The role of adhesion molecules in osteocalcin-induced effects on glucose and lipid metabolism in adipocytes.

Recent findings suggest that uncarboxylated osteocalcin (GluOC) promotes glucose and lipid metabolism via its putative receptor GPRC6A; however, its direct effect on adipocytes remains elusive. In this study, we elucidated the effects of GluOC on adipocytes, with an emphasis on the role of cell adhesion molecules. We determined that GluOC promoted the expression of adipocyte adhesion molecule (ACAM) and its transcription factor Krüppel-like factor 4 and enhanced the cortical actin filament assembly, which ameliorated lipid droplet hypertrophy. Additionally, GluOC upregulated the expression of integrin αVß3 and activation of focal adhesion kinase (FAK) and prevented insulin receptor substrate 1 (IRS1) degradation by inhibiting the ubiquitin-proteasome system via the FAK-PLC-PKC axis, which activated IRS1-Akt-mediated glucose transporter 4 (GLUT4) transport. Furthermore, we showed that GluOC elevated the expression of the insulin-independent glucose transporters GLUT1 and GLUT8, which facilitated insulin stimulation-independent glucose transport. The GluOC-induced activation of integrin αVß3 signaling promoted microtubule assembly, which improved glucose and lipid metabolism via its involvement in intracellular vesicular transport. GluOC treatment also suppressed collagen type 1 formation, which might prevent adipose tissue fibrosis in obese individuals. Overall, our results imply that GluOC promotes glucose and lipid metabolism via ACAM, integrin αVß3, and GLUT1 and 8 expression, directly affecting adipocytes.

Cyclic anthraquinone derivatives, unique G-quadruplex binders, selectively induce cancer cell apoptosis and inhibit tumor growth.

Cyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder.

Phospholipase C-related but catalytically inactive protein acts as a positive regulator of insulin signalling in adipocytes.

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.

Spectrally blue hydrated parent body of asteroid (162173) Ryugu.

Ryugu is a carbonaceous rubble-pile asteroid visited by the Hayabusa2 spacecraft. Small rubble pile asteroids record the thermal evolution of their much larger parent bodies. However, recent space weathering and/or solar heating create ambiguities between the uppermost layer observable by remote-sensing and the pristine material from the parent body. Hayabusa2 remote-sensing observations find that on the asteroid (162173) Ryugu both north and south pole regions preserve the material least processed by space weathering, which is spectrally blue carbonaceous chondritic material with a 0-3% deep 0.7-µm band absorption, indicative of Fe-bearing phyllosilicates. Here we report that spectrally blue Ryugu's parent body experienced intensive aqueous alteration and subsequent thermal metamorphism at 570-670 K (300-400 °C), suggesting that Ryugu's parent body was heated by radioactive decay of short-lived radionuclides possibly because of its early formation 2-2.5 Ma. The samples being brought to Earth by Hayabusa2 will give us our first insights into this epoch in solar system history.

Substituent effects of cyclic naphthalene diimide on G-quadruplex binding and the inhibition of cancer cell growth.

Interaction of cyclic naphthalene diimide derivatives (cNDIs), 1-4, with TA-core and c-myc as G-quartet (G4) DNA was studied under dilute or molecular crowding condition. Binding study for TA-core based on an isothermal titration calorimetry showed that 1-4 has 106 M-1 order of binding affinity with the following order: 1 > 4 > 2 > 3 under both conditions. Meting temperature (Tm) of TA-core obtained from the temperature dependence of circular dichroism spectra shows that TA-core was most stabilized by 4, which is in agreement with the result of PCR stop assay and the stabilization effect for 1-3 was correlated with their binding affinity under dilute condition. 3 showed specific growth inhibition of cancer cell line Ca9-22 at <0.03 µM of IC50, with no inhibitory effect against normal bone marrow cells. 3, which has highest value of ΔH/ΔG, shows the highest inhibition ability for Ca9-22, carrying a highest expression level of telomerase mRNA.

Enhanced x-ray emission coinciding with giant radio pulses from the Crab Pulsar.

Giant radio pulses (GRPs) are sporadic bursts emitted by some pulsars that last a few microseconds and are hundreds to thousands of times brighter than regular pulses from these sources. The only GRP-associated emission outside of radio wavelengths is from the Crab Pulsar, where optical emission is enhanced by a few percentage points during GRPs. We observed the Crab Pulsar simultaneously at x-ray and radio wavelengths, finding enhancement of the x-ray emission by 3.8 ± 0.7% (a 5.4σ detection) coinciding with GRPs. This implies that the total emitted energy from GRPs is tens to hundreds of times higher than previously known. We discuss the implications for the pulsar emission mechanism and extragalactic fast radio bursts.

Osteocalcin promotes proliferation, differentiation, and survival of PC12 cells.

Involvement of the bone matrix protein osteocalcin (OC) in the development of learning and memory, and the prevention of anxiety-like behaviors in mice. However, the direct effects of OC on neurons are still unknown comparing to the mechanism how OC affects systemic energy expenditure and glucose homeostasis. In this study, we investigated the effect of OC on proliferation, differentiation, and survival of neurons using the rat pheochromocytoma cell line PC12. RT-PCR analysis for OC receptor candidates revealed that Gpr158, but not Gprc6a, mRNA was expressed in PC12 cells. The growth of PC12 cells cultured in the presence of 5-50 ng/mL of either uncarboxylated (GluOC) or carboxylated (GlaOC) OC was increased compared to cells cultured in the absence of OC. In addition, NGF-induced neurite outgrowth was enhanced by OC, and H2O2-induced cell death was suppressed by pretreatment with OC. All of these results were observed for both GluOC and GlaOC at comparable levels, suggesting that OC may directly affect cell proliferation, differentiation, and survival by binding to its candidate receptor, GPR158.

Adipocyte-specific GPRC6A ablation promotes diet-induced obesity by inhibiting lipolysis.

The G protein-coupled receptor GPRC6A regulates various physiological processes in response to its interaction with multiple ligands, such as extracellular basic amino acids, divalent cations, testosterone, and the uncarboxylated form of osteocalcin (GluOC). Global ablation of GPRC6A increases the susceptibility of mice to diet-induced obesity and related metabolic disorders. However, given that GPRC6A is expressed in many tissues and responds to a variety of hormonal and nutritional signals, the cellular and molecular mechanisms underlying the development of metabolic disorders in conventional knockout mice have remained unclear. On the basis of our previous observation that long-term oral administration of GluOC markedly reduced adipocyte size and improved glucose tolerance in WT mice, we examined whether GPRC6A signaling in adipose tissue might be responsible for prevention of metabolic disorders. We thus generated adipocyte-specific GPRC6A knockout mice, and we found that these animals manifested increased adipose tissue weight, adipocyte hypertrophy, and adipose tissue inflammation when fed a high-fat and high-sucrose diet compared with control mice. These effects were associated with reduced lipolytic activity because of downregulation of lipolytic enzymes such as adipose triglyceride lipase and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of adipose triglyceride lipase in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells.

OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Effect of aging on bone metabolism: the involvement of complement C1q.

Purpose Impairment of normal bone remodeling affects the successful osseointegration of dental implants. Recently, it has been reported that complement C1q level increases with age and delays wound healing by modulating Wnt signaling. As Wnt signaling is known to play an essential role in bone remodeling, we hypothesized that aging-dependent increases in C1q affect bone remodeling. In this study, we examined whether C1q affects the differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts, and investigated whether C1q could modify cellular signaling, including the Wnt/ß-catenin pathway in these cells.Methods Osteogenic differentiation of MC3T3-E1 cells was assessed using alkaline phosphatase staining. Differentiation of osteoclasts from mouse bone marrow cells was assessed using tartrate-resistant acid phosphatase staining. Activation of canonical Wnt signaling and protein phosphorylation was monitored using Western blotting.Results C1q, at 5-15 µg/mL promoted osteoclast fusion, whereas it did not affect the differentiation of osteoblasts. On the other hand, a higher concentration of C1q (50 µg/mL) suppressed both bone morphogenetic protein-2-induced osteogenic differentiation and osteoclast formation. C1q did not induce an obvious activation of Wnt/ ß-catenin signaling in either pre-osteoblasts or pre-osteoclasts, contrary to previous reports using other tissues. Instead, C1q upregulated the receptor activator of nuclear factor-kappa B ligand (RANKL)-induced phosphorylation of Akt.Conclusions C1q could affect cellular signaling and modify the differentiation of osteoblasts and osteoclasts, depending on the concentration. Therefore, an increase in C1q with age could be one of the factors that determine the prognosis of treatment of elderly patients.

The roles of osteocalcin in lipid metabolism in adipose tissue and liver.

Bone provides skeletal support and functions as an endocrine organ by producing osteocalcin, whose uncarboxylated form (GluOC) increases the metabolism of glucose and lipid by activating its putative G protein-coupled receptor (family C group 6 subtype A). Low doses (≤10 ng/ml) of GluOC induce the expression of adiponectin, adipose triglyceride lipase and peroxisome proliferator-activated receptor γ, and promote active phosphorylation of lipolytic enzymes such as perilipin and hormone-sensitive lipase via the cAMP-PKA-Src-Rap1-ERK-CREB signaling axis in 3T3-L1 adipocytes. Administration of high-dose (≥20 ng/ml) GluOC induces programmed necrosis (necroptosis) through a juxtacrine mechanism triggered by the binding of Fas ligand, whose expression is induced by forkhead box O1, to Fas that is expressed in adjacent adipocytes. Furthermore, expression of adiponectin and adipose triglyceride lipase in adipocytes is triggered in the same manner as following low-dose GluOC stimulation; these effects protect mice from diet-induced accumulation of triglycerides in hepatocytes and consequent liver injury through the upregulation of nuclear translocation of nuclear factor-E2-related factor-2, expression of antioxidant enzymes, and inhibition of the c-Jun N-terminal kinase pathway. Evaluation of these molecular mechanisms leads us to consider that GluOC might have potential as a treatment for lipid metabolism disorders. Indeed, there have been many reports demonstrating the negative correlation between serum osteocalcin levels and obesity or non-alcoholic fatty liver disease, a common risk factor for which is dyslipidemia in humans. The present review summarizes the effects of GluOC on lipid metabolism as well as its possible therapeutic application for metabolic diseases including obesity and dyslipidemia.

Modification of TRPV4 activity by acetaminophen.

N-Acetyl-p-aminophenol (APAP/acetaminophen) is a widely used analgesic/antipyretic with weaker inhibitory effects on cyclooxygenase compared to those of non-steroidal anti-inflammatory drugs. The effect of APAP is mediated by its metabolites, N-arachidonoyl-phenolamine and N-acetyl-p-benzoquinone imine, which activate transient receptor potential (TRP) channels, including TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or cannabinoid receptor type 1. However, the exact molecular mechanism underlying the cellular actions of APAP remains unclear. Recently, we observed that APAP promotes cell migration through TRPV4; in this study, we examined the effect of APAP on Ca2+-channel activity of TRPV4. In the rat cell line PC12 expressing TRPV4, GSK1016790A (GSK), a TRPV4 agonist, stimulated an increase in [Ca2+]i; these effects were abrogated by HC-067047 treatment. This GSK-induced Ca2+ entry through TRPV4 was inhibited by APAP in a dose-dependent manner, whereas APAP alone did not affect [Ca2+]i. The specificity of the effect of APAP on TRPV4 was further confirmed using HeLa cells, which lack endogenous TRPV4 but stably express exogenous TRPV4 (HeLa-mTRPV4). GSK-induced [Ca2+]i elevation was only observed in HeLa-mTRPV4 cells compared to that in the control HeLa cells, indicating the specific action of GSK on TRPV4. APAP dose-dependently suppressed this GSK-induced Ca2+ entry in HeLa-mTRPV4. However, it is unlikely that the metabolites of APAP were involved in these effects as the reaction in this study was rapid. The results suggest that APAP suppresses the newly identified target TRPV4 without being metabolized and exerts antipyretic/analgesic and/or other effects on TRPV4-related phenomena in the body. The effect of APAP on TRPV4 was opposite to that on TRPV1 or TRPA1, as the latter is activated by APAP.

GLP-1 signaling is required for improvement of glucose tolerance by osteocalcin.

Osteocalcin is a bone-derived hormone that in its uncarboxylated form (GluOC) plays an important role in glucose and energy metabolism by stimulating insulin secretion and pancreatic ß-cell proliferation through its putative receptor GPRC6A. We previously showed that the effect of GluOC on insulin secretion is mediated predominantly by glucagon-like peptide-1 (GLP-1) released from intestinal endocrine cells in response to GluOC stimulation. Moreover, oral administration of GluOC was found to reduce the fasting blood glucose level, to improve glucose tolerance, and to increase the fasting serum insulin concentration and ß-cell area in the pancreas in wild-type mice. We have now examined the effects of oral GluOC administration for at least 4 weeks in GLP-1 receptor-knockout mice. Such administration of GluOC in the mutant mice triggered glucose intolerance, enhanced gluconeogenesis and promoted both lipid accumulation in the liver as well as adipocyte hypertrophy and inflammation in adipose tissue. Furthermore, inactivation of GLP-1 receptor signaling in association with GluOC administration induced activation of the transcription factor FoxO1 and expression of its transcriptional coactivator PGC1α in the liver, likely accounting for the observed upregulation of gluconeogenic gene expression. Our results thus indicate that the beneficial metabolic effects of GluOC are dependent on GLP-1 receptor signaling.

Determination of sterigmatocystin in foods in Japan: method validation and occurrence data.

A survey of the contamination of foods by sterigmatocystin (STC) was performed by an analytical method based on LC-MS/MS. STC was extracted from samples with acetonitrile/water (85/15, v/v) and then purified with immunoaffinity columns. The method was validated by a small-scale inter-laboratory study using spiked wheat samples. Mean recoveries of STC were 100.3% and 92.5% from two samples spiked at 0.5 and 5.0 µg/kg, respectively. A total of 583 samples were analysed between 2016 and 2018, and STC was detected in 19.9% of all samples at >0.05 µg/kg (limit of quantification). The foods that were contaminated by STC were wheat flour, Job's tears products, rye flour, rice, buckwheat flour, white sorghum, barley products, azuki bean and corn flour. STC was not found in beer or wine. The occurrence of STC in domestic wheat flour (44.4%), Job's tears products (41.7%) and rye flour (29.9%) accounted for the three highest values. The highest mean concentrations were obtained for Job's tears products (0.3 µg/kg) and rye flour (0.3 µg/kg). The maximum contamination level was present in a sample of rye flour (7.1 µg/kg). Although the contamination levels were low, these results indicate that STC frequently contaminates Japanese retail foods. A continuous survey is required to assess exposure to STC in Japan.

Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300.

The uncarboxylated form of osteocalcin (GluOC) regulates glucose and lipid metabolism in mice. We previously showed that low-dose (≤10 ng/ml) GluOC induces the expression of adiponectin and peroxisome proliferator-activated receptor γ (PPARγ) via a cAMP-PKA-ERK-CREB signaling pathway in 3T3-L1 adipocytes. We also noticed that high-dose (≥20 ng/ml) GluOC inhibits the expression of adiponectin and PPARγ in these cells. We have here explored the mechanism underlying these effects of high-dose GluOC. High-dose GluOC triggered morphological changes in 3T3-L1 adipocytes suggestive of the induction of cell death. It activated the putative GluOC receptor GPRC6A and thereby induced the production of cAMP and activation of protein kinase A (PKA), similar to signaling by low-dose GluOC with the exception that the catalytic subunit of PKA also entered the nucleus. Cytosolic PKA induced phosphorylation of cAMP response element-binding protein (CREB) at serine-133 via extracellular signal-regulated kinase (ERK). Nuclear PKA appeared to mediate the inhibitory phosphorylation of salt-inducible kinase 2 (SIK2) at serine-358 and thereby to alleviate the inhibitory phosphorylation of the CREB co-activator p300 at serine-89. The activation of CREB and p300 resulted in increased expression of the transcription factor FoxO1 and consequent upregulation of Fas ligand (FasL) at the plasma membrane. The interaction of FasL with Fas on neighboring adipocytes triggered the phosphorylation at threonine-357/serine-358 and homotrimerization of mixed-lineage kinase domain-like protein (MLKL), a key regulator of necroptosis, as well as Ca2+ influx via transient receptor potential melastatin 7 (TRPM7), the generation of reactive oxygen species and lipid peroxides, and dephosphorylation of dynamin-related protein 1 (DRP1) at serine-637, resulting in mitochondrial fragmentation. Together, our results indicate that high-dose GluOC triggers necroptosis through upregulation of FasL at the plasma membrane in a manner dependent of activation of CREB-p300, followed by the activation of Fas signaling in neighboring adipocytes.

Brief multifamily Psychoeducation for family members of patients with chronic major depression: a randomized controlled trial.

BACKGROUND: Major depressive disorder (MDD) is a common and often chronic problem. Patients with chronic MDD often have negative impacts on the health of their families. Family psychoeducation is recognized as part of the optimal treatment for patients with psychotic disorder, and has been shown to reduce the rate of relapse in individuals with schizophrenia and to reduce the burden on their caregivers. Thus, we predict that family psychoeducation has the potential to reduce the burden on the caregivers of patients with chronic MDD. In the present study, we aimed to investigate the effects of brief multifamily psychoeducation (BMP) on the mental health status of family members of patients with chronic MDD. METHODS: We conducted a clinical trial consisting of 49 chronic MDD patients and their families. Each family was randomly assigned to either the BMP intervention group or the control group. The intervention group received four BMP sessions, once every two weeks for eight weeks. The control group received one counseling session administered by a nurse. All patients received standard treatment administered by physicians. The primary outcome measurement was the Kessler Screening Scale for Psychological Distress (K6) score of family members at 16- weeks after the first BMP session. Secondary outcomes were depressive symptoms of both family members and patients at multiple time points, as well as family functioning as evaluated by the patients. Intention-to-treat analyses were conducted. RESULTS: There was no statistically significant effect of BMP on K6 scores at 16- weeks (mean difference 1.17, 95% confidence interval: - 0.63 to 2.98, P = 0.19). Exploratory analyses revealed that BMP reduced depressive symptoms in family members at 8- weeks (difference = - 3.37, 95%CI -6.32 to - 0.43, P = 0.02) and improved family functioning at multiple time points (Role; 8 W, difference = - 0.13, 95%CI -0.26 to - 0.00, P = 0.04, Affective Responsiveness; 8 W, difference = - 0.24, 95%CI -0.43 to - 0.05, P = 0.01, 32 W, difference = - 0.22, 95%CI -0.41 to - 0.03, P = 0.02, Behavior Control; 16 W, difference = - 0.17, 95%CI -0.34 to - 0.00, P = 0.04). CONCLUSIONS: Four BMP sessions did not significantly reduce the psychological distress of family members of patients with chronic MDD. TRIAL REGISTRATION: Clinical Trials. gov NCT01734291 , retrospectively registered (Registration date: November 21, 2012).

Size-guided multi-seed heuristic method for geometry optimization of clusters: Application to benzene clusters.

Since searching for the global minimum on the potential energy surface of a cluster is very difficult, many geometry optimization methods have been proposed, in which initial geometries are randomly generated and subsequently improved with different algorithms. In this study, a size-guided multi-seed heuristic method is developed and applied to benzene clusters. It produces initial configurations of the cluster with n molecules from the lowest-energy configurations of the cluster with n - 1 molecules (seeds). The initial geometries are further optimized with the geometrical perturbations previously used for molecular clusters. These steps are repeated until the size n satisfies a predefined one. The method locates putative global minima of benzene clusters with up to 65 molecules. The performance of the method is discussed using the computational cost, rates to locate the global minima, and energies of initial geometries. © 2018 Wiley Periodicals, Inc.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.

Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

BACKGROUND: N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. METHODS: Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. RESULTS: APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. CONCLUSION: APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions.

Celecoxib inhibits osteoblast differentiation independent of cyclooxygenase activity.

Non-steroidal anti-inflammatory drugs (NSAIDs) exert their effects primarily by inhibiting the activity of cyclooxygenase (COX), thus suppressing prostaglandin synthesis. Some NSAIDs are known to perform functions other than pain control, such as suppressing tumour cell growth, independent of their COX-inhibiting activity. To identify NSAIDs with COX-independent activity, we examined various NSAIDs for their ability to inhibit osteoblastic differentiation using the mouse pre-osteoblast cell line MC3T3-E1. Only celecoxib and valdecoxib strongly inhibited osteoblastic differentiation, and this effect was not correlated with COX-inhibiting activity. Moreover, 2,5-dimethyl (DM)-celecoxib, a celecoxib analogue that does not inhibit COX activity, also inhibited osteoblastic differentiation. Celecoxib and DM-celecoxib inhibited osteoblastic differentiation induced by bone morphogenetic protein (BMP)-2 in C2C12 mouse myoblast cell line. Although celecoxib suppresses the growth of some tumour cells, the viability and proliferation of MC3T3-E1 cells were not affected by celecoxib or DM-celecoxib. Instead, celecoxib and DM-celecoxib suppressed BMP-2-induced phosphorylation of Smad1/5, a major downstream target of BMP receptor. Although it is well known that COX plays important roles in osteoblastic differentiation, these results suggest that some NSAIDs, such as celecoxib, have targets other than COX and regulate phospho-dependent intracellular signalling, thereby modifying bone remodelling.