Bone scan index (BSI) scoring by using bone scintigraphy and circulating tumor cells (CTCs): predictive factors for enzalutamide effectiveness in patients with castration-resistant prostate cancer and bone metastases.

Reports of Bone Scan Index (BSI) calculations as imaging biomarkers to predict survival in patients with metastatic castration-resistant prostate cancer (mCRPC) have been mainly from retrospective studies. To evaluate the effectiveness of enzalutamide (ENZ) in Japanese patients with mCRPC and bone metastases using BSI (bone scintigraphy) and circulating tumor cell (CTC) analysis. Prospective, single-arm study at Juntendo University affiliated hospitals, Japan. Patients were administered 160 mg ENZ daily, with 3 monthly assessments: BSI, prostate specific antigen (PSA), CTC and androgen receptor splicing variant-7 (AR-V7) status. Primary endpoint: BSI-decreasing rate after ENZ treatment. Secondary endpoints: PSA-decreasing rate and progression free survival (PFS). Statistical analyses included the Wilcoxon t-test, Cox proportional hazard regression analysis, and log-rank test. Median observation period: 17.9 months, and median PFS: 13.8 (2.0-43.9) months (n = 90 patients). A decrease in BSI compared to baseline as best BSI change on ENZ treatment was evident in 69% patients at the end of the observation period (29% patients showed a complete response, BSI 0.00). At 3 months 67% patients showed a ≥ 50% PSA reduction, and 70% after ENZ treatment. PSA decline (3 months) significantly associated with a prolonged median PFS: 18.0 (estimated) versus 6.4 months (HR 2.977 [95% CI 1.53-5.78], p = 0.001). Best BSI decline response significantly associated with a prolonged PFS: 18.1(estimated) versus 7.8 months (HR 2.045 [95% CI: 1.07-3.90], p = 0.029). CTC negative status (n = 20) significantly associated with a prolonged PFS: 13.4 [estimated] vs 8.6 months (HR 2.366, 95% CI 0.97-5.71, p = 0.041). CTC positive/AR-V7 positive status significantly associated with a shorter PFS: 5.9 months (HR 8.56, 95% CI 2.40-30.43, p = 0.0087). -reduction (3 months) and BSI-reduction (on ENZ treatment) were significant response biomarkers, and a negative CTC status was a predictive factor for ENZ efficacy in patients with mCRPC.

HSP70 inhibitors upregulate prostaglandin E1-induced synthesis of interleukin-6 in osteoblasts.

Interleukin-6 (IL-6) is a pro-inflammatory and bone-resorptive cytokine that also regulates bone formation. We previously showed that prostaglandin E1 (PGE1) induces the synthesis of IL-6 by activating p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAPK in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether heat shock protein 70 (HSP70), a molecular chaperone that coordinates protein folding and homeostasis, affects PGE1-stimulated IL-6 synthesis in MC3T3-E1 cells through the MAPK activation. The osteoblast-like MC3T3-E1 cells were treated with HSP70 inhibitors-VER-155008 and YM-08-, PD98059, SB203580 or SP600125 and then stimulated with PGE1. IL-6 synthesis was evaluated using an IL-6 enzyme-linked immunosorbent assay kit. IL-6 mRNA expression was measured by real-time RT-PCR. The phosphorylation of p38 MAPK was evaluated by Western blotting. We found that VER-155008, an HSP70 inhibitor, enhanced the PGE1-stimulated IL-6 release and IL-6 mRNA expression. YM-08, another HSP70 inhibitor, also enhanced PGE1-stimulated IL-6 release. PD98059, a p44/p42 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, upregulated PGE1-stimulated IL-6 release. On the other hand, SB203580, a p38 MAPK inhibitor, suppressed PGE1-stimulated IL-6 release. YM-08 stimulated the PGE1-induced phosphorylation of p38 MAPK. SB203580 suppressed the amplification by YM-08 of the PGE1-stimulated IL-6 release. Our results suggest that HSP70 inhibitors upregulate the PGE1-stimulated IL-6 synthesis through p38 MAPK in osteoblasts and therefore affect bone remodeling.

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.

The coral reef-dwelling Peneroplis spp. shows calcification recovery to ocean acidification conditions.

Large Benthic Foraminifera are a crucial component of coral-reef ecosystems, which are currently threatened by ocean acidification. We conducted culture experiments to evaluate the impact of low pH on survival and test dissolution of the symbiont-bearing species Peneroplis spp., and to observe potential calcification recovery when specimens are placed back under reference pH value (7.9). We found that Peneroplis spp. displayed living activity up to 3 days at pH 6.9 (Ωcal < 1) or up to 1 month at pH 7.4 (Ωcal > 1), despite the dark and unfed conditions. Dissolution features were observed under low Ωcal values, such as changes in test density, peeled extrados layers, and decalcified tests with exposed organic linings. A new calcification phase started when specimens were placed back at reference pH. This calcification's resumption was an addition of new chambers without reparation of the dissolved parts, which is consistent with the porcelaneous calcification pathway of Peneroplis spp. The most decalcified specimens displayed a strong survival response by adding up to 8 new chambers, and the contribution of food supply in this process was highlighted. These results suggest that porcelaneous LBF species have some recovery abilities to short exposure (e.g., 3 days to 1 month) to acidified conditions. However, the geochemical signature of trace elements in the new calcite was impacted, and the majority of the new chambers were distorted and resulted in abnormal tests, which might hinder the specimens' reproduction and thus their survival on the long term.

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.

Decrease in volume and density of foraminiferal shells with progressing ocean acidification.

Rapid increases in anthropogenic atmospheric CO2 partial pressure have led to a decrease in the pH of seawater. Calcifying organisms generally respond negatively to ocean acidification. Foraminifera are one of the major carbonate producers in the ocean; however, whether calcification reduction by ocean acidification affects either foraminiferal shell volume or density, or both, has yet to be investigated. In this study, we cultured asexually reproducing specimens of Amphisorus kudakajimensis, a dinoflagellate endosymbiont-bearing large benthic foraminifera (LBF), under different pH conditions (pH 7.7-8.3, NBS scale). The results suggest that changes in seawater pH would affect not only the quantity (i.e., shell volume) but also the quality (i.e., shell density) of foraminiferal calcification. We proposed that pH and temperature affect these growth parameters differently because (1) they have differences in the contribution to the calcification process (e.g., Ca2+-ATPase and Ω) and (2) pH mainly affects calcification and temperature mainly affects photosynthesis. Our findings also suggest that, under the IPCC RCP8.5 scenario, both ocean acidification and warming will have a significant impact on reef foraminiferal carbonate production by the end of this century, even in the tropics.

HSP22 (HSPB8) positively regulates PGF2α-induced synthesis of interleukin-6 and vascular endothelial growth factor in osteoblasts.

BACKGROUND: Heat shock protein 22 (HSP22) belongs to class I of the small HSP family that displays ubiquitous expression in osteoblasts. We previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling factor, induces the synthesis of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether HSP22 is implicated in the PGF2α-induced synthesis of IL-6 and VEGF and the mechanism of MC3T3-E1 cells. METHODS: MC3T3-E1 cells were transfected with HSP22-siRNA. IL-6 and VEGF release was assessed by ELISA. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was detected by Western blotting. RESULTS: The PGF2α-induced release of IL-6 in HSP22 knockdown cells was significantly suppressed compared with that in the control cells. HSP22 knockdown also reduced the VEGF release by PGF2α. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was attenuated by HSP22 downregulation. CONCLUSIONS: Our results strongly suggest that HSP22 acts as a positive regulator in the PGF2α-induced synthesis of IL-6 and VEGF in osteoblasts.

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.

Tramadol enhances PGF2α-stimulated osteoprotegerin synthesis in osteoblasts.

Osteoprotegerin (OPG) synthesized by osteoblasts is currently considered a crucial regulator to suppress the formation and function of osteoclasts. We previously showed that the synthesis of OPG is stimulated by prostaglandin F2α (PGF2α) in the involvement of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. We also found that Rho-kinase is involved in the signaling of PGF2α upstream of p38 MAPK in these cells. Tramadol is widely used to treat chronic pain, such as low back pain associated with osteoporosis. We investigated whether or not tramadol affects the OPG release induced by PGF2α in osteoblast-like MC3T3-E1 cells. The levels of OPG in the conditioned medium were measured by an enzyme-linked immunosorbent assay. The mRNA expression of OPG was determined with real-time reverse transcription polymerase chain reaction. The phosphorylation of target protein was determined with a Western blot analysis. PGF2α induced the release and the mRNA expression of OPG, which tramadol significantly enhanced. Morphine, a selective µ-opioid receptor (MOR) agonist, also enhanced the PGF2α-induced OPG release. In addition, naloxone, a MOR antagonist, suppressed the enhancement by tramadol or morphine of the PGF2α-induced OPG synthesis. Tramadol upregulated the phosphorylation of SAPK/JNK and p38 MAPK stimulated by PGF2α but not that of p44/p42 MAPK or myosin phosphatase targeting protein (MYPT), a substrate of Rho-kinase. The inhibitors of both p38 MAPK and SAPK/JNK, SB203580 and SP600125, respectively, reduced the tramadol amplification of OPG release stimulated by PGF2α. The present results strongly suggest that tramadol enhances the synthesis of OPG stimulated by PGF2α through MOR in osteoblasts, and that the amplifying effect is exerted at upstream of p38 MAPK and SAPK/JNK but downstream of Rho-kinase.

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.

Incretin accelerates platelet-derived growth factor-BB-induced osteoblast migration via protein kinase A: The upregulation of p38 MAP kinase.

Incretins, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine cells after food ingestion, are currently recognized to regulate glucose metabolism through insulin secretion. We previously demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces the migration of osteoblast-like MC3T3-E1 cells through mitogen-activated protein (MAP) kinases, including p38 MAP kinase. In the present study, we investigated whether or not incretins affect the osteoblast migration. The PDGF-BB-induced cell migration was significantly reinforced by GLP-1, GIP or cAMP analogues in MC3T3-E1 cells and normal human osteoblasts. The upregulated migration by GLP-1 or cAMP analogues was suppressed by H89, an inhibitor of protein kinase A. The amplification by GLP-1 of migration induced by PDGF-BB was almost completely reduced by SB203580, a p38 MAP kinase inhibitor in MC3T3-E1 cells and normal human osteoblasts. In addition, GIP markedly strengthened the PDGF-BB-induced phosphorylation of p38 MAP kinase. Exendin-4, a GLP-1 analogue, induced Rho A expression and its translocation from cytoplasm to plasma membranes in osteoblasts at the epiphyseal lines of developing mouse femurs in vivo. These results strongly suggest that incretins accelerates the PDGF-BB-induced migration of osteoblasts via protein kinase A, and the up-regulation of p38 MAP kinase is involved in this acceleration. Our findings may highlight the novel potential of incretins to bone physiology and therapeutic strategy against bone repair.

HSP90 inhibitors diminish PDGF-BB-induced migration of osteoblasts via suppression of p44/p42 MAP kinase.

Migration of osteoblasts to the sites resorbed by osteoclasts is an essential step in bone remodeling. However, the exact mechanism of osteoblast migration is still not known. We have shown that platelet-derived growth factor (PDGF)-BB induces the migration of osteoblast-like MC3T3-E1 cells through the activation of p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase. Evidence is accumulating that heat shock protein 90 (HSP90) acts as a central regulator of proteostasis under stress conditions and physiological cell functions. In the present study, using transwell cell migration assay and wound-healing assay, we investigated the involvement of HSP90 in the PDGF-BB-stimulated migration of MC3T3-E1 cells, and the underlying signaling mechanism estimated by Western blot analyses. Onalespib, an HSP90 inhibitor, significantly reduced the PDGF-BB-stimulated migration evaluated by the two types of migration assays. The cell migration was also suppressed by geldanamycin, another type of HSP90 inhibitor. Onalespib markedly attenuated the PDGF-BB-elicited phosphorylation of p44/p42 MAP kinase without affecting that of p38 MAP kinase or JNK. In addition, the phosphorylation of p44/p42 MAP kinase by PDGF-BB was reduced by geldanamycin. Taken together, these results strongly suggest that HSP90 inhibitors suppress the PDGF-BB-induced osteoblast migration through the attenuation of p44/p42 MAP kinase activity.

Wnt3a downregulates thyroid hormone-induced osteocalcin expression in osteoblasts.

Wnt3a is a crucial modulator of bone metabolism through the canonical Wnt/ß-catenin signaling pathway in bone-forming osteoblasts. We previously reported that the expression of osteocalcin is stimulated by triiodothyronine (T3) at least in part through the activation of p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on the T3-induced osteocalcin expression in these cells. Wnt3a suppressed the release of osteocalcin induced by T3. The inhibitory effect of Wnt3a was dose-dependent between 0.3 and 30 ng/ml. SB216763, an inhibitor of glycogen synthase kinase-3ß, that reduces the phosphorylation of ß-catenin, inhibited the T3-induced osteocalcin release. Wnt3a, as well as SB216763, reduced the expression of osteocalcin mRNA induced by T3. The transcriptional activity induced by T3, assessed by a luciferase activity, was also suppressed by both Wnt3a and SB216763. In contrast, Wnt3a did not markedly affect the T3-stimulated phosphorylation of p38 MAP kinase. These results suggested that Wnt3a downregulates the T3-stimulated osteocalcin expression in MC3T3-E1 cells, and the suppressive effect of Wnt3a is independent of p38 MAP kinase.

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.

[MAKING OF THE DISEASE SEVERITY PREDICTION INDEX FOR ACUTE PYELONEPHRITIS ASSOCIATED WITH URINARY OBSTRUCTION DUE TO URETERAL CALCULI].

(Purpose) Both administration of antibiotics and drainage of urine are necessary for the treatment of acute pyelonephritis associated with urinary obstruction by the ureteral calculi. Though most patients get better after the treatment, some patients deteriorate accompanying with low blood pressure, and need the intensive care. Such patients sometimes visit small hospital, even at night with a few medical staffs. It is sometimes difficult to predict the patient's outcome. The disease severity prediction index for the patients was investigated. (Object and method) We examined 134 patients, who visited our hospital from 2001 to 2013, retrospectively. Ureteral stenting or nephrostomy was undergone within 24 hours in principle. If the blood pressure became under 90 mmHg, or lowered more than 40 mmHg than usual, the case was defined as serious. Blood data and physical findings were compared between serious and non-serious cases. The factors affecting the seriousness were found. Multiple logistic analysis was done to make a disease severity prediction index. (Result) 42 cases were judged as serious and 92 cases as non-serious. Six factors consisting of heart rate, serum creatinine, platelets, ages, PS and CRP affected the consequence significantly (p<0.05), however, white blood cells did not.Multiple logistic analysis was done, four factors consisting of serum creatinine, platelets, PS and CRP affected the consequence significantly (p<0.05), and the standardizing coefficients of each points were found to be 2, 2, 1, 1, respectively.The disease severity prediction index was proposed. If the index was 4 or more, the sensitivity and specificity were found to be 73.8% and 82.6%, respectively. (Conclusion) This index is useful and reliable for the prediction of the outcome of the disease.

HSP70 Inhibitors Reduce The Osteoblast Migration By Epidermal Growth Factor.

BACKGROUND: We have recently reported that epidermal growth factor (EGF) induces migration of osteoblast-like MC3T3-E1 cells through the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) and Akt. Furthermore, we demonstrated that heat shock protein 70 (HSP70) down-regulates the transforming growth factor-ß- stimulated vascular endothelial growth factor synthesis via suppression of p38 MAP kinase in osteoblast-like MC3T3-E1 cells. However, the exact role of HSP70 underlying osteoblast migration is not fully elucidated. OBJECTIVE: The aim of this study is to investigate the effects of HSP70 inhibitors on the EGF-stimulated osteoblast migration, and the underlying mechanism. METHODS: Osteoblast-like MC3T3-E1 cells were treated with two types of HSP70 inhibitors, VER-155008 or YM-08. Transwell cell migration assay and wound-healing assay were analyzed for osteoblast migration. The expression levels of HSP70 and the phosphorylation of p38 MAP kinase, p44/p42 MAP kinase, SAPK/JNK or Akt were evaluated by a Western blot analysis. RESULTS: EGF hardly affected the expression levels of HSP70 at the present or absent of VER-155008. EGF-stimulated migration was significantly reduced by both HSP70 inhibitors, VER-155008 and YM-08, determined by a transwell cell migration assay. The suppressive effects of both HSP70 inhibitors on the migration stimulated by EGF were also observed by a wound-healing assay. VER-155008 inhibited the EGF-induced phosphorylation of p44/p42 MAP kinase and AKT, but not p38 MAP kinase or SAPK/JNK. CONCLUSION: This study provides new evidence that HSP70 inhibitors reduce the EGFstimulated migration of osteoblasts through the suppression of p44/p42 MAP kinase and Akt.

HSP70 Inhibitor Suppresses IGF-I-Stimulated Migration of Osteoblasts through p44/p42 MAP Kinase.

Heat shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone in a variety of cells including osteoblasts. We previously showed that insulin-like growth factor-I (IGF-I) elicits migration of osteoblast-like MC3T3-E1 cells through the activation of phosphatidylinositol 3-kinase/Akt and p44/p42 mitogen-activated protein (MAP) kinase. In the present study, we investigated the effects of HSP70 inhibitors on the IGF-I-elicited migration of these cells and the mechanism involved. The IGF-I-stimulated osteoblast migration evaluated by a wound-healing assay and by a transwell cell migration was significantly reduced by VER-155008 and YM-08, which are both HSP70 inhibitors. VER-155008 markedly suppressed the IGF-I-induced phosphorylation of p44/p42 MAP kinase without affecting that of Akt. In conclusion, our results strongly suggest that the HSP70 inhibitor reduces the IGF-I-elicited migration of osteoblasts via the p44/p42 MAP kinase.

(-)-Epigallocatechin gallate but not chlorogenic acid suppresses EGF-stimulated migration of osteoblasts via attenuation of p38 MAPK activity.

Phenolic compounds provide health benefits in humans. A previous study by our group has indicated that the epidermal growth factor (EGF)­induced migration of osteoblast­like MC3T3­E1 cells is mediated by the phosphorylation of p44/p42 mitogen­activated protein (MAPK), p38 MAPK, stress­activated protein kinase (SAPK)/c­Jun N­terminal kinase (JNK) and Akt, and that resveratrol, a major polyphenol in grape skin, suppresses the EGF­induced migration by attenuating Akt and SAPK/JNK activation. In the present study, the effects of chlorogenic acid, a major phenolic acid in coffee, and (­)­epigallocatechin gallate (EGCG), a major flavonoid in green tea, on the EGF­induced migration of MC3T3­E1 cells were investigated. EGCG significantly reduced the EGF­induced migration as evaluated by a Transwell migration assay and by a wound healing assay. However, chlorogenic acid failed to affect the EGF­induced migration. The phosphorylation of p38 MAPK induced by EGF was significantly suppressed by EGCG; however, the EGF­induced phosphorylation of p44/p42 MAP kinase, SAPK/JNK or Akt was not affected by EGCG. These results suggest that EGCG, but not chlorogenic acid, suppresses EGF­induced osteoblast migration through inhibiting p38 MAPK activation.

Repression of IGF-I-induced osteoblast migration by (-)-epigallocatechin gallate through p44/p42 MAP kinase signaling.

Polyphenolic compounds in beverages may have benefits in the prevention of osteoporosis. It has been demonstrated previously that insulin-like growth factor-I (IGF-I) could stimulate the migration of osteoblasts. In the present study, it was investigated whether chlorogenic acid, a major polyphenol in coffee, and (-)-epigallocatechin gallate (EGCG), a major polyphenol in green tea, could affect this IGF-I-stimulated migration of osteoblast-like MC3T3-E1 cells. The IGF-I-stimulated osteoblast migration, evaluated by Transwell cell migration and wound-healing assays, was inhibited by EGCG but not chlorogenic acid. IGF-I induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p70 S6 kinase and Akt. The IGF-I-induced migration was suppressed by PD98059, a MAP kinase kinase 1/2 inhibitor, and deguelin, an Akt inhibitor, but not rapamycin, an inhibitor of the upstream kinase of p70 S6 kinase (mammalian target of rapamycin). EGCG attenuated the IGF-I-induced phosphorylation of p44/p42 MAP kinase but not Akt. Taken together, the present results suggest that EGCG inhibits IGF-I-induced osteoblast migration via p44/p42 MAP kinase.