Anterior Cruciate Ligament-Injured Knees With Meniscal Ramp Lesions Manifest Greater Anteroposterior and Rotatory Instability Compared With Isolated Anterior Cruciate Ligament-Injured Knees.

PURPOSE: To investigate the effects of ramp lesion (RL) and its repair on knee instability in patients with anterior cruciate ligament (ACL) injury by quantitatively assessing anteroposterior and rotational knee instability before and after ACL reconstruction. METHODS: All primary double-bundle ACL reconstructions using hamstring autografts between 2016 and 2021 were evaluated retrospectively. Patients with RLs without other meniscal injuries were included in group R, whereas those with isolated ACL injuries constituted group C. RL was repaired using all-inside devices in all patients in group R. Knee instability, including the amount of anterior tibial translation (ATT), and the acceleration and external rotational angular velocity of the knee joint (ERAV) during the pivot-shift test were assessed at the time of surgery. The pivot-shift test grade was recorded. RESULTS: A total of 73 patients were included in this study. Preoperatively, group R (n = 23) had significantly greater pivot-shift grades (P = .039), ATT (6.0 mm, group R; 4.5 mm, group C, P < .001), acceleration (6.8, 2.8; P = .037), and ERAV (3.9, 2.8; P = .001) than group C (n = 50). Intraoperatively, ATT (-1.0 mm, -1.0 mm; P < .001), acceleration (1.2, 1.1; P < .001), and ERAV (1.4, 1.2; P < .001) were significantly decreased compared with the preoperative values in both groups. No significant differences in these values were observed between groups R and C. CONCLUSIONS: ACL-injured knees accompanied by RLs exhibited significantly greater anteroposterior and rotatory instability than knees with isolated ACL injuries; increased knee instability can be effectively addressed by performing RL repair in conjunction with ACL reconstruction. The quantitative assessments employed-specifically measuring ATT, acceleration, and ERAV during the pivot-shift test-have allowed us to delineate these aspects of knee instability with greater precision. LEVEL OF EVIDENCE: Level Ⅲ, retrospective comparative study.

Efficacy of all-inside devices in reducing gap and step-off in knee extension for ramp lesion repair: A cadaveric study.

PURPOSE: The aim of this study is to assess the dynamics of the tear site of meniscal ramp lesions, particularly considering knee flexion angles, and validate anchor fixation using an all-inside device. METHODS: Eight Thiel-embalmed paired cadaveric knees with their whole bodies were used in this study. The ramp lesions were created arthroscopically, and ramp lesion dynamics were evaluated by gradually extending the knee from 90° of knee flexion. Changes in the gap and step-off (0: no step-off; 1: cross-sectional overlap exists; and 2: tibial articular surface exposed) were evaluated at 90°, 60°, 30°, and 10° of knee flexion. After dynamic evaluation, all-inside repairs of the ramp lesions using all-inside devices were conducted. Dissection was performed to confirm the position of anchor fixation. RESULTS: As the knee was extended, the gap significantly decreased at all knee flexion angles. Similarly, the step-off grade decreased as the knee was extended, and the step-off completely disappeared in all cases when the knee was extended from 30° to 10°. The average knee flexion angle at which the gap and step-off completely disappeared was 22.5°. After suturing the ramp lesion, arthroscopic evaluation showed that the gap had disappeared and the step-off had been repaired in all cases. Anchor fixation locations were not found within the joint but were fixed to the semimembranosus tendon or its surrounding articular capsule. Overall, 31% (5/16) anchors were fixed to the attachment site of the semimembranosus tendon, whereas the remaining were fixed to the articular capsule, located peripherally to the semimembranosus tendon. CONCLUSION: Suturing with an all-inside device for ramp lesions is a good option, and the repair in knee extension was found to be reasonable, considering the dynamics of ramp lesions in this study. LEVEL OF EVIDENCE: Level IV.

Endosteal strut using a hydroxyapatite/poly-L-lactide mesh tube with a proximal humeral locking plate for the treatment of proximal humeral fractures.

PURPOSE: Proximal humeral fractures cause large intramedullary bone defects after humeral-head reduction. Hydroxyapatite/poly-L-lactide (HA/PLLA) materials are widely used for various fractures. However, the efficacy of endosteal strut using a HA/PLLA mesh tube (ES-HA/PLLA) with a locking plate for treating proximal humeral fractures was not reported. The purpose of this study is to examine the efficacy of ES-HA/PLLA with a proximal humeral locking plate in proximal humeral fractures. METHODS: Seventeen patients with proximal humeral fractures treated using ES-HA/PLLA with a locking plate from November 2017 to November 2021 were evaluated. The range of motion of the shoulder and postoperative complications were assessed at the final follow-up. Radiographs were evaluated to assess bone union and loss of reduction by measuring humeral-head height (HHH) and humeral neck-shaft angle (NSA). RESULTS: The average flexion and external rotation of the shoulder at the final follow-up were 137° (range, 90-180°) and 39° (range, - 10 to 60°), respectively. All fractures were united. The average HHH and NSA just after the surgery and final follow-up were 12.5 mm and 11.6 mm and 129.9° and 127.4°, respectively. Two patients presented screw perforation of the humeral head. One patient underwent implant removal due to infection. Avascular necrosis of the humeral head was observed in one patient with arthritis mutilans. CONCLUSIONS: The use of ES-HA/PLLA with a proximal humeral locking plate resulted in bone union in all patients and prevented postoperative loss of reduction. ES-HA/PLLA is one of the treatment options for proximal humeral fractures.

Oncostatin M attenuates tumor necrosis factor-α-induced synthesis of macrophage-colony stimulating factor via suppression of Akt in osteoblasts.

BACKGROUND: Oncostatin M produced by osteal macrophages, a cytokine that belongs to the interleukin-6 family, is implicated in bone fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts plays an important role in osteoclastogenesis. We have previously reported that tumor necrosis factor-α (TNF-α), a potent bone resorptive agent, stimulates the activation of p44/p42 mitogen-activated protein (MAP) kinase, Akt, and p70 S6 kinase in osteoblast-like MC3T3-E1 cells, and induces the synthesis of M-CSF at least in part via Akt. OBJECTIVE: In the present study, we investigated whether oncostatin M affects the TNF-α-induced M-CSF synthesis in MC3T3-E1 cells and the underlying mechanisms. METHODS: Clonal osteoblast-like MC3T3-E1 cells were treated with oncostatin M or rapamycin and then stimulated with TNF-α. M-CSF release was assessed by ELISA. M-CSF mRNA expression level was assessed by real-time RT-PCR. Phosphorylation of Akt, p44/p42 MAP kinase, and p70 S6 kinase was detected by Western blot analysis. RESULTS: Oncostatin M dose-dependently reduced the TNF-α-stimulated M-CSF release. The expression of M-CSF mRNA induced by TNF-α was significantly suppressed by oncostatin M. Rapamycin, an inhibitor of mTOR/p70 S6 kinase, had little effect on the M-CSF release by TNF-α. Oncostatin M significantly reduced the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. However, the p70 S6 kinase phosphorylation by TNF-α was not affected by oncostatin M. CONCLUSION: These results strongly suggest that oncostatin M attenuates TNF-α-stimulated synthesis of M-CSF in osteoblasts, and the inhibitory effect is exerted at a point upstream of Akt and p44/p42 MAP kinase but not p70 S6 kinase.

Resveratrol inhibits basic fibroblast growth factor-induced macrophage colony-stimulating factor synthesis via the PI3-kinase/Akt pathway in osteoblasts.

Resveratrol is a natural polyphenol found in grapes and beneficial for human health. Resveratrol regulates basic fibroblast growth factor (bFGF)-induced osteoprotegerin synthesis through Akt pathway in osteoblast-like MC3T3-E1 cells. In this study, we investigated resveratrol effects on bFGF-induced macrophage colony-stimulating factor (M-CSF) synthesis in MC3T3-E1 cells. bFGF significantly stimulated release and mRNA expression of M-CSF, which was reduced by resveratrol and SRT1720, sirtuin 1 (SIRT1) activator. Inauhzin, SIRT1 inhibitor, reversed inhibitory effects of resveratrol on bFGF-induced mRNA expression of M-CSF. Deguelin, Akt inhibitor, and LY294002, phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, reduced bFGF-induced M-CSF synthesis. Inauhzin reversed inhibitory effects of resveratrol on bFGF-induced Akt phosphorylation. Suppressive effect of resveratrol on bFGF-induced osteoprotegerin mRNA expression was confirmed in the identical samples using in experiment of M-CSF mRNA expression. Therefore, resveratrol reduces bFGF-induced M-CSF synthesis in addition to osteoprotegerin synthesis by inhibiting PI3-kinase/Akt pathway and suppressive effects are mediated through SIRT1 activation in osteoblasts.

Clinical and radiographic outcomes of primary total hip arthroplasty with the revelation hip system using density mapping.

The Revelation Hip System is a cementless stem with a lateral flare concept. Stable fixation is achieved by fitting the stem to the medullary cavity of the proximal lateral femoral cortex. Patients who have undergone total hip arthroplasty using the Revelation Hip System show good postoperative clinical and radiographic outcomes. However, to the best of our knowledge, no study has reported the relationship between stem fitting and clinical or radiological outcomes after the surgery. In the present study, we investigated the relationship between stem fitting and clinical or radiological outcomes after total hip arthroplasty (THA) using the Revelation Hip System. In this study, 28 hips of 26 patients who were treated with the Revelation Hip System for osteoarthritis, osteonecrosis of the femoral head, rheumatoid arthritis, and rapidly destructive coxarthropathy and were followed up for > 5 y were enrolled. These patients were divided into two groups, including the rest fit group (11 hips, group R) and the control group (17 hips, group C), according to the results of the density mapping analysis. In group R, the lateral side of the stem fits on the medullary cavity of the proximal lateral femoral cortex, while in group C, the lateral side of the stem did not fit. Radiographic results showed no significant differences between the groups in terms of stem alignment, subsidence, and stress shielding around the cup. The incidence of stress shielding around the stem in zone 7 was not significant but tended to be higher in group R than in group C (p = 0.052). Clinical outcomes showed no significant differences between group R and group C in terms of the Harris hip score, the Japanese Orthopaedic Association (JOA) score, and the Japanese Orthopaedic Association Hip Disease Evaluation Questionnaire (JHEQ) total score. However, pain complaints that were assessed by patient-reported outcomes using the 36-Item Short Form Health Survey (SF-36) bodily pain and vitality subscales and the JHEQ pain subscale were significantly higher in group R than in group C at the final follow-up. These results suggest that some patients had pain complaint even if the stems were inserted as per the concept after THA with the Revelation Hip System.Trial Registration911.

Incretins Enhance PGF2α-Induced Synthesis of IL-6 and Osteoprotegerin in Osteoblasts.

Incretins including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), which are secreted from the small intestine after oral food ingestion, are currently well-known to stimulate insulin secretion from pancreatic ß-cells and used for the treatment of type 2 diabetes mellitus. We have previously reported that prostaglandin F2α (PGF2α) stimulates the synthesis of interleukin-6 (IL-6) and osteoprotegerin in osteoblast-like MC3T3-E1 cells, and that IL-6 and osteoprotegerin release are mediated through the p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways. In the present study, we investigated the effects of incretins including GLP-1 and GIP, on the PGF2α-induced synthesis of IL-6 and osteoprotegerin and examined the detailed mechanism in osteoblast-like MC3T3-E1 cells. We found that GIP and GLP-1 significantly stimulated the PGF2α-induced synthesis of IL-6 in osteoblast-like MC3T3-E1 cells. In addition, GIP and GLP-1 significantly enhanced the PGF2α-induced mRNA expression levels of IL-6. On the other hand, GIP and GLP-1 markedly stimulated the PGF2α-induced synthesis of osteoprotegerin. However, the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or JNK induced by PGF2α was not affected by GIP or GLP-1. Therefore, these results strongly suggest that incretins enhance the PGF2α-induced synthesis of IL-6 and osteoprotegerin in osteoblast-like MC3T3-E1 cells. However, these syntheses are not mediated through p44/p42 MAP kinase, p38 MAP kinase, or JNK pathways.

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.

Diagnosis of medial meniscal ramp lesion is difficult by pre-operative magnetic resonance imaging evaluation and needs a methodical arthroscopic exploration.

BACKGROUND: Meniscal ramp lesion (RL) is the peripheral lesion of the posterior horn of the medial meniscus (PHMM) associated with anterior cruciate ligament (ACL) tear. The purpose of this study was to evaluate the accuracy of pre-operative magnetic resonance imaging (MRI) evaluation in diagnosing RL and to identify whether the difficulty in diagnosis differs depending on the location of RL. METHODS: ACL-injured patients undergoing ACL reconstruction from January 2017 to January 2019 were enrolled. A methodical arthroscopic exploration to identify RL was conducted intra-operatively using three steps, namely, the anterior visualization step, the inter-condylar visualization step, and the posteromedial step. The location of the RLs was evaluated and classified into two types as follows: Red-red zone (RR) - a meniscal tear of the red-red zone of the PHMM. Menisco-capsular junction (MCJ) - a lesion at the menisco-capsular junction of the PHMM, which is more peripheral than RR. Furthermore, the accuracy of 1.5-T MRI evaluation to diagnose RL by two testers using sagittal proton-density fat-saturated images was calculated. RESULTS: Of the 81 patients enrolled, 11 had RL: 5 cases each were at the MCJ and RR, and 1 case was at both locations. The sensitivity of MRI for detecting RL was 27.3-45.5%, whereas the specificity was 84.3-95.7% in total. The sensitivity of MRI in detecting RL at the RR and MCJ was 40.0-80.0%, 0-20.0%, respectively. The intra-observer reliability of the MRI evaluation was moderate (κ coefficient: 0.40-0.46), while the inter-observer reliability was fair to moderate (κ coefficient: 0.27-0.41). CONCLUSIONS: A low sensitivity of the MRI in detecting RL at the MCJ was observed, and the reliability of the MRI evaluation for diagnosis of RL was not high. Therefore, methodical arthroscopic exploration is essential to diagnose RL even when it is not suspected on pre-operative MRI.

Impact of social support on severity of depressive symptoms by remission status in patients with rheumatoid arthritis.

OBJECTIVES: We aimed to examine the psychosocial characteristics of patients with rheumatoid arthritis (RA) by remission status and determine the impacts of social support on severity of depressive symptoms. METHODS: We enrolled RA patients aged 40-79 years who visited university hospitals' outpatient clinics. Severity of depressive symptoms (Beck Depression Inventory-II), physical disability (Health Assessment Questionnaire), and support were evaluated. Furthermore, RA disease activity was evaluated by 28-point Disease Activity Score (DAS28) calculation. The independent impacts of instrumental and emotional social support on depressive symptoms by remission status defined as DAS28 score < 2.6 were estimated by multivariable regression analysis. RESULTS: This study included 360 RA patients. In the remission group, emotional support showed a statistically significant negative impact on depressive symptoms, whereas instrumental support had an extremely limited contribution to severity of depressive symptoms. In the non-remission group, instrumental support showed a negative tendency of impact on severity of depressive symptoms, whereas emotional support had a wide range of influence. CONCLUSIONS: Favourable association between emotional support and depressive symptoms is confirmed only among RA patients in remission status. The influence of emotional support in non-remission patients and that of instrumental support regardless of remission status are inconclusive.

Depression, physical function, and disease activity associated with frailty in patients with rheumatoid arthritis.

OBJECTIVES: To investigate the clinical and psychosocial backgrounds of frailty in rheumatoid arthritis (RA) patients. METHODS: Patients with RA between 40 and 79 years of age who visited university hospitals in an urban area were recruited. Well-validated self-reported questionnaires were used to evaluate patient physical function (Health Assessment Questionnaire, HAQ), depressive symptoms (Beck Depression Inventory-II, BDI-II), and frailty (Kihon Checklist). A 28-point Disease Activity Score (DAS-28) was calculated to evaluate RA disease activity. RESULTS: A total of 375 RA patients, 323 of whom were women, were enrolled (average age: 65.2 ± 9.7 years; average disease duration: 16.6 ± 11.9 years). The prevalence rates of frailty, working-age (40-64 years), young-old (65-74 years), and old-old (≥75 years) patients were 18.5, 28.8, and 36.6%, respectively. Higher age and longer disease duration were associated with frailty. Multivariable logistic regression analysis revealed that HAQ, DAS-28, and BDI-II scores were independently associated with frailty in RA patients. CONCLUSION: Frailty is common, even among working-age RA patients. Physical function, disease activity, and depressive symptoms were independently associated with frailty. A multidisciplinary intervention approach, along with adequate pharmacological therapy, may promote successful aging in patients with RA.

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.

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.

(-)-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.

Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients.

It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9-25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25-50 µm), large aggregates (50-70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients.

Regulation by heat shock protein 22 (HSPB8) of transforming growth factor-α-induced ovary cancer cell migration.

Accumulating evidence suggests that heat shock proteins (HSPs) are implicated in progression of cancer. HSP22 (HSPB8), a small HSP, is recognized to be ubiquitously expressed in various tissues. However, the expression and the role of HSP22 in ovarian cancer remain to be clarified. In the present study, we investigated the involvement of HSP22 in transforming growth factor (TGF)-α-induced migration of ovarian cancer cells. The expression of HSP22 was detected in a serous ovarian cancer cell line, SKOV3.ip1. The migration was reduced by down-regulation of HSP22 expression. The TGF-α-induced migration was reduced by SB203580 (a p38 MAP kinase inhibitor), SP600125 (a SAPK/JNK inhibitor) and Y27632 (a Rho-kinase inhibitor). However, down-regulation of HSP22 had little effect on the TGF-α-induced phosphorylation of p38 MAP kinase, SAPK/JNK and MYPT, a target protein of Rho-kinase. The HSP22 expression was further analyzed in 20 resected specimens of human ovarian serous carcinoma. The expression of HSP22 was detected in all the twenty tissues (8.24-109.22 pg/mg protein), and the cases with highly expression of HSP22 showed a tendency to acquire the progressive ability. Our results strongly suggest that HSP22 acts as a positive regulator in TGF-α-induced migration of ovarian cancer cells, subsequently directing ovarian cancer toward progression.

Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: suppression of stress-activated protein kinase/c-Jun N-terminal kinase.

Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts.

Resveratrol suppresses prostaglandin F(2α)-induced osteoprotegerin synthesis in osteoblasts: inhibition of the MAP kinase signaling.

Resveratrol, a natural polyphenol abundantly found in grape skins and red wine, possesses various beneficial properties for human health. In the present study, we investigated the mechanism underlying the effects of prostaglandin F2α (PGF2α) on osteoprotegerin (OPG) synthesis and of resveratrol on the OPG synthesis in osteoblast-like MC3T3-E1 cells. PGF2α stimulated both the release of the OPG protein and the expression of OPG mRNA. Treatment with PD98059, SB203580 and SP600125, specific inhibitors of MEK1/2, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) all suppressed the OPG release induced by PGF2α. Resveratrol also significantly reduced the PGF2α-stimulated OPG release and the mRNA levels of OPG. Similarly, treatment with SRT1720, an activator of SIRT1, also suppressed the PGF2α-stimulated OPG release. Resveratrol and SRT1720 both attenuated the phosphorylation of p44/p42 MAP kinase, MEK1/2, Raf-1, p38 MAP kinase and SAPK/JNK induced by PGF2α. These findings strongly suggest that resveratrol suppresses PGF2α-stimulated OPG synthesis by inhibiting the MAP kinase pathways in osteoblasts, and that the effect is mediated via SIRT1 activation.

Down-regulation by resveratrol of basic fibroblast growth factor-stimulated osteoprotegerin synthesis through suppression of Akt in osteoblasts.

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation.