Exploring the genetic diversity of the Japanese population: Insights from a large-scale whole genome sequencing analysis.

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. We analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective from the genomic data of 9,287 samples obtained by high-coverage whole-genome sequencing (WGS) by the National Center Biobank Network. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Haplotype analysis suggested increased allele frequencies for genes related to alcohol and fatty acid metabolism, which were reported as loci that had experienced positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to increase in the allele frequency; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

Cross-sectional association of continuous glucose monitoring-derived metrics with cerebral small vessel disease in older adults with type 2 diabetes.

AIM: To examine cross-sectional associations between continuous glucose monitoring (CGM)-derived metrics and cerebral small vessel disease (SVD) in older adults with type 2 diabetes. MATERIALS AND METHODS: In total, 80 patients with type 2 diabetes aged ≥70 years were analysed. Participants underwent CGM for 14 days. From the CGM data, we derived mean sensor glucose, percentage glucose coefficient of variation, mean amplitude of glucose excursion, time in range (TIR, 70-180 mg/dl), time above range (TAR) and time below range metrics, glycaemia risk index and high/low blood glucose index. The presence of cerebral SVD, including lacunes, microbleeds, enlarged perivascular spaces and white matter hyperintensities, was assessed, and the total number of these findings comprised the total cerebral SVD score (0-4). Ordinal logistic regression analyses were performed to examine the association of CGM-derived metrics with the total SVD score. RESULTS: The median SVD score was 1 (interquartile range 0-2). Higher hyperglycaemic metrics, including mean sensor glucose, TAR >180 mg/dl, TAR >250 mg/dl, and high blood glucose index and glycaemia risk index, were associated with a higher total SVD score. In contrast, a higher TIR (per 10% increase) was associated with a lower total SVD score (odds ratio 0.73, 95% confidence interval 0.56-0.95). Glycated haemoglobin, percentage glucose coefficient of variation, mean amplitude of glucose excursions, time below range and low blood glucose index were not associated with total cerebral SVD scores. CONCLUSIONS: The hyperglycaemia metrics and TIR, derived from CGM, were associated with cerebral SVD in older adults with type 2 diabetes.

Oncostatin M suppresses bone morphogenetic protein-4-induced osteoprotegerin synthesis in MC3T3-E1 osteoblast-like cells: p70 S6 kinase attenuation.

Evidence is accumulating that osteal macrophages, in addition to bone-resorbing osteoclasts and bone-forming osteoblasts, participate vitally in bone remodeling process. Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodeling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We have reported that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in MC3T3-E1 osteoblast-like cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase are involved in the OPG synthesis. The present study aims to investigate the effect of OSM on the synthesis of OPG stimulated by BMP-4 in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells. Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK was affected by OSM. On the other hand, the phosphorylation of p70 S6 kinase stimulated by BMP-4 was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblast-like cells.

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.

Cross-sectional association of metrics derived from continuous glucose monitoring with cognitive performance in older adults with type 2 diabetes.

AIM: To examine the association between continuous glucose monitoring (CGM)-derived metrics and cognitive performance in older adults with type 2 diabetes (T2D). MATERIALS AND METHODS: A total of 100 outpatients with T2D aged 70 years or older were analysed. Participants underwent CGM for 14 days. As CGM-derived metrics, mean sensor glucose (SG), glucose coefficient of variation (CV), time in range (TIR; 70-180 mg/dl), time above range (TAR; > 180 mg/dl) and time below range (TBR; < 70 mg/dl), were calculated. Participants underwent cognitive tests, including the Japanese version of the Montreal Cognitive Assessment (MoCA-J), a delayed word-recall test from the Alzheimer's Disease Assessment Scale-cognitive subscale, a digit symbol substitution test, a letter word fluency test, a trail-making test (TMT) and digit span test (DSP). RESULTS: In multiple regression analyses adjusted for confounders, a higher mean SG was associated with a lower performance in MoCA-J and TMT part B (TMT-B) (P < .05). A higher TAR was associated with a lower performance in TMT-B and DSP-backward (P < .05). By contrast, a higher TIR was associated with better function in TMT-B and DSP-backward (P < .05). Furthermore, CV and TBR were not associated with any cognitive function. CONCLUSION: Hyperglycaemia metrics and TIR derived from CGM are associated with cognitive functions, especially with executive function and working memory, in older adults with T2D.

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.

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

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.

Use of heated tobacco products, moderate alcohol drinking, and anti-SARS-CoV-2 IgG antibody titers after BNT162b2 vaccination among Japanese healthcare workers.

The effect of heated tobacco products (HTPs) use and moderate alcohol drinking on immunogenicity to coronavirus disease (COVID-19) vaccines remain elusive. This study aimed to examine the association of tobacco product use and alcohol consumption with anti-SARS-CoV-2 spike IgG antibody titers after the BNT162b2 vaccine. Participants were 3433 healthcare workers receiving two vaccine doses in the 4 national centers for advanced medical and research in Japan. Smoking status and alcohol consumption were assessed via a questionnaire, and anti-SARS-CoV-2 spike IgG titers were measured with chemiluminescent enzyme immunoassay using serum collected on the median of 64 days after the second vaccination. Multilevel linear regression models were used to estimate the geometric mean titers (GMT) and the ratios of means (RoM) between groups with adjustment for covariates. Compared with never-smokers (GMT = 118), IgG antibody titers were significantly lower among HTPs users (including those who also smoked cigarettes) (GMT = 105; RoM = 0.89 [95%CI: 0.78-0.99]) and exclusive cigarettes smokers (GMT = 98; RoM = 0.81 [95%CI: 0.71-0.92]). Compared with non-drinkers of alcohol (GMT = 123), alcohol drinkers consuming <1 go/day (GMT = 113; RoM = 0.93 [95%CI: 0.88-0.98]), 1-1.9 go/day (GMT = 104; RoM = 0.85 [95%CI: 0.78-0.93]), and ≥ 2 go/day (GMT = 103; RoM = 0.84 [95%CI: 0.74-0.96]) had significantly lower antibody titers (P for trend<0.01). Spline analysis showed a large reduction of antibody until around 1 go/day of alcohol consumption, and then they gradually decreased. Results suggest that in addition to conventional cigarette smoking and heavy alcohol drinking, HTPs use and moderate alcohol drinking may be predictors of lower immunological response to COVID-19 vaccine.

Amyloid ß protein negatively regulates human platelet activation induced by thrombin receptor-activating protein.

Amyloid ß protein deposition in cerebral vessels, a characteristic of Alzheimer's disease, is a risk factor for intracerebral hemorrhage. Amyloid ß protein directly modulates human platelet function; however, the exact mechanism of action is unclear. Therefore, we investigated the effects of amyloid ß protein on human platelet activation using an aggregometer with laser scattering. Amyloid ß protein decreased platelet aggregation induced by thrombin receptor-activating protein, but not by collagen and ADP. Amyloid ß protein also suppressed platelet aggregation induced by SCP0237 and A3227. Platelet-derived growth factor-AB secretion and phosphorylated-heat shock protein 27 release by thrombin receptor-activating protein were inhibited by amyloid ß protein. Additionally, thrombin receptor-activating protein-induced phosphorylation of JNK and p38 MAP kinase was reduced by amyloid ß protein. Collectively, our results strongly suggest that amyloid ß protein negatively regulates protease-activated receptor-elicited human platelet activation. These findings may indicate a cause of intracerebral hemorrhage due to amyloid ß protein.

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.

Relationship between the Responsiveness of Amyloid ß Protein to Platelet Activation by TRAP Stimulation and Brain Atrophy in Patients with Diabetes Mellitus.

Type 2 DM is a risk factor for dementia, including Alzheimer's disease (AD), and is associated with brain atrophy. Amyloid ß protein (Aß) deposition in the brain parenchyma is implicated in the neurodegeneration that occurs in AD. Platelets, known as abundant storage of Aß, are recognized to play important roles in the onset and progression of AD. We recently showed that Aß negatively regulates platelet activation induced by thrombin receptor-activating protein (TRAP) in healthy people. In the present study, we investigated the effects of Aß on the TRAP-stimulated platelet activation in DM patients, and the relationship between the individual responsiveness to Aß and quantitative findings of MRI, the volume of white matter hyperintensity (WMH)/intracranial volume (IC) and the volume of parenchyma (PAR)/IC. In some DM patients, Aß reduced platelet aggregation induced by TRAP, while in others it was unchanged or rather enhanced. The TRAP-induced levels of phosphorylated-Akt and phosphorylated-HSP27, the levels of PDGF-AB and the released phosphorylated-HSP27 correlated with the degree of platelet aggregability. The individual levels of not WMH/IC but PAR/IC was correlated with those of TRAP-stimulated PDGF-AB release. Collectively, our results suggest that the reactivity of TRAP-stimulated platelet activation to Aß differs in DM patients from healthy people. The anti-suppressive feature of platelet activation to Aß might be protective for brain atrophy in DM patients.

Proximity Proteomics Has Potential for Extracellular Vesicle Identification.

Extracellular vesicles (EVs) are biomarkers and mediators of intercellular communication. In biological samples, EVs are secreted by various types of cells. The proteomic identification of proteins expressed in EVs has potential to contribute to research and clinical applications, particularly for cancer. In this study, the proximity-labeling method-based proteomic approach was used for EV identification, labeling membrane components proximal to a given molecule on the EV membrane surface. Due to the small labeling range, proteins on the surface of the same EVs are likely to be labeled by selecting a given EV surface antigen. The protein group of cancer cell-secreted EV (cEV), which abundantly expresses a close homologue of L1 (CHL1), was examined using a model mouse for lung cancer (LC). cEV-expressed proteins were identified by proteomic analysis of enzyme-mediated activation of radical sources by comparing serum EVs from wild-type and LC mice. SLC4A1 was found to be co-expressed in CHL1-expressing EVs, highlighting EVs expressing both CHL1 and SLC4A1 as candidates for cEVs. Serum EVs expressing both CHL1 and caspase 14 were significantly elevated in LC patients compared with healthy individuals. Thus, the combination of proximity labeling and proteomic analysis allows for effective EV identification.

Duloxetine suppresses BMP-4-induced release of osteoprotegerin via inhibition of the SMAD signaling pathway in osteoblasts.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway 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.

Duloxetine strengthens osteoblast activation by prostaglandin E1: Upregulation of p38 MAP kinase.

Duloxetine, a serotonin-norepinephrine reuptake inhibitor, is currently recommended as a useful medicine to chronic pain including low back pain. However, as the analogy of classical selective serotonin reuptake inhibitors, there is a concern to deteriorate osteoporosis with remaining to clarify the exact mechanism of duloxetine in bone metabolism. We have previously reported that prostaglandin E1 (PGE1) induces the synthesis of both osteoprotegerin (OPG) and interleukin-6 (IL-6), essential regulators of bone metabolism, in osteoblast-like MC3T3-E1 cells. Based upon them, we herein investigated the mechanism whereby the effect of duloxetine on the synthesis of OPG and IL-6 induced by PGE1 in these cells. Duloxetine enhanced the release from MC3T3-E1 cells of both OPG and IL-6 stimulated by PGE1. However, reboxetine, a selective and specific inhibitor of norepinephrine reuptake, failed to affect the PGE1-induced release of OPG or IL-6. Oppositely, fluvoxamine and sertraline, agents belonging to the class of selective serotonin reuptake inhibitor, upregulated the PGE1-stimulated release of both OPG and IL-6. Duloxetine amplified the expression of OPG mRNA and IL-6 mRNA stimulated by PGE1. Duloxetine strengthened the PGE1-induced p38 MAP kinase phosphorylation, which was amplified by fluvoxamine as well. SB203880, an inhibitor of p38 MAP kinase, suppressed the amplifying effects by duloxetine or fluvoxamine on the PGE1-stimulated release of OPG and IL-6. These results strongly suggest that duloxetine could strengthen osteoblast activation by PGE1 through the upregulation of p38 MAP kinase, leading to increasing the synthesis of OPG and IL-6.

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.

Olive polyphenol reduces the collagen-elicited release of phosphorylated HSP27 from human platelets.

Hydroxytyrosol (HT) and oleuropein (OLE) are natural polyphenols found in extra virgin olive oil. Accumulating evidence indicates that ingestion of olive oil contributes to reduce the risk of cardiovascular diseases and stroke. It has been reported that HT and OLE inhibit human platelet aggregation. We have shown that collagen induces the phosphorylation of heat shock protein 27 (HSP27) in human platelets, resulting in the release of HSP27, an extracellular pro-inflammatory agent. In this study, we investigated the effects of HT and OLE on the collagen-stimulated human platelet activation. The PDGF-AB secretion and the soluble CD40 ligand (sCD40L) release by collagen were reduced by HT or OLE. HT and OLE significantly suppressed the phosphorylation of HSP27 and the release of phosphorylated-HSP27. These findings suggest that olive polyphenol reduces the collagen-stimulated phosphorylation of HSP27 in human platelets and the release. Our results may provide a novel anti- inflammatory effect of olive polyphenol.

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.