasive alternative to open curettage for ABC treatment. We aimed to describe the use of ESC for the treatment of ABCs and to report our clinical outcomes, including the incidence rate of recurrence, radiological appearance at final follow-up, time to solid union, complications, and postoperative function. METHODS: Between 1998 and 2015, 30 patients (18 men and 12 women; mean age, 17.4 years) underwent ESC for the treatment of primary ABCs at our hospital (mean postoperative follow-up, 55 months). ESC was performed under arthroscopic guidance for direct visualization, and curettage extended until normal bone was observed in the medullary cavity. To investigate bone healing after ESC, we evaluated the consolidation of cysts at the final evaluation (based on the modified Neer classification) and time to solid union after surgery, which was defined as sufficient cortical bone thickness to prevent fracture and allow physical activities. RESULTS: Recurrence was identified in 3 cases (10%). Curative outcomes were obtained after repeated ESC or open curettage. A log-rank analysis indicated that age < 10 years (p = 0.004) and contact of the tumour with the physis (p = 0.01) increased the risk of tumour recurrence. Residual tumours were identified in 9 cases (30%); these lesions remained inactive over the extended follow-up period. The average time to solid union after endoscopic curettage was 3.2 months. Transient radial nerve palsy was identified in 1 case. Good postoperative functional recovery occurred in all cases. CONCLUSIONS: ESC is a minimally invasive technique for the treatment of ABCs, and the tumour recurrence rate is comparable to that of other standard procedures. However, the application of this method should be carefully considered, especially for patients < 10 years and when the tumour comes in contact with the physis.

Spontaneous shrinkage of solitary osteochondromas.

OBJECTIVE: Osteochondromas are the most common benign bone tumors, and thus far, their spontaneous shrinkage is considered a rare phenomenon. This study was designed to investigate the exact ratio of remission to progressive or stable cases and analyze the mechanism of tumor regression on the basis of existing theories. MATERIALS AND METHODS: We retrospectively collected images of solitary osteochondromas in patients from 1992 to 2013, excluding cases involving short-term follow-up periods and follow-up periods that ended before growth plate closure. A total of 121 patients were diagnosed and screened for study inclusion. Tumor shrinkage was measured by assessing three points on tumor contours to determine if they had regressed or vanished. Patterns of shrinkage were further divided on the basis of mechanisms described as incorporation, absorption, and fracture. RESULTS: Seventeen patients (mean age at initial diagnosis 13.1 years) met the study inclusion criteria. Tumor morphological classifications were pedunculated (10 cases) and sessile (7 cases). Osteochondroma shrinkage was the most common outcome (8 cases), followed by stable osteochondromas (6 cases), and osteochondromas that had progressed (3 cases). Tumors with sessile morphology were more prone to shrinkage (6 of 7 cases) compared with those of pedunculated morphology (2 of 10 cases; p = 0.015). Among pedunculated cases, tumor shrinkage was via absorption. The timing of tumor growth cessation was related to the pattern of tumor shrinkage. Absorption mostly followed tumor growth cessation, whereas incorporation mostly preceded tumor growth cessation. CONCLUSION: The shrinkage of osteochondromas appears less rare than was originally thought.

Post-operative rotator cuff integrity, based on Sugaya's classification, can reflect abduction muscle strength of the shoulder.

PURPOSE: Magnetic resonance (MR) imaging is common in structural and qualitative assessment of the rotator cuff post-operatively. Rotator cuff integrity has been thought to be associated with clinical outcome. The purpose of this study was to evaluate the inter-observer reliability of cuff integrity (Sugaya's classification) and assess the correlation between Sugaya's classification and the clinical outcome. It was hypothesized that Sugaya's classification would show good reliability and good correlation with the clinical outcome. METHODS: Post-operative MR images were taken two years post-operatively, following arthroscopic rotator cuff repair. For assessment of inter-rater reliability, all radiographic evaluations for the supraspinatus muscle were done by two orthopaedic surgeons and one radiologist. Rotator cuff integrity was classified into five categories, according to Sugaya's classification. Fatty infiltration was graded into four categories, based on the Fuchs' classification grading system. Muscle hypotrophy was graded as four grades, according to the scale proposed by Warner. The clinical outcome was assessed according to the constant scoring system pre-operatively and 2 years post-operatively. RESULTS: Of the sixty-two consecutive patients with full-thickness rotator cuff tears, fifty-two patients were reviewed in this study. These subjects included twenty-three men and twenty-nine women, with an average age of fifty-seven years. In terms of the inter-rater reliability between orthopaedic surgeons, Sugaya's classification showed the highest agreement [ICC (2.1) = 0.82] for rotator cuff integrity. The grade of fatty infiltration and muscle atrophy demonstrated good agreement, respectively (0.722 and 0.758). With regard to the inter-rater reliability between orthopaedic surgeon and radiologist, Sugaya's classification showed good reliability [ICC (2.1) = 0.70]. On the other hand, fatty infiltration and muscle hypotrophy classifications demonstrated fair and moderate agreement [ICC (2.1) = 0.39 and 0.49]. Although no significant correlation was found between overall post-operative constant score and Sugaya's classification, Sugaya's classification indicated significant correlation with the muscle strength score. CONCLUSIONS: Sugaya's classification showed repeatability and good agreement between the orthopaedist and radiologist, who are involved in the patient care for the rotator cuff tear. Common classification of rotator cuff integrity with good reliability will give appropriate information for clinicians to improve the patient care of the rotator cuff tear. This classification also would be helpful to predict the strength of arm abduction in the scapular plane. LEVEL OF EVIDENCE: IV.

MRI appearance in the early stage of Legg-Calvé-Perthes disease to predict lateral pillar classification: A retrospective analysis of the labral horizontalization.

BACKGROUND: The Herring lateral pillar classification is widely used for the classification of Legg-Calvé-Perthes disease, but is not applied at the early stage of Legg-Calvé-Perthes disease because it is typically applied at the late fragmentation stage. The purpose of this study was to investigate the correlation between the early appearance on magnetic resonance imaging of the acetabular labrum and lateral pillar involvement in Legg-Calvé-Perthes disease. METHODS: Non-contrast magnetic resonance images of 26 hips in 25 children with early-stage Legg-Calvé-Perthes disease were retrospectively reviewed. The extent of labral horizontalization was quantitatively evaluated with a new method, the labral angle, on T2*-weighted magnetic resonance images. A small labral angle indicates strong labral horizontalization. Calculation of the teardrop distance and acetabular head index on radiographs was modified for application to magnetic resonance imaging, and the extent of cartilaginous lateral subluxation (cartilaginous tear drop distance) and cartilaginous lateral extrusion (cartilaginous acetabular head index) were evaluated. The outcome measure was the lateral pillar classification. RESULTS: There were statistically significant correlations between the labral angle and the cartilaginous tear drop distance (p = 0.002, ɤ = -0.58) and the cartilaginous acetabular head index (p < 0.001, ɤ = 0.65) on magnetic resonance images. The labral angle was small in order of groups C, B, and A, and there were significant differences between groups A and C (p < 0.001) and B and C (p = 0.006). CONCLUSION: Greater labral horizontalization observed on magnetic resonance imaging at the early stage of Legg-Calvé-Perthes disease correlated with strong cartilaginous lateral subluxation and extrusion, and with increased lateral pillar collapse at the maximum fragmentation stage. Our finding suggests that a quantitative evaluation of labral horizontalization using magnetic resonance imaging in the early-stage of Legg-Calvé-Perthes disease can predict the later lateral pillar classification.

Mithramycin has inhibitory effects on gliostatin and matrix metalloproteinase expression induced by gliostatin in rheumatoid fibroblast-like synoviocytes.

OBJECTIVES: Gliostatin (GLS) has angiogenic and arthritogenic activities and enzymatic activity as thymidine phosphorylase. Aberrant GLS production has been observed in the synovial membranes of patients with rheumatoid arthritis (RA). Matrix metalloproteinases (MMPs) are involved in joint destruction. Promoters of GLS and some MMP genes contain Sp1 binding sites. We examined the inhibitory effect of the Sp1 inhibitor mithramycin on GLS-induced GLS and MMP expression in cultured fibroblast-like synoviocytes (FLSs). METHODS: Synovial tissue samples were obtained from patients with RA. FLSs pretreated with mithramycin were cultured with GLS. The mRNA expression levels of GLS and MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13 were determined using reverse transcription polymerase chain reactions. Protein levels were measured using enzyme immunoassay and gelatin zymography. RESULTS: GLS upregulated the expression of GLS itself and of MMP-1, MMP-3, MMP-9, and MMP-13, an effect significantly reduced by treatment with mithramycin. GLS and mithramycin had no effect on MMP-2 expression. CONCLUSIONS: Mithramycin downregulated the increased expression of GLS and MMP-1, MMP-3, MMP-9, and MMP-13 in FLSs treated with GLS. Because GLS plays a pathological role in RA, blocking GLS stimulation using an agent such as mithramycin may be a novel approach to antirheumatic therapy.

Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

BACKGROUND/AIMS: Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. METHODS: The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. RESULTS: The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. CONCLUSION: Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1.

Heat Shock Protein 70 Negatively Regulates TGF-ß-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts.

BACKGROUND/AIMS: We previously demonstrated that transforming growth factor-ß (TGF-ß) stimulates the synthesis of vascular endothelial growth factor (VEGF) through the activation of p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70) is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-ß-stimulated VEGF synthesis and the underlying mechanism in these cells. METHODS: Culture MC3T3-E1 cells were stimulated by TGF-ß. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. RESULTS: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-ß-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-ß was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-ß-induced phosphorylation of p38 MAP kinase. The TGF-ß-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-ß-induced VEGF release. CONCLUSION: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-ß-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

(-)-Epigallocatechin gallate synergistically potentiates prostaglandin E2-stimulated osteoprotegerin synthesis in osteoblasts.

(-)-Epigallocatechin gallate (EGCG), the most abundant flavonoid in green tea, and chlorogenic acid, the main polyphenol found in coffee, attract significant attention owing to health benefits. We have previously demonstrated that prostaglandin E2 (PGE2) stimulates osteoprotegerin synthesis through the activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of EGCG or chlorogenic acid on the PGE2-stimulated osteoprotegerin synthesis in MC3T3-E1 cells. EGCG significantly amplified the PGE2-induced release. EGCG markedly enhanced the expression levels of osteoprotegerin mRNA induced by PGE2. On the contrary, chlorogenic acid had no effect on the PGE2-stimulated release of osteoprotegerin. EGCG significantly strengthened the PGE2-induced phosphorylation of p38 MAP kinase and SAPK/JNK, whereas chlorogenic acid failed to affect them. BIRB0796 and SP600125, a p38 MAP kinase inhibitor and a SAPK/JNK inhibitor, respectively, markedly reduced the amplification by EGCG of the PGE2-stimulated osteoprotegerin release. These results strongly suggest that EGCG synergistically enhances the PGE2-stimulated osteoprotegerin synthesis via potentiation of p38 MAP kinase and SAPK/JNK in osteoblasts. Our present findings could present a new significant aspect in the favorable effect of EGCG on the prevention of osteoporotic bone loss and fracture especially in elderly people since osteoprotegerin secreted from osteoblasts is well-recognized to act as a suppressor of osteoclastic bone resorption.