Background: Cirrhosis is a condition come from excessive liver fibrosis and followed by serious secondary diseases, but there is no effective therapeutic medicine. α7 nicotinic acetylcholine receptor (α7nAChR), initially found as a receptor related to neurotransmission on neural cells. This receptor also expresses on immune cells to do anti-inflammatory action. However, there is few reports showing the relationship between α7nAChR and fibrosis.

Aim: We investigated whether α7nAChR has any effects on liver fibrosis and what is the mechanism.

Methods: Liver fibrosis model mice were established with CCl₄. The pro-fibrotic mRNA expressions and collagen content in livers were measured at 1.5 and 4 weeks. Moreover, we performed immunohistochemical staining and RT-PCR to determine which cells were involved in the mechanism.

Results: α7nAChR KO mice treated with CCl₄ showed significant decrease in pro-fibrotic mRNA expressions at 1.5 weeks and liver fibrosis at 4 weeks compared to WT mice. Furthermore, hepatocytes around fibrosis area expressed ACh transferase and activated hepatic stellate cells expressed α7nAChR.

Conclusion: The severity of fibrosis was significantly decreased in α7nAChR KO mice. Moreover, it is suggested that ACh produced by hepatocytes might stimulate hepatic stellate cells to promote collagen production.

We previously presented that bezafibrate (BF), a PPAR-pan agonist, has preventing and therapeutic effects on high fat and high cholesterol (HFHC) diet-induced nonalcoholic steatohepatitis (NASH) model in mice. In this study, we examined the effects of BF to high trans-fat diet (HTD) -induced NASH model in mice. HTD was fed to 7-week-old ob (spontaneously obese, hyperglycemia model) mice for 12 weeks to induce NASH. Half of ob mice were orally administered BF (100 mg/kg/day) for 12 weeks. Remaining ob mice were served as controls and administered the vehicle. Normal C57BL/6J mice were fed normal diet and administered the vehicle. After 12 weeks, plasma levels of AST, ALT, TC, glucose, and concentrations of hepatic TG, TC in ob mice fed HTD were markedly higher than those of normal mice. Fatty droplets, inflammation and fibrosis were observed in histopathologic examination of liver. BF significantly lowered plasma TC level, histopathologic fibrosis area and fibrosis scoring without effecting to plasma ALT levels. These results suggested that bezafibrate may suppress the progression of fibrosis in mice NASH model induced by HTD through a decrease in plasma cholesterol level or directly.

The role of nitric oxide (NO) on quality of life (QOL) and mucosal injury induced by a single and a consecutive administration of methotrexate was investigated. Rats received methotrexate intraperitoneally either a single administration (50 mg/kg) or a consecutive administration (12.5 mg/kg/day) for 4 days. N^G-nitro-L-arginine methyl ester (L-NAME) was given subcutaneously to inhibit NO synthase (NOS). 96 h after a first administration of methotrexate, ileal tissues were collected to analyze. Both a single administration and a consecutive administration of methotrexate decreased the QOL. When methotrexate was administered consecutively, L-NAME further worsened the QOL. A consecutive, but not a single, administration of methotrexate caused a significant mucosal injury and inflammation. A consecutive, but not a single, administration of methotrexate significantly induced a constitutive NOS expression in the ileal tissue. The role of NO derived from constitutive NOS in the reduction of QOL and small intestinal by methotrexate administration may be more important in a consecutive administration than a single administration.

Background Smooth muscle contraction is regulated by the balance between MLC kinase and MLC phosphatase (MLCP). Phosphorylated CPI-17 can inhibit MLCP resulted in induce contraction. Aim This study was planned to elucidate the effect of CPI-17 on contraction in myometrium from non-pregnant and pregnant mice. Wild type C57BL6/J mice (WT), CPI-17 deficient mice (KO) and phospho-inactive mutant CPI-17 at T38 to alanine knocked-in mice (TA) were used. Methods Isometric force stimulated with high concentration (65.4 mM) of KCl (high K), oxytocin (Oxy; 100 nM) and Carbachol (CCh, 5-100 µM) from non-pregnant and pregnant mice. Results The spontaneous contraction and absolute contractile ability stimulated with CCh and high K⁺ were not difference in non-pregnant and pregnant myometrium isolated from WT, KO and TA. In non-pregnant myometrium, Oxy induced spontaneous rhythmic contraction with small sustained one in WT. Oxy induced persistent weaker contraction in KO and TA than WT. On the other hand in pregnant myometrium, the Oxy-induced contractions were tended to be smaller in TA and KO than WT. Conclusion PKC/CPI-17 pathway induces an important role to regulate contraction by Oxy but not by CCh in both non-pregnant and pregnant myometrium. CPI-17 may be important in normal parturition induction.

Background: Dipeptidyl peptidase-4 (DPP-4) inhibitors not only improve impaired glucose tolerance in diabetes, but also have pleiotropic extra-pancreatic effects such as preconditioning effect for myocardial ischemia-reperfusion injury. Here, we investigated the anti-remodeling effects of linagliptin, a DPP-4 inhibitor, by use of DPP-4-deficient rats.

Methods and Results: After the induction of myocardial infarction (MI), Fischer 344 rats with inactivating mutation of DPP-4 were orally administrated with a DPP-4 inhibitor, linagliptin (5 mg·kg^-1·day^-1), or vehicle in drinking water for 4 weeks. Linagliptin did not affect hemodynamic status, body weight, and infarct size. In echocardiography, linagliptin tended to improve left ventricular (LV) systolic function, and significantly improved LV diastolic function, surprisingly. Interstitial fibrosis and macrophage infiltration were significantly lower in the linagliptin group than those in the vehicle group. Fibrosis-related gene expressions, such as collagen I and transforming growth factor-β1 (TGF-β1), and inflammation-related expressions, such as macrophage chemotactic protein 1 and matrix metalloproteinase-2 (MMP-2), were significantly suppressed in marginal area of the linagliptin-treated rats compared with the vehicle rats. The TGF-β1 and MMP-2 protein levels were attenuated by linagliptin in DPP-4-deficient cardiac fibroblasts.

Conclusions: Linagliptin can attenuate MI-induced cardiac remodeling via a DPP-4-independent pathway.

Background: Doxorubicin (DOX) is an anti-cancer drug which develops heart failure.  SIRT1, an NAD⁺-dependent deacetylase, affords cellular protection under various stresses.  Here, we investigated whether and how SIRT1 protects the heart from DOX-induced cardiotoxicity in mice.

Methods and Results: Wild type (WT) and tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (cKO) mice were treated with vehicle (Veh) or DOX (4 IP injections of 5 mg/kg/week) starting at 3 months of age. Echocardiography at 1 week after final vehicle or DOX showed that left ventricular fractional shortening (FS), an index of cardiac function, was similar in vehicle treated WT and cKO. Although DOX reduced FS in both genotypes, cKO showed lower FS after DOX than that in WT.  Cardiac ANP mRNA level was also higher in cKO than WT after DOX.  TUNEL-positive nuclei were unchanged in Veh-treated but were more increased by DOX in cKO than WT, indicating increased apoptosis in cKO after DOX.  A long-range PCR method for analysis of mtDNA deletion, which indicates mtDNA damage, demonstrated an increase in level of mtDNA with deletion by DOX in cKO but not in WT.  Immunoblotting showed that DOX significantly reduced cardiac levels of an autophagosome marker LC3-II and p62 protein which is degraded by autophagy in WT but not in cKO, suggesting activation of cardiac autophagy only in WT.

Conclusions: SIRT1 deletion worsens DOX-induced cardiotoxicity probably through enhanced mitochondrial damage via impaired autophagy.

Abnormal Ca²⁺ handling is essential in the pathophysiology of dilated cardiomyopathy (DCM). One of Ca²⁺ permeable channels, transient receptor potential cation channel, subfamily V, member 2 (TRPV2) has been suggested as a principal candidate for Ca²⁺ entry pathways and a potential therapeutic target for DCM. In this study, we produced selective antibodies recognizing TRPV2 from the outside of cell. One of antibodies inhibited the Ca²⁺ influx via TRPV2 in cultured cells and caused TRPV2 to disappear from the plasma membrane via cellular internalization. The antibody epitope existed in the turret of pore-forming outer gate of TRPV2. We tested the therapeutic efficacy of the antibody in DCM developed in the δ-sarcoglycan-deficient hamsters (J2N-k). Intraperitoneal administration of the antibody (0.5 mg/kg) for 2 weeks (once a week) prevented the progression of cardiac dysfunction as evaluated by echocardiography and improved the abnormal Ca²⁺ handling. Further, the antibody was also effective in preventing heart failure of the murine 4C30 model with end-stage DCM. Interestingly, endogenous TRPV2 accumulated in the cardiac muscle sarcolemma disappeared upon antibody administration. Thus, the produced antibody is capable of ameliorating DCM through enhanced cellular internalization, and may be a promising treatment for patients with DCM.

Currently available human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been known to exert a negative force-frequency relationship as one of their immature properties. In this study, we examined whether controlling the direction of contraction process and/or supplying the higher oxygen tension may overcome such limitation of the contraction movement. We prepared one layered, higher cell-density sheets of hiPSC-CMs, and simultaneously recorded the motion vectors and field potentials. In a cell sheet under spontaneous activity, a synchronous movement consisted of multiple contractions which started from various sites. During electrical stimulation, the contraction started around the pacing electrodes and we observed the positive force-frequency relationships in contraction as well as relaxation along with the frequency-dependent shortening of the field potential durations. The use of fractional analysis of motion vectors demonstrated that contraction as well as relaxation processes consisted of fast and slow phases.  Increase in oxygen tension from 20 to 95% in mixed gas accelerated the fast phase of relaxation. β-Stimulation accelerated the timing of fast phase of relaxation, whereas a tyrosine kinase inhibitor dasatinib delayed it. Thus, these observations can indicate that the currently proposed procedure may become a new tool for integrating the drug-induced biological phenomena in vitro extrapolating to clinically observed cardiac efficacy and adverse effects.

Gicerin/CD146 is a cell adhesion molecule which belongs to the immunoglobulin (Ig) superfamily. We have reported the existence of Gicerin/CD146 in the heart, lung and smooth muscles of blood vessels. It is expected that Gicerin/CD146 expressed in these tissues may have a function different from those in the nervous system or tumors. WE have reported that Gicerin/CD146 is involved in the hypertrophy of vessel smooth muscles. We speculate that in the heart, Gicerin/CD146 may also have some role in the hypertrophy of the cardiac muscle cells. In this study, we make a cardiac hypertrophy model rat by constricting the rat aorta (TAC, transverse aortic constriction) and examined the effect on the expression of Gicerin/CD146.We confirmed the status of hypertrophy by an expression of the β-MHC gene (β myosin heavy chain), which has been reported to be elevated during cardiac hypertrophy. Next, stretch stimulation was applied to myocardial cell line H9c2 cells. We confirmed the gene expression level of Gicerin/CD146 was influenced by TAC treatment. In cultured myocardial cells, the expression level of Gicerin/CD146 was also influenced by the stretch stimulation. Based on the above, it was suggested that the expression of Gicerin/CD146 is involved in cardiac hypertrophy as well as the stretch stimulation.

Hsp90 is a highly conserved molecular chaperone involved stabilization of client proteins. Hsp90 clients are various signal transducers including protein kinases. It is well known that the cardiac remodeling such as cardiac hypertrophy and fibrosis is involved in the development of chronic heart failure. The cardiac remodeling is regulated by many signaling pathways. c-Raf and JNK are signal transducers involved in cardiac remodeling, and are also Hsp90 client proteins. Therefore, to clarify the roles of Hsp90 and its clients in progression to the chronic heart failure, we examined effects of Hsp90 inhibitor on the signal transducers in the development of chronic heart failure in animal models. Treatment of the animals with Hsp90 inhibitor resulted in a suppression of cardiac remodeling and a preservation of cardiac pump function. Furthermore, c-Raf/Erk signaling was attenuated by an administration of Hsp90 inhibitor. These results suggest that Hsp90 contributes to stabilization of several cardiac remodeling-associated protein kinases during the development of chronic heart failure.

Oxidative stress is known to be involved in various pathologies such as heart failure and cancer. Mitochondria, which produce oxygen species (ROS) as a type of oxidative stress, are abundantly expressed especially in the cardiac cells. Once mitochondrial damage occurs, produced ROS is accumulating and causes mitochondria damage and subsequently lead to cardiac dysfunction. Recently, cardiac dysfunction caused by anticancer drugs and cancer itself has become a concern, and it has been suggested that oxidative stress is involved in one of these causes. The anthracycline antitumor antibiotic doxorubicin (DOX) induces severe adverse effects to non-tumor tissues including cardiac cells. DOX causes cardiomyopathy in a dose-dependent manner and consequent heart failure often limits DOX-based chemotherapy. The Japanese Kampo medicine ninjinyoeito is known to improve the quality of life (QOL) in cancer patients because of its antioxidative effects. However, molecular mechanisms of ninjinyoeito for the preventive effects of oxidative stress remain unclear. In the present study, we sought to determine the effect of ninjinyoeito on oxidative stress in the heart and DOX-induced cardiotoxicity in in vitro experimental system. The effects of ninjinyoeito on the oxidative toxicity in cultured cardiac cells induced by DOX will be presented.

Developmental changes in contraction and relaxation mechanisms were examined in the ventricular myocardium from fetal, neonatal, and 1, 2 and 4 week-old mice. In isolated tissue, the negative inotropy by ryanodine increased with age, while that by nifedipine decreased with age. The prolonging effect on the relaxation by cyclopiazonic acid, a SR Ca²⁺ ATPase (SERCA) inhibitor, increased with age, while that by SEA0400, a Na⁺/Ca²⁺ exchanger inhibitor, decreased with age. Carboxyeosin, a plasma membrane Ca²⁺ ATPase (PMCA) inhibitor, had no effect on the relaxation in all developmental stages. In the presence of cyclopiazonic acid and SEA0400, carboxyeosin slightly prolonged the relaxation, and its prolonging effect decreased with age. In cardiomyocytes, fluorescence imaging revealed that the SR increases with age. t-Tubules, which were absent in the cell center region until 1 week, were present throughout the cytoplasm after 2 weeks. Until 1 week, Ca²⁺ at the cell center showed slower rise than the subsarcolemmal region, but after 2 weeks, Ca²⁺ increased simultaneously across the entire width of the cell. Ca²⁺ decay time decreased with age. These results indicate that the contraction and relaxation mechanisms in the mouse ventricular myocardium convert from membrane dependent to SR dependent during the postnatal development.

Endothelial cells play an important role in regulation of vascular function, which are affected by various bioactive substances. Angiotensin (Ang)Ⅱ induces vasoconstriction and is suggested to cause vascular dysfunction. In this study, we compared the endothelium-dependent vasorelaxation response of the thoracic aorta and mesenteric artery in AngⅡ-induced hypertensive model mice.

In thoracic aorta, stimulation of endothelial a₂-adrenoceptor with clonidine induced a PI3-K-dependent relaxation, whereas acetylcholine (ACh) induced the relaxation via Gq-PLC-Ca²⁺ pathway. In mesenteric artery, ACh-induce relaxation was remarkable, but clonidine-induced relaxation was hardly observed. AngⅡ-induced hypertension significantly impaired the endothelium-dependent relaxation in aorta, but not in mesenteric artery. Experiment using NOS inhibitor revealed that vasorelaxation responses to clonidine and ACh in aorta were largely dependent on NOS activity, whereas NO played minor role in endothelial-dependent vasorelaxation in the mesenteric artery. These results suggest that NOS-dependent endothelial regulation of vascular tone is more sensitive to AngⅡ-induced vascular dysfunction. In addition, the difference in endothelium-dependent relaxation mechanism may be related to the susceptibility of distinct blood vessel to AngⅡ-induced vascular injury.

Vascular deposition of amyloid-bprotein (Aβ), known as cerebrovascular amyloid angiopathy (CAA), is associated with vascular dysfunction. In the previous meeting (WCP2018), we reported amylospheroids (ASPD, 30-mers highly neurotoxic Aβ assemblies in average isolated from Alzheimer's disease brain) suppressed the vasorelaxation via endothelial eNOS inactivation through mitochondrial ROS/PKC pathway. Neuronal toxicity of ASPD was reported to be exerted by impairing the activity of Na⁺, K⁺-ATPase α3 (NAKα3) via the direct binding (Ohnishi et al. PNAS2015). Here, we sought to elucidate whether NAKα3 was involved with the eNOS inactivation by ASPD. We first detected and found the protein and mRNA of NAKα3 in cerebrovascular endothelial cells. Furthermore, we found NAKα3 was expressed in innermost endothelial layer of vascular vessels. We then examined whether the eNOS inactivation by ASPD was abolished by siRNA transfection. Remarkably the knockdown of ASPD-binding target NAKα3 by ATP1A3-siRNA transfection blocked the eNOS inactivation by ASPD. Taken together, these results suggest the endothelial toxicity of ASPD was mediated by NAKα3.

Pathological retinal angiogenesis contributes to the development and progression of vision-threatening eye diseases, such as retinopathy of prematurity and diabetic retinopathy. Valproic acid, a widely used antiepileptic drug, exerts anti-angiogenic effects by inhibiting histone deacetylase (HDAC). We previously reported that valproic acid and vorinostat, a HDAC inhibitor, suppress pathologic retinal angiogenesis in mice with oxygen-induced retinopathy. In this study, using neonatal mouse retina, we examined the mechanisms of anti-angiogenic effects of valproic acid and vorinostat. Mice were subcutaneously injected with valproic acid, vorinostat, or vehicle once a day from postnatal day (P) 0 to P3. At P4, the delayed retinal angiogenesis was observed in mice treated with valproic acid or vorinostat. The expression level of vascular endothelial growth factor (VEGF) was reduced 2 or 6 hours after a single injection of valproic acid or vorinostat in P4 mice. Both drugs suppressed the VEGF-mediated activation of mammalian target of rapamycin pathway in proliferating endothelial cells. The proteasome inhibitor MG132 prevented valproic acid- and vorinostat-induced reduction in the VEGF expression level.These results suggest that valproic acid suppresses retinal angiogenesis by decreasing retinal VEGF levels by a proteasome-dependent mechanism.

Νeuronal and glial cells play an important role in the development of vasculature in the retina. In this study, we investigated the role of glial cells in angiogenesis in retinas with neurodegenerative injuries. To induce retinal neurodegenerative injuries, N-methyl-D-aspartic acid (NMDA, 200 nmol) was injected into the vitreous chamber of the eye on postnatal day (P)7. Morphological changes in retinal neurons and vasculature were assessed on P14, P21, and P35. Prevention of angiogenesis and regression of some capillaries were observed on P14 in retinas of NMDA-treated eyes. However, angiogenesis started on P21, and the retinal vascular network was established by P35 in retinas with neurodegenerative injuries. The results of mechanistic analyses suggest that astrocytes activated by the injury produce and secrete fibronectin to form a scaffold for endothelial cell migration. Vascular endothelial growth factor (VEGF) released mainly from glial cells stimulates the process of angiogenesis. These results suggest that glial cells play an important role in angiogenesis in neonatal rat models of retinal neurodegeneration.

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in maintaining BBB barrier function. Oxidative stress induces accumulation of excessive reactive oxygen species (ROS) and facilitates brain capillary cell death, leading to damage BBB. However, it remains still unclear how oxidative stress induces the cell death of BCECs. In this study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under oxidative stress with 30 µM H₂O₂ for 24 hr. The protein expressions of Orai1 and STIM1 were not affected by oxidative stress in t-BBEC117 cells. However, the oxidative stress inhibited store-operated Ca²⁺ (SOC) entry and the suppression was rescued by the application of 10 mM N-acetyl-cycteine (NAC), a ROS scavenger. Ca²⁺ imaging study with Orai1 siRNAs revealed that SOC entry was mainly mediated by Orai1 channels under oxidative stress in t-BBEC117 cells. The application of 5 µM 2-Aminoethoxydiphenylborane (2-APB), an Orai channel activator, enhanced SOC entry under oxidative stress in t-BBEC117 cells and rescued H₂O₂-induced cell　death. We show here that oxidative stress inhibits Orai1-mediated Ca²⁺ entry, and thereby facilitates the death of BCECs.

The blood-brain barrier (BBB) contributes to the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) are a major component of the BBB and, thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying mechanisms remain unclear. In this study, the molecular components of Ca²⁺-activated Cl^- (Cl_Ca) channels and their physiological role were examined using the cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was predominantly expressed in t-BBEC117 cells. Whole-cell Cl^- currents were sensitive to Cl_Ca channel blockers, niflumic acid and T16A_inh-A01, and markedly reduced by the siRNA knockdown of TMEM16A. The blockade of Cl_Ca channel activity with Cl_Ca channel blockers or TMEM16A siRNA induced a significant membrane hyperpolarization. The treatment with TMEM16A siRNA caused an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) at the resting level. T16A_inh-A01 inhibited cell viability in a dose-dependent manner, and Cl_Ca channel blockers and TMEM16A siRNA also blocked cell proliferation. In addition, Cl_Ca channel blockers and TMEM16A siRNA clearly attenuated cell migration. These results indicate that TMEM16A contributes to Cl_Ca channel conductance and its activity regulates the resting membrane potential of and [Ca²⁺]_cyt in BCECs, TMEM16A Cl_Ca channel are involved in the maintenance of BBB functions, including the proliferation and migration of BCECs.

The tightness of the endothelial barrier is tissue-dependent. We expect that tissue-specific barrier function can be induced in immature vascular endothelial cells by co-culturing with tissue-specific cells. The purpose of this study is to construct tissue-specific vascular endothelial models on a collagen vitrigel membrane (CVM) composed of high-density collagen fibrils equivalent to connective tissues in vivo and compare the responsivity to histamine. We used human microvascular endothelial cells (HMVECs) derived from a newborn foreskin. HMVECs were cultured in a CVM chamber with or without human dermal fibroblasts (HDFs), C6 cells (a rat glioma cell line) or HepG2-NIAS cells (a human hepatocellular carcinoma cell line) cultured on the reverse-side of CVM for up to 6 days. The endothelial barrier function was evaluated by transendothelial electric resistance (TEER). TEER values of a HMVEC monolayer were 15-20 Ω/cm² during culture periods. It significantly increased up to 40-60 Ω/cm² by co-culturing with HDFs, C6 cells and HepG2-NIAS cells. Also, TEER value was clearly decreased in the co-culture model composed of HMVECs and HDFs treated with 1 μM histamine while HMVEC monolayer model showed slight response to 100 μM histamine. Now we are investigating tissue-specific responses to histamine among the models.

In patients with liver fibrosis, muscle mass and muscle strength tend to decline, which affects their prognosis. In 2016, the Sarcopenia Criteria for Liver Disease (First Edition) was established. However, it is unclear why hepatic fibrosis leads to muscle weakness. We induced hepatic fibrosis by performing bile duct ligation (BDL) in mice and investigated the pathogenesis of muscle atrophy caused by hepatic fibrosis. In BDL mice, the weight and cross-sectional area of the tibialis anterior muscle decreased from the first week after surgery in the early stage of fibrosis. We performed forelimb grip tests to confirm a significant decrease in muscle strength. From these results, we considered that hepatic fibrosis-dependent muscle sarcopenia model had been established.

Using this model, we investigated the cause of hepatic fibrosis-dependent muscle atrophy. We applied BDL mouse serum to cultured myotube cells in vitro, and myotube atrophy was induced. This suggested the possibility of multi-organ linkage in which atrophy-inducing factor was transmitted via blood as a mechanism of muscle atrophy dependent on liver fibrosis. Currently, in order to search for atrophy-inducing factors present in the blood, we are analyzing several cytokines that have been reported to be involved in muscle atrophy.

INTRODUCTION: The effect of unsaturated fatty acids on skeletal muscle differentiation in an intrauterine hyperglycemic environment has not been clarified. We explored the effect of maternal nutrition on myotubes formation using the L6 rat myoblast cell model exhibiting hyperglycemia during pregnancy.

METHODS: Rat L6 skeletal myoblasts were grown in DMEM medium containing 100 mg/dL glucose (control), and subsequently it examined the effect of palmitate against the differentiation into myotubes in differentiation medium containing control or 450 mg/L (high glucose). Phospho-Akt was detected by western blotting and the expressions of muscle differentiation markers (myf5 and myoD) were evaluated by real-time PCR.

RESULTS: The gene expression of myf5 and myoD and the level of the Phospho-Akt were significantly higher in high glucose than in control with myogenic differentiation. Palmitate decreased the expression level of myf5 and myoD in 24 hours, however, their expression level increased again after 48 hours. Palmitate also was decreased the Phospho-Akt with differentiation.

CONCLUSION: We showed that palmitate suppressed myogenic differentiation in hyperglycemic condition. We would like to explore the influence of other unsaturated fatty acid against myogenic differentiation.

Calcineurin B homologous protein3 (CHP3) is an EF-hand calcium-binding protein. In our previous study, we found that the expression level of endogenous CHP3 protein was increased in an early differentiation step in C2C12 mouse myoblasts. In this study, we aimed to elucidate the role of CHP3 in skeletal muscle differentiation. Live-cell time-lapse imaging revealed that CHP3 over-expression inhibited cell division of undifferentiated C2C12. Therefore, CHP3 may negatively regulate the proliferation of C2C12.

We previously demonstrated that PDZRN3 is an important protein for myogenic differentiation from myoblasts to myotubes. In regeneration of injured skeletal muscle in vivo, stem cells induce MyoD expression and differentiate into myoblasts, which expand through proliferation. We reported that PDZRN3 is upregulated along with MyoD during regeneration of injured muscle. In this study, we aimed to clarify a role of PDZRN3 in proliferation of myoblasts. When exposed to serum deprivation stress, PDZRN3-depleted C2C12 myoblasts by RNAi showed higher levels of apoptotic markers as compared with those of control cells. PDZRN3-depletion also suppressed the activation of anti-apoptotic Akt, indicating the involvement of PDZRN3 in apoptotic regulation. We found that the abundance of cyclin A2 was reduced in PDZRN3-depleted C2C12 myoblasts, as was that of Mre11, which plays an important role in the repair of DNA damage. The activation of p53 was enhanced in PDZRN3-depleted cells due to the DNA damage accumulation. Overexpression of cyclin A2 restored the expression of Mre11 and attenuated caspase-3 cleavage in PDZRN3-depleted C2C12 cells subjected to serum deprivation. These results thus indicate that PDZRN3 restrains apoptosis in myoblasts through maintenance of cyclin A2 expression.

Bone matrix protein osteocalcin (OC) was recently reported to support the development of learning and memory and also prevent anxiety-like behaviors in mice. Although the mechanism through which OC affects systemic energy expenditure and glucose homeostasis has been relatively well studied, the direct actions of OC on neurons in detail are still uncovered. Therefore, we here investigated the effect of OC on neurons using rat pheochromocytoma cell line PC12, with special reference to the neurite outgrowth, cell proliferation and survival, as well as intracellular signaling.

The number of PC12 cells cultured for four days in the presence of 5 to 50 ng/mL of OC was increased compared to the cells cultured in the absence of OC. The length, but not the number of NGF-induced neurite outgrowth was enhanced by OC. NGF-induced phosphorylation of Akt and ERK was both affected by pretreatment of the cells with OC. RT-PCR analysis for candidates of OC receptor revealed that mRNA expression of Gpr158, but not Gprc6a, was detected in PC12 cells. These results suggested that OC may exerts direct effect on cell growth and differentiation by binding to Gpr158 and modulation of downstream intracellular signaling.

Gingival overgrowth is caused in response to the antiepileptic drug, phenytoin (PHT). PHT-induced gingival overgrowth is characterized by the proliferation of fibroblasts and increased collagen formation in gingiva. Interleukin-1α (IL-1α) increases basic fibroblast growth factor (bFGF) production and influences the release of bFGF in human gingival fibroblasts (hGFs). We have previously reported that PHT induced gingival overgrowth to promote cell proliferation by ETS-1 expression and reduced apoptosis through SAPK/JNK pathway, since PHT enhanced Bcl-2 mRNA and protein expression in hGFs. The present study investigated the effect of PHT on PI3K/Akt signaling pathway in hGFs to clarify the mechanism of PHT-induced gingival overgrowth. Cultured hGFs were purchased from ScienCell Research Laboratories. hGFs were cultured to semi-confluence and treated with PHT and/or IL-1α for 1, 6, 24, and 96 hours. The expression and phosphorylation of Akt，GSK-3β，PTEN，PDK1，p21Waf1/Cip1，and p27 Kip1 were measured by Western blot analysis. IL-1α increased the phosphorylation of Akt (Ser473), however, PHT did not affect that of Akt (Ser473) and GSK-3β. On the other hand, PHT decreased the expression of p21Waf1/Cip1 and p27 Kip1. These results suggest that PHT may affect the expression of p21Waf1/Cip1 and p27 Kip1 through another pathway except Akt and GSK-3β signaling.

The many patients complain about orthodontic force-induced pain. It has reported that jaw-opening reflex (JOR) excitability is increased in 1 (D1) day and is decreased in 7 days (D7) after orthodontic force application in rats. In this model, potential analgesic role of Receptor Potential Vanilloid 1 (TRPV1) antagonism in orthodontic force-induced pain and related features were investigated.

Rats were applied continuous orthodontic force to right maxillary first molar. TRPV1 antagonists (A-889425: 5-10 mmol/kg, AMG9810: 10-15 mmol/kg) or aspirin (560 mmol/kg) was applied to D1-D7. Inflammatory cytokines were measured by antibody arrays. Excitation of trigeminal ganglia (TG) was evaluated by expression of Glial fibrillary acidic protein (GFAP) in satellite glial cells. And, expression of mature osteoclasts was measured by TRAP staining.

All chemicals significantly reduced JOR excitability at D1. Temporal alteration of JOR excitability was associated with GFAP expression and that was significantly reduced by TRPV1 antagonists and aspirin. Although these chemicals reduced expression of mature osteoclasts at D7 significantly, distance of tooth movement was not altered. Significant increase of CINC2 and IL-6 was induced by orthodontic force application. Both TRPV1 antagonists significantly reduced CINC2 and IL-6, however, aspirin failed to reduce CINC2.

Taken together, TRPV1 antagonism, in both peripheral and central, induced broad effects on orthodontic force-induced physiological and morphological alterations.

Dysfunction of unilateral salivary glands causes compensatoryhypertrophy of the contralateral salivary gland. To examine a functional change of the hypertrophiedsubmandibular gland (SMG) after the ligation of main excretory duct (MED) of unilateral SMGs, we monitored Ca²⁺responses and salivary secretion in rat SMG using the intravital Ca²⁺imaging system and the fibre-optic pressure sensor, simultaneously. Submaximal dose of ACh (<120 nmol/min) induced 2 times larger increase in intracellular Ca²⁺concentration and salivary secretion in hypertrophied SMGs than in controlSMGs, whereas the maximal dose of ACh (360 nmol/min) induced comparable responses in these SMGs. These results indicate that the ligation of MED of unilateral SMGs increased the sensitivity of contralateral SMGs to ACh. To clarify the molecular mechanism for inducing "the compensatory hyperfunction", we examined gene expression of the hypertrophiedSMG by a comprehensive analysis using the next-generation sequencing and a quantitative RT-PCR analysis. Currently, we identified 57 candidate genes of which 6 genes showed significant up- or down-regulation after MED ligation in the contralateralSMGs. Our data suggest the increase in acinar cell by the proliferation and transition from ductal cells, and the involvement of some growth factors and clock genes.

Purpose: Globo-series glycosphingolipids have not only been used as markers for stem cells and tumors but are also considered to regulate maintain stemness and immune system. However, there have no reports on the involvement of globo-series glycosphingolipids in bone metabolism. In this study, we investigated the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-seires glycosphingolipids on bone metabolism.

Material & Methods: We examined expression levels of globo-series glycosphingolipids (Gb3, Gb4, and Gb5) in MC3T3 E1 mouse osteoblast-like cells and SaM-1 human osteoblast cells using flow cytometry. To determine whether globo-series glycosphingolipids are involved in bone metabolism, we analyzed bone phenotype of Gb3 shynthase-knockout (Gb3S KO) mice using μCT. Furthermore, we conducted a calcein double labeling method to evaluate bone formation.

Results & Conclusion: Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. μCT analysis revealed that femoral cancellous bone mass in Gb3S KO mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was lower that that in WT mice. We demonstrated that Gb4 is expressed in osteoblasts, and globo-series glycosphingolipids regulate bone mass through bone formation.

Sphingosine-1-phosphate (S1P) is known as a signaling sphingolipid that regulates many cellular responses, including cellular differentiation. Signaling through the specific cell surface G-protein-coupled receptors (subtypes S1PR1 to S1PR5) mediates most of the biological action of S1P. In this study, we investigated the roles of S1PR2 signaling for bone formation on the rat apicoectomy model.

We used 10 week-old male Wistar rat, and created the apicoectomy model with bone cavity (diameter 2 mm, depth 1 mm) on a mesial root of mandibular left first molar. Then we injected S1PR2 agonist (CYM-5520) mixed with scaffold (atelocollagen). Rat sacrificed after 3 weeks, and then subjected to micro-computed tomography analysis. The handling rats and all procedures were approved by the Animal Committee of Fukuoka Dental College (No. 18014).

Injection of S1PR2 agonist promoted bone properties such as bone volume density, and trabecular number compared with control. Interestingly, we found that S1PR2 agonist showed the osteoinductive effect. 

We conclude that S1PR2 signaling accelerate bone formation and induce osteoblast differentiation.

Osteoporosis characterized by a decrease in bone mass, is thought to be one of the chronic and lifestyle-related diseases. We aimed to elucidate the bone protective effects of petunidin, considering its potent antioxidative activity. Seven-week-old female C57BL/6J mice were divided into three groups: control, sRANKL-induced osteopenic mice (vehicle) and 7.5 mg/kg/day petunidin-treated osteopenic mice (petunidin). Bone morphometric parameters and microarchitectural properties of the femur were determined using a micro-CT system. The vacant area observed in the marrow cavity of vehicle group reduced in size and filled with trabeculae by petunidin administration. Quantitative analyses showed that petunidin significantly increased BV/TV, Tb.Th, Tb.N, reflecting the increase in trabecular bone mass. Furthermore, bone histomorphometry analyses showed that petunidin administration significantly increased OV/TV, O.Th, OS/BS, Ob.S/BS and N.Ob/BS, suggesting that bone formation was accelerated by petunidin. In contrast, major resorption-parameters (ES/BS, Oc.S/BS and N. c/BS) were decreased in the petunidin-treated group. Histological sections of the distal femurs demonstrated that both of osteoid thickness and height of the osteoblasts were increased by petunidin administration. In conclusion, the present study showed that oral administration of petunidin improved sRANKL-induced osteopenia in mice through increased osteoid formation, reflecting accelerated osteoblastogenesis, concomitant with suppressed bone resorption.

First-generation antiepileptic drugs (AEDs) increase the risk of fracture in patients with epilepsy. Although phenytoin has been reported to adversely influence bone metabolism, little is known about the effects of recent AEDs. In this study, we examined the effects of newer AED, vigabatrin on bone metabolism in rats. Male Wistar rats were treated orally with phenytoin (20 mg/kg) or vigabatrin (50 or 200 mg/kg) daily for 6 weeks. Bone histomorphometric analysis was performed, and bone strength was evaluated using a three-point bending method. Bone mineral density (BMD) was measured using quantitative computed tomography. Administration of phenytoin significantly decreased BMD. In contrast, vigabatrin treatment did not affect BMD.  However, a significant decrease in bone microstructure parameters were observed in the vigabatrin 200 mg/kg treated group. The bone formation parameters decreased after vigabatrin 200 mg/kg treatment, whereas the bone resorption parameters increased. Our data suggest that vigabatrin-induced trabecular bone rarefaction, which is associated with decreased bone formation and enhanced bone resorption may affect bone strength after chronic exposure.

Acute lung injury is one of the common lethal complications in sepsis. It is clinically characterized by refractory hypoxemia, diffuse pulmonary infiltrates, and high permeability pulmonary edema. Several molecules could contribute to increased vascular permeability during sepsis. In this study, we investigated whether VEGF, originally known as a vascular permeability factor, plays a possible role in sepsis-associated lung vascular hyperpermeability. Initially, we examined time-dependent expression of VEGF and its receptors, Flt1 and KDR, in human pulmonary endothelial cells (HPMEC-ST1.6R) when stimulated with LPS + INFγ. Following stimulation, VEGF expression was significantly increased, but Flt1 and KDR remained unchanged. VEGF release by HPMEC-ST1.6R after stimulation was significantly increased, and it was suppressed by JNK, MEK, and p38 MAPK inhibitors. Next, when experimental mouse models of sepsis were used, septic conditions resulted in enhanced lung vascular permeability, as assessed by IgM concentrations in bronchoalveolar lavage fluid, and led to a significant increase in blood VEGF concentrations. Bevacizumab, an anti-VEGF antibody, significantly suppressed pulmonary hyperpermeability in sepsis. These results suggest that VEGF is involved as one of the factors that increase lung vascular permeability in sepsis.

It has been known that 5-10% of asthma patients are resistant to the steroid therapy. However, mechanisms underlying the acquisition of steroid resistance remain unclear. Objective of this study is to elucidate whether IL-5/eosinophils are involved in the acquisition of steroid resistance. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 5 or 500 µg/animal 4 times. Infiltration of eosinophils into the lung, and development of airway remodeling and airway hyperresponsiveness (AHR) were evaluated 1 day after the 4th challenge. Dexamethasone and/or anti-IL-5 mAb was i.p. administered during the multiple challenges. AHR was evaluated by forced oscillation technique using FlexiVent. Infiltration of eosinophils into the lung, and the development of airway remodeling and AHR in the 5 µg OVA-induced model were significantly suppressed by dexamethasone, whereas those asthmatic responses to 500 µg OVA were not inhibited by dexamethasone. Under treatment with anti-IL-5 mAb, in which the eosinophil infiltration was strongly reduced, dexamethasone showed significant inhibition on the development of airway remodeling. However, the steroid resistance in AHR was not restored by the anti-IL-5 mAb. It was suggested that IL-5/eosinophils are involved in the acquisition of steroid resistance in the severe asthma.

Patients with chronic spontaneous urticarial (CSU) are often resistant to treatment with H1 antihistamines and have an increased response to intradermal injection of histamine. In this study, we evaluated the changes of responsiveness of vascular endothelial cells treated with Lipopolysaccharide (LPS) and Tumor necrosis factor (TNF)-α, known as exacerbating factors of CSU, to histamine using the impedance sensor, which can analyze the gap formation of cells in real time. Umbilical cord blood-derived vascular endothelial cells (HUVECs) were used as human vascular endothelial cells. HUVECs were cultured on the electrodes of sensor and the effects of LPS and TNFαtreatment on the gap formation of cells in response to histamine were monitored. Moreover, the effects of H1 antihistamines on the gap formation of LPS- or TNF- treated HUVECs were examined. When LPS and TNFα were added simultaneously with histamine, the response of HUVECs to histamine did not increase. On the other hand, when LPS and TNFα were added a day before measurement of impedance, the responsiveness to histamine was increased and the response became difficult to be suppressed by H1 antihistamines. The increase of histamine responsiveness in vascular endothelial cells by LPS and TNFα may contribute to resistance to antihistamines in patients with CSU.

Systemic sclerosis (SSc: scleroderma) is an autoimmune disorder characterized by progressive dermal fibrosis with diffusion to multiple organs which could be fatal. SSc could be initiated by anti-cancer therapy or graft-versus-host disease (GVHD) after bone marrow transplantation. However, the current treatment methods for SSc are only conservative. Therefore the novel agents which make fundamental treatment possible are waited. The murine chronic GVHD model induced by allogenic cell transplantation from B10.D2 mice (donner) to BALB/c mice (recipient) is known as one of the experimental scleroderma models. The aim of this study is to confirm the pathologic state of the chronic GVHD scleroderma model and evaluate the effects of STAT inhibitor. We validated the model by inspection of skin score, skin hydroxyproline (HYP) content and histopathologic examinations. The skin score and skin HYP content increased in all cell transplanted groups compared to control group. Thus, it was indicated that the SSc-like symptoms with dermal fibrosis developed on model animals. Effects of STAT inhibitor were evaluated on this model. The results indicate usefulness of the chronic GVHD scleroderma model in evaluating the anti-fibrosis effect of therapeutic agents, which leads to fundamental treatment of SSc.

Intelectin-1 (ITLN) is a secretory protein that exists in blood, thoracis, and gut lumen. ITLN doesn't induce obvious physiologic and immunological reaction. Therefore, ITLN would induce no harmful effect when it injects into the body. In previous study, we found that ITLN specifically bound to 1,2-diol residues. The 1,2-diol residue is chemically stable, low-toxic, and is not bound with almost other proteins. Accordingly, we hoped that we could develop a retrievable molecular target drug by combining an ITLN-fused antibody and a blood purification device with a diol-coated resin.

In this study, we used ITLN-fused TNF receptor (TNFR-ITLN) instead of antibody because it doesn't need humanization. TNFR-ITLN is a soluble TNF receptor replacing Fc of etanercept with ITLN. TNFR-ITLN bound and neutralized to TNF-α as well as etanercept. In addition, TNFR-ITLN was specifically eliminated from the blood with a diol-silica gel column. On an apheresis model using LPS-treated rats (sepsis model), we measured the concentration of plasma TNF-α. When the TNFR-ITLN-injected LPS-treated rats were treated by apheresis using diol column, the transient increase of TNF-α was clearly suppressed in the blood. Furthermore, these rats were prevented from death by endotoxin shock.

According to these results, we considered that the apheresis using TNFR-ITLN plus diol column could selectively remove plasma TNF-α. A Fab-fused ITLN may specifically eliminate any pathogenic antigen from the body. This medical technology can applicable to various autoimmune diseases and it is expected to clinical application for a new blood purification method.

An increase in red blood cells (RBCs) is believed to improve exercise performance, because RBCs transport O₂ from the lungs to the tissues and deliver metabolically produced CO₂ to the lungs for expiration. In this study, we examined the effects of increased or decreased RBCs on exercise performance in mice. In order to vary the volume percentage of RBCs in blood (hematocrit levels), trained FVB mice were administered darbepoetin alfa (DPA), a long-acting erythropoiesis-stimulating agent, or phenylhydrazine (PHZ), a reagent inducing hemolytic anemia. The exercise performance was evaluated using a forced swimming pool.  The administration of DPA or PHZ caused a significant increase or decrease in hematoctit levels, respectively. However, the partial improvement in exercise performance due to increased RBCs was observed only when higher intensity exercise was applied to mice whose hematocrit levels exceeded 70%. In addition, the decrease in exercise performance due to decreased RBCs was limited, even when the hematocrit levels was about 35%. These results suggested that the increase or decrease in RBCs had little effect on exercise performance in mice.

Stress is thought to be a risk factor for psychiatric disorders, such as major depression. Previously, we reported that mice exposed to repeat excessive restraint stress showed emotional abnormality. Corticosterone (CT) elevated by repeated stress have been reported to increases the ubiquitination in the brain. The present study was designed to investigate the levels of expression of ubiquitin ligase proteins in the prefrontal cortex and hippocampus of stress-maladaptive mice and to examine whether CT alters the proteins expression levels in a mouse hippocampal HT22 cells.

Male ICR mice were chronically exposed to inadaptable stress, i.e. repeated restraint stress for 240 min/day for 14 days. After the final exposure to stress, brains of mice were rapidly removed and the hippocampus was dissected. HT22 cells were cultured in DMEM media supplemented with 10% FBS. Cell viability was measured by using MTT Assay.

Western blot analysis revealed that no change in the level of expression of E3 ubiquitin ligase Nedd4-2 was observed in among all mice. In contrast, a significant decrease in the expression level of phosphorylated Nedd4-2 (p-Nedd4-2) was observed in the hippocampus, but not the prefrontal cortex, of stressed mice. Although CT had no effect on cell survival at concentrations of less than 3 μM, pretreatment with 3 μM CT down-regulated p-Nedd4-2 in HT22 cells. These results suggested that CT is an important effector for activation of Nedd4-2 in hippocampal neurons, and Nedd4-2 plays a pivotal role in the emotional abnormality in stress-maladaptive mice.

Background:It is known that there is a function of cell survival such as oxidant stress resistance and NAD + dependent histone deacetylation by SIRT1 activitots. We previously reported that administration of activator of SIRT1, resveratorol (Rsv) ,ameliorates muscular pathophysiology of dystrophin-deficient mdx mice, and inhibition of SIRT1 suppresses membrane repair process of C2C12 myoblasts and myotubes. However, it remains uncrear whether cell membrane repair in muscle cells is accelerated by SIRT1 activators. The purpose of this study is to prove that SIRT1 activators improve cell membrane repair ability as a factor in cell survival.

Method:In the present study, we used C2C12 myoblasts and myotubes. After treating these with Rsv (50μM) and nicotinamide mononucleotide (NMN) (5mM) (24h), we damaged the cell surface by local laser irradiation using a confocal microscope (Nikon A1). And membrane resealing process was monitored by an influx of fluorescent dye FM1-43 in cells surface after laser irradiation.

Result:We found that levels of fluorescence FM1-43 dye intake after laser-induced membrane destruction in C2C12 cells were significantly decreased in NMN (54.9 ± 18.9%) and Rsv  (48.0 ± 20.4%)  respectively.

Conclusion:Our data indicate a possibility that SIRT1 activators ameliorate muscular pathophysiology of muscular dystrophies through promoting plasma membrane repair.

Sarcopenia is defined as a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, poor quality of life and death, as a common negative consequence of aging. The present study is undertaken to investigate the correlation between muscle atrophy and aging-related immune deterioration by using a sciatic nerve dissected sarcopenic mouse model. The fraction of immunological cells, including T cells, B cells, macrophages, natural killer cells, and neutrophils, was observed. The systemic inflammatory responses were evaluated in lipopolysaccharide-induced sepsis animals. The T and B cell lineages were significantly suppressed in the sarcopenic model compared with control mice, and the fraction of macrophages and natural killer cells also tended to increase. The survival proportions in sarcopenic mice were significantly decreased while comparing to control mice. In conclusion, sarcopenia induces a functional impairment in immune cells. The mechanism of impaired immunological system would be assessed in our future planned study.

Purpose

Dexmedetomidine (DXM) is clinically used for sedation in perioperative patients. Previous studies reported that DXM is preventive against oxidative stress. Thus we hypothesized that DXM directly scavenges free radicals thereby acting as antioxidant.

Methods

Direct scavenging activity of DXM was evaluated against nine species of free radicals by electron spin resonance spectroscopy with the spin-trapping method. Fluorescence-based assays of cellular viability and intracellular free radical production were conducted using Alamar Blue and MitoROS 580.

Results

DXM significantly scavenged the following free radicals in dose-dependent manners; hydroxyl radical, superoxide anion, t-butoxyl radical, singlet oxygen and ascorbyl free radical. However, no scavenging activity was observed against t-butyl peroxyl radical, nitric oxide, DPPH and tyrosyl radical. Cellular viability of MRC-5 cells exposed to hydrogen peroxide was significantly improved in the presence of 0.1 μM DXM. 10 μM DXM significantly inhibited mitochondrial superoxide anion by Antimycin A. DXM showed no cytotoxicity up to 100 μM.

Conclusions

Although no effect was observed on nitrogen-centered radicals including nitric oxide, it was confirmed that DXM directly scavenges multiple oxygen-centered free radicals including hydroxyl radical (one of the strongest free radicals in living body) and superoxide anion (the most upstream of ischemia-reperfusion injury), which probably contributes to its antioxidative activity.

Serotonin (5-HT) can induce hepatocyte DNA synthesis and proliferation by autocrine secretion of transforming growth factor (TGF)-α through 5-HT_2B receptor/phospholipase C (PLC)/Ca²⁺. In the present study, we investigated whether 5-HT or a selective 5-HT_2B receptor agonist BW723C86, would stimulate phosphorylation of TGF-α receptor tyrosine kinase (RTK) and ribosomal p70 S6 kinase (p70 S6K) in primary cultures of adult rat hepatocytes by using Western blotting analysis. Our results showed that 5-HT- or BW723C86-induced phosphorylation of EGF/TGF-α RTK peaked at between 5 and 10 min. On the other hand, 5-HT- or BW723C86 -induced phosphorylation of p70 S6K peaked at about 30 min. Furthermore, a selective 5-HT_2B receptor antagonist LY272015, a specific PLC inhibitor U-73122, a membrane-permeable Ca²⁺ chelator BAPTA/AM, an L-type Ca²⁺ channel blocker verapamil, somatostatin, and a specific p70 S6K inhibitor LY2584702 completely abolished the phosphorylation of p70 S6K induced by both 5-HT and BW723C86. These results indicate that phosphorylation of p70 S6K is dependent on the 5-HT_2B-receptor-mediated autocrine secretion of TGF-α. In addition, these results demonstrate that the hepatocyte proliferating action of 5-HT and BW723C86 are mediated by phosphorylation of p70 S6K, a downstream element of the EGF/TGF-α RTK signaling pathway.

Recent investigations of human immunological regulation have suggested that mast cells may play an important role in maintaining homeostasis. Some of these studies have suggested that mast cell cytokines may play key roles in prevention of both viral and bacterial infections, and the development of tumors. The present study focused on the enzymes for melatonin synthesis in mast cells because of their rolls in immune response. mRNA expression from LAD2 cells, a human mast cell-derived cell line, was examined for aralkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransfase (HIOMT), key enzymes in melatonin synthesis. LAD2 were positive for mRNA expression of both enzymes. The mRNA levels were enhanced by stimulation with db-cAMP (500 μM) with no β-hexosaminidase (β-Hex) release; in contrast, A23187 (2 μM) did not enhanced mRNA levels but did induce β-Hex release. These results suggest that melatonin release from mast cells is involved in maintaining homeostasis, and is not involved in allergic responses.

O-GlcNAcylation of the stromal interaction molecule 1 (STIM1) is known to impair store-operated Ca²⁺ entry (SOCE). Because it has not been identified the O-GlcNAcylation sites of STIM1, we examined whether the Serine/Threonine (Ser/Thr) residues (Ser-575, Ser-608, Ser-621, and Thr-626) of STIM1 S/P-rich domain and the adjacent region were O-GlcNAcylated or not using co-immunoprecipitation. The result showed that Ser-621 and Thr-626 residues were O-GlcNAcylated. To examine the role of the O-GlcNAcylation on SOCE activity, we established the STIM1 knockout HEK293 cells by the CRISPR/Cas9 system, and then, transfected T626A (Thr-626 substituted to Ala) to the cells. Surprisingly, the SOCE activity was reduced via reduced phosphorylation at Ser-621 residue in T626A transfected cells. It was also shown that the SOCE activity was reduced with the treatment of O-GlcNAcase inhibitor Thiamet G via reduced phosphorylation at Ser-621 residue in STIM1 wild-type transfected cells. These data may indicate that both decreased O-GlcNAcylation of STIM1 at Thr-626 and/or increased STIM1 O-GlcNAcylation at Ser-621 results in impaired SOCE activity. This is the first report showing the dual role of O-GlcNAcylation in regulating the SOCE activity of STIM1.

A549 cells are an immortalized alveolar epithelial cell line, which has been employed to investigate alveolar epithelial cell responses to several treatments. In the present study, we found that treatment of A549 cells with interleukin 1 beta (IL-1β) induced the activation of p38 mitogen-activated protein kinase (MAP kinase) and MAP kinase-activated protein kinase-2 (MAPKAPK-2), and the phosphorylation of epidermal growth factor receptor (EGFR) at serine 1047. The activation of MAPKAPK-2 and phosphorylation of EGFR were inhibited by SB203580, an inhibitor of p38 MAP kinase. In addition, MK2a inhibitor, an inhibitor of MAPKAPK-2, inhibited the phosphorylation of EGFR. Biotinylation of cell surface proteins indicated that IL-1β treatment induced the internalization of EGFR. Furthermore, the long-term treatment of A549 cells with IL-1β changed the cell morphology, with the loss of cell-cell contacts. In addition, IL-1β augmented the effects of transforming growth factor beta 1 on changes in the epithelial-mesenchymal transition. These results suggested that IL-1β regulates the functions of EGFR and induces morphological changes in alveolar epithelial cells.

The G protein-coupled receptors (GPCRs) transduce their signaling through the activation of trimeric G proteins, but their associated mechanisms have remained unclear. It has been shown that the proteins that interact with carboxyl (C)-termini of GPCRs regulate the GPCRs-mediated signal transduction by modulating intracellular localization of the receptors. Parathyroid hormone (PTH)/PTH-related protein receptor (PTHR) is a G_s- and G_q-coupled GPCR. We previously showed that the C-terminus of PTHR directly binds to a subcortical cytoskeletal protein 4.1G. Cell surface expression of PTHR and its G_q/[Ca²⁺]_i signaling were increased by 4.1G, whereas its G_s/adenylyl cyclase (AC)/cyclic AMP (cAMP) signaling was reduced by 4.1G through unknown mechanisms. In the present study, we first found that AC type 6 (AC6) interacted with 4.1G in HEK293 cells and the N-terminus of AC6 (AC6-N) directly and selectively bound to the 4.1/ezrin/radixin/moesin (FERM) domain of 4.1G (4.1G-FERM) in vitro. Association of AC6-N with the plasma membrane was disturbed by the knockdown of 4.1G. Next, AC6-N was overexpressed to competitively inhibit the interaction of endogenous AC6 and 4.1G in the cells. Overexpression of AC6-N, as well as 4.1G-knockdown, augmented the cAMP production induced by forskolin, a direct AC activator, and PTH-(1-34). Taken together, our results demonstrate a model in which AC6-N associates with the plasma membrane through binding to 4.1G-FERM, resulting in low AC6 activity. The mechanism is responsible for the attenuation of PTHR-mediated G_s/AC6/cAMP signaling.

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor associated with numerous redox reactions including energy production. NAD also serves as a substrate for ADP-ribosylation by poly(ADP-ribose) polymerase and protein deacetylation by sirtuins. Interventions using NAD precursors have been reported to have various beneficial effects on aging-associated diseases, such as obesity, diabetes, and Alzheimer disease. In mammals, NAD is synthesized from niacin (nicotinamide and nicotinic acid) and tryptophan. It is known that deficiency of niacin causes Pellagra featured by diarrhea, inflamed skin, and dementia. However, it is not fully understood why niacin deficiency causes Pellagra. Here we found that deficiency of NAD synthetase (NASD) caused the shortening of villi length in small intestine in mice. This photocopies the colitis observed in Pellagra patients. Furthermore, these mice are more sensitive to colitis induced by 5-fluorouracil treatment. These results suggest that NADS is involved in mechanism of Pellagra onset and is important for maintaining intestinal homeostasis.

Three receptor subtypes are known for neurohypophyseal hormone, Arginine vasopressin (V1aR, V1bR, and V2R). We previously reported the decrease of litter size and the delayed labor in V1aR knockout (KO) mice. In order to further elucidate the mechanism of the decreased litter size in V1aR KO mice, we counted the number of implantation sites at embryonic day 5.5. Unexpectedly, there was no difference in the number of implantation sites between WT and V1aR KO mice. This observation suggests that the pups' number decreases after the implantation. We also measured the body weight of pregnant mothers and newborns. Both WT and V1aR KO mothers showed the similar weight gain during pregnancy. The body weight of newborns at the parturition also showed no difference between WT and V1aR KO mice. However, at 3 weeks old, when pups were weaned from their mothers, the body weights of V1aR KO litters were significantly lower than those of WT. These results suggest that the pups of the V1aR KO mice show growth disorder at the time of the transition from juvenile to adult. Therefore, V1aR should be requisite for the normal growth.

The Developmental Origin of Health and Disease (DOHaD) hypothesis, advocating long-term effect of fetal origins on adult disease, suggests that adverse environmental exposure during fetal and neonatal development might increase the susceptibility for developing a wide range of lifestyle-related diseases in later life. Indeed, our previous study demonstrated that maternal high-fat and high-sucrose feeding during gestational period deteriorated offspring's glucose and lipid parameters and triggered obesity in mice.

We recently clarified that osteocalcin, one of bone matrix proteins with placental transportability, is a mediator between glycolipid metabolism and bone metabolism through multiple mechanisms improving glycolipid metabolism. In this study, we aimed to investigate whether maternal osteocalcin administration during gestational period may ameliorate the offspring's metabolic statusthrough the function of changing the nutritional response in utero in mice, from an epigenetic perspective, because the biological mechanism underlying the DOHaD hypothesis is supposed to be mainly related to alteration in epigenetically regulated gene expression.

As a result, we revealed that maternal osteocalcin intake could avoid the ameliorable effects of gestational overnutrition on pups by regulating epigenomic nutritional responses.

Progesterone is a sex steroid hormone synthesized by the ovary, and plays a pivotal role for reproductive functions such as ovulation and the maintenance of pregnancy. Although these effects of progesterone have been implicated in nuclear progesterone receptors (PRs)-mediated classical signaling pathway, key pathways involved in non-classical progesterone signaling has been provided by the identification of membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ, and mPRε). Interestingly, mPRs may have been related to progesterone-mediated unknown rapid non-genomic action that cannot be currently explained by their genomic action through PRs. However, the structure, intracellular signaling, and physiological functions of these progesterone receptors are still unclear. In this study, we confirmed that mPRε, among mPRs receptors, is specifically expressed in the white adipose tissue (WAT), liver, and kidney of adult male and female mice. Furthermore, progesterone- mPRε signaling may contribute to suppression of glucose uptake and impair glucose tolerance in WAT. These findings provide new insights of regarding the non-genomic action of progesterone in metabolic homeostasis and novel therapeutic targets and strategies for metabolic disorder such as obesity and type 2 diabetes mellitus.