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Caloric restriction (CR) improves whole-body metabolism, suppresses various age-related pathophysiological changes, and extends lifespan. The beneficial actions of CR are regulated in growth hormone (GH)/insulin-like growth factor-1 (IGF-1) signal-dependent and -independent manners. To clarify the GH/IGF-1-independent mechanism, we compared gene expression profiles in white adipose tissue (WAT) between CR and GH/IGF-1 suppression, and found that CR upregulated sterol regulatory element-binding protein 1c (SREBP-1c) regulatory gene expression. To validate the impact of SREBP-1c as a beneficial mediator of CR, we compared the responses to CR between wild-type and SREBP-1c knockout (KO) mice. CR extended lifespan, upregulated gene expression involved in FA biosynthesis, activated mitochondrial biogenesis, and suppressed oxidative stress predominantly in WAT. In contrast, most of these findings were not observed in KO mice. Furthermore, SREBP-1c was implicated in CR-associated mitochondrial activation through upregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Sirtuin-3 (SIRT3) regulates mitochondrial quality and is also involved in the beneficial actions of CR. We observed that CR upregulated the mature form of SIRT3 protein and mitochondrial intermediate peptidase (MIPEP), a mitochondrial signal peptidase (MtSPase), in WAT. MIPEP cleaved precursor form of SIRT3 to mature form, and activated certain mitochondrial matrix proteins, suggesting that MIPEP might contribute to maintenance of mitochondrial quality during CR via SIRT3 activation. Taken together, CR induces SREBP-1c-dependent metabolic remodeling, including enhancement of FA biosynthesis and mitochondrial activation, via PGC-1α, and improvement of mitochondria quality via Mipep in WAT, resulting in beneficial actions.

The combination treatment of radiotherapy with anti-PD-1/PD-L1 antibody has been shown to significantly improve the clinical outcome of various cancers. Recent studies showthat radiotherapy has multiple functions in modifying the tumor microenvironment, by inducing both immunostimulation and immunosuppression. The upregulation of PD-L1 expression in cancer cells interferes with the effector functions of interacting T cells. Preclinical studies demonstrate that radiotherapy induces PD-L1 upregulation by 4 pathways; (1)IFN-γ signaling,(2)EGFR pathway,(3)DNA damage signaling pathway, and(4)cGAS-STING pathway. All of these mechanisms are involved in the upregulation of PD-L1 expression in cancer cells via JAK/STAT pathway. Because the PD-1/PD-L1 interaction has been shown to be one of the major mechanisms of cancer immune escape, which leads to treatment failure, anti-PD-1/PD-L1 antibody may improve the efficacy of radiotherapy by enhancement of anti-tumor activity. In addition, PD-L1 expression is one of the biomarkers of good response to anti-PD-1/ PD-L1 antibody. Therefore, the comprehensive understanding of the mechanism underlying PD-L1 expression in response to radiotherapy is important for the establishment of optimal combination strategy. This approach could help to provide the basis for the combined therapies and promote personalized immuno-radiotherapy, although the signaling of PD-L1 upregulation induced by radiotherapy in tumors could be intricately regulated. In this article, we review previous researches which revealed the mechanisms of PD-L1 upregulation induced by radiotherapy.

Angiotensin (Ang) II, the main bioactive component of the renin-angiotensin system, is reported to participate in either the inhibition or the facilitation of pain transmission depending on brain area. Although Ang II is known to exist in the superficial dorsal horn of the spinal cord, the role of Ang II in spinal pain transmission remains unclear. In order to elucidate that role, we examined the effect of intrathecal administration of Ang II to mice. We found that Ang II produced pain-related behavior accompanied by the phosphorylation of p38 mitogen-activated protein kinase (MAPK) via Ang II type 1 (AT1) receptors, which are highly expressed in the superficial dorsal horn (laminae I and II). In addition, pain-related behavior was caused by acting on spinal neurons and astrocytes that express AT1 receptors. We next examined whether the spinal Ang II system is responsible for diabetic neuropathic pain. In streptozotocin-induced diabetic mice, neuronal Ang-converting enzyme was upregulated in the superficial dorsal horn, which led to an increase in spinal Ang II levels. Furthermore, this increase in Ang II caused mechanical hypersensitivity accompanied by the activation of p38 MAPK via AT1 receptors. In conclusion, our findings suggest that Ang II may act as a neurotransmitter and/or neuromodulator in spinal pain transmission. This review describes our recent efforts regarding the role of spinal Ang II in spinal pain transmission.

Reelin is a large secreted glycoprotein that regulates embryonic neuronal lamination and adult synaptic function. Secreted Reelin binds to lipoprotein receptors expressed on neurons. The Reelin-receptor interaction induces phosphorylation of an intracellular adaptor protein Dab1, which is required for normal embryonic brain development and adult brain functions. It has been suggested that Reelin hypofunction plays a role in the pathogenesis of several neuropsychiatric diseases, such as schizophrenia, autism, and Alzheimer's disease. Thus upregulation of Reelin activity may ameliorate the symptoms of neuropsychiatric diseases. However, the regulatory mechanism underlying the functions of Reelin is largely unknown and there are no good animal models of Reelin malfunction; thus, causal relations between Reelin and neuropsychiatric diseases remain unclear. Recently, our studies have shown that proteolytic cleavage of the Reelin protein regulates its activity. Herein, we will review recent findings about relations between Reelin and Alzheimer's disease, and the mechanism underlying the regulation of Reelin function by proteolytic cleavage. Also, we will discuss the prospect of treating neuropsychiatric diseases by upregulation of Reelin activity.

In chronic kidney disease (CKD), progressive nephron loss causes tubulointerstitial fibrosis and progressive tubular injury. Recent identification of the major cell populations of myofibroblast precursors in the kidney has enabled us to dissect the fibrogenic process after tubular injury. Kidney pericytes are a possible precursor of myofibroblasts, and may be promising targets for treating fibrogenesis. Our recent studies have shown that pericytes activate Toll-like receptor (TLR) 2/4- and myeloid differentiation primary response 88 (MyD88)-dependent proinflammatory signaling in response to renal tubular injury. We also found active roles of inflammasomes in kidney pericytes, leading to interleukin (IL)-1β and IL-18 secretion. Genetic ablation of MyD88 in pericytes, or pharmacological inhibition of MyD88 signaling by an IL-1 receptor-associated kinase 4 (IRAK4) inhibitor, halted interstitial fibrosis after renal tubular injury. Our data indicate that pericytes not only contribute to interstitial fibrosis by aberrant wound-healing responses, but also serve as innate immune surveillance cells that regulate the inflammatory process, exacerbating tubular injury by the release of cytokines and chemokines. On the other hand, our recent study using a microarray analysis aimed to identify molecular changes that reflected the histopathological progression of renal tubulointerstitial fibrosis and tubular cell damage in patients with CKD. The results indicated that 5 genes were up-regulated in the kidney of CKD patients, and that their expression levels correlated with the extent of tubulointerstitial fibrosis and tubular cell injury. These findings provide important information for the development of diagnostic tools and therapeutic agents for predicting and preventing progressive renal disease.

A growing body of evidence indicates that extracellular ATP released or leaked from nonexcitable cells as well as neurons plays important roles in the regulation of neuronal and glial functions in the entire body through ATP receptors. ATP receptors (ionotropic P2X and metabotropic P2Y receptors) are the most abundant receptor families in living organisms. In the central nervous system, these receptors participate in the synaptic transmission and intercellular communications between neurons and glia. The glia cells are classified into three types: astrocytes; oligodendrocytes; and microglia. There are many reports that spinal microglia express ATP receptors (P2X4, P2X7, P2Y6, and P2Y12 receptors) that have very important roles. We reported that several molecules of microglia are activated after peripheral nerve injury in a neuropathic pain model. In particular, P2X4 receptors (P2X4Rs) expressed in microglia play a critical role in evoking neuropathic pain. P2X4Rs are upregulated in spinal microglia after nerve injury by several factors such as the CC chemokine receptor CCR2, fibronectin in the spinal cord, interferon regulatory factor (IRF) 8, and IRF5 expressed in microglia. The inhibition of P2X4R action suppresses the functions of microglia and neuropathic pain. These results indicate that overexpressing P2X4Rs on microglia are a central player in evoking neuropathic pain.

Peripheral neuropathy is a common adverse effect of paclitaxel and oxaliplatin treatment. The major dose-limiting side effect of these drugs is peripheral sensory neuropathy. The symptoms of paclitaxel-induced neuropathy are mostly sensory and peripheral in nature, consisting of mechanical allodynia/hyperalgesia, tingling, and numbness. Oxaliplatin-induced neurotoxicity manifests as rapid-onset neuropathic symptoms that are exacerbated by cold exposure and as chronic neuropathy that develops after several treatment cycles. Although many basic and clinical researchers have studied anticancer drug-induced peripheral neuropathy, the mechanism is not well understood. In this review, we focus on (1) analysis of transient receptor potential vanilloid 1 (TRPV1) channel expression in the rat dorsal root ganglion (DRG) after paclitaxel treatment and (2) analysis of transient receptor potential ankyrin 1 (TRPA1) channel in the DRG after oxaliplatin treatment. This review describes that (1) paclitaxel-induced neuropathic pain may be the result of up-regulation of TRPV1 in small- and medium-diameter DRG neurons. In addition, paclitaxel treatment increases the release of substance P, but not calcitonin gene-related peptide, in the superficial layers of the spinal dorsal horn. (2) TRPA1 expression via activation of p38 mitogen-activated protein kinase in small-diameter DRG neurons, at least in part, contributes to the development of oxaliplatin-induced acute cold hyperalgesia. We suggest that TRPV1 or TRPA1 antagonists may be potential therapeutic lead compounds for treating anticancer drug-induced peripheral neuropathy.

Emerging evidence has been unveiled that major oncogenic pathways, which serve as a main focus for the molecular targeting therapies, play a pivotal role in establishing immunosuppressive tumor microenvironments. The modulation of various immune modulatory pathways, such as upregulation of PD-L1 by various oncogenic mutations such as EGFR, BRAF, activation of PI3K and JAK-Stat3 on tumor cells, may be critical in modulating tumor immune responses. Given the potential clinical impact of immune checkpoint blockade and other immunotherapeutic regimens as a future anticancer regimen, it is critical to clarify the mechanisms by which molecularly targetable oncogenes and tumor microenvironments influence quality of tumor immunosurveillance, which should provide a new strategy for suitable combinatorial approaches composing of molecular targeting and immunotherapies.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are classified to a multigene family of channel proteins that mediate Ca2+ release from endoplasmic reticulum, and are one of regulators to modify intracellular Ca2+ concentration. Little is known about functional relationship between rewarding effects due to drugs of abuse and IP3Rs. This report reviews the roles and regulatory mechanisms of intracellular Ca2+ channels, especially type 1 IP3Rs (IP3Rs-1), in brain of animals with rewarding effects produced by drugs of abuse. Our recent studies have reported that the blockade of IP3Rs suppresses the development of rewarding effects on methamphetamine or cocaine, suggesting that functional up-regulation of IP3R-1 occurs during the development of rewarding effects. Moreover, the critical expression of IP3R-1 in the development of methamphetamine- and cocaine-induced rewarding effects are regulated by Ca2+ participating in signal transduction pathways via both dopamine D1 and D2 receptors. Taken together these results it is suggested that the changes in IP3R-1 play an essential role in the development of drug dependence.

Injury to the nervous system results in debilitating chronic pain states (called neuropathic pain) whose mechanisms remain unclear. Emerging lines of evidence indicate the pivotal role of spinal glial cells in neuropathic pain. Spinal microglia rapidly respond to peripheral nerve injury (PNI) and become activated with changing expression of a variety of genes. The best known example is purinergic P2X4 receptors, an ATP-gated cation channel. The expression of P2X4 receptors is upregulated in spinal microglia after PNI, and inhibition of P2X4 activity suppresses neuropathic pain. Furthermore, interferon regulatory factor-8 (IRF8) and IRF5 are identified as microglial transcription factors whose expression is upregulated in spinal microglia after PNI, and the IRF8-IRF5 transcriptional cascade is the core process for shifting spinal microglia toward a state with high expression of P2X4 receptors. Astrocytes in the spinal cord show a delayed onset of activation and play an important role in the maintenance of neuropathic pain via the transcription factor STAT3 and the intracellular kinase JNK. These results obtained from glial cell research will advance our understanding of the development and maintenance of neuropathic pain and provide a new target for treating this pain.

Niacin, also known as vitamin B3, is required for the synthesis of coenzymes, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Niacin binds with G protein-coupled receptor (GPR) 109A on cutaneous Langerhans cells and causes vasodilation with flushing in head and neck area. Niacin deficiency due to excessive alcohol consumption, certain drugs or inadequate uptake in diet causes pellagra, a photosensitivity dermatitis. Recently several studies have revealed the mechanism of photosensitivity in niacin deficiency, which may pave a way for new therapeutic approaches. The expression level of prostaglandin E synthase (PTGES) is up-regulated in the skin of both pellagra patients and niacin deficient pellagra mouse models. In addition, pellagra is mediated through prostaglandin E₂-EP4 (PGE₂-EP4) signaling via reactive oxygen species (ROS) production in keratinocytes. In this article, we have reviewed the role of niacin in immunity and the mechanism of niacin deficiency-induced photosensitivity.

Ca2+ influx into neuron through L-type voltage-gated Ca2+ channels (VDCCs) plays an important role in psychostimulant-induced behaviroal and neuronal plasticity. On the other hand, Ca2+ release from ryanodine receptors in the endoplasmic reticulum is one mechanism altering the intracellular Ca2+ concentration. Little is known about functional relationship between psychological dependence due to drugs of abuse and L-type VDCCs or ryanodine receptors. In this paper, we review the roles and regulatory mechanisms of intracellular Ca2+ channels, especially L-type VDCCs and ryanodine receptors in brain of animals with drug dependence. Our recent study have reported that blockade of L-type VDCCs inhibits the development of rewarding effects on drugs of abuse (methamphetamine, cocaine and morphine), suggesting that up-regulation of L-type VDCCs (alpha 1C and alpha 1D subunits) occurs during the development of psychological dependence. Moreover, the critical expression of type-1 and -2 ryanodine receptors in the development of methamphetamine- and cocaine-induced rewarding effects are regulated by dopamine D1 receptors. These results suggest that changes in intracellular Ca2+ dynamics play an essential role in the development of drug dependence.

It has been reported that hyaluronan tetrasaccharide (HA4) has an ability to accelerate the healing process of spinal cord injuries and to mitigate the symptoms of experimental autoimmune encephalitis (EAE) in animal models. However, the precise mechanisms of the effect of HA4 are unclear. This study examined the in vitro effects of HA4 on the differentiation of PC12 cells and neuronal progenitor cells from mouse embryo. The effect of HA4 on the proliferation and differentiation of rat oligodendrocyte precursor cells was examined. HA4 upregulated neurite-outgrowth of cultured PC12 cells in the presence of Nerve Growth Factor (NGF), which induces neuronal differentiation in PC12. HA4 also upregulated beta III-tubulin positive cells and increased the expression of neurofilament M, two of the marker proteins of nerve differentiation. The activation was observed in an HA4 dose-dependent manner, and the effect was observed at levels as low as femtogram/L. Furthermore, the enhancement of neurite-outgrowth was not observed in the presence of hyaluronan disaccharide, hexasaccharide, 12 mer or high molecular weight hyaluronan. Upregulation of neurite-outgrowth was observed only in the presence of NGF. While HA4 accelerated neurite-outgrowth of primary mouse neural progenitor cells, it did not enhance the proliferation or differentiation of oligodendrocyte precursor cells derived from rat primary neuronal progenitor cells in culture. These results suggest that the effects of HA4 alleviating some neuronal diseases may be ascribable, at least in part, to its capacity to enhance differentiation of neurons.

Active studies of skin science have gradually clarified the underlying mechanisms of skin problems regarding skin beauty. The major skin problems are the alterations in appearance such as the hyperpigmentation and wrinkling caused by age. Those skin alterations are accelerated by solar light, particularly by ultraviolet rays, and it has been reported that reactive oxygen species (ROS) also involves in most of those processes. Thus, the reduction of oxidative stress induced by intracellular ROS is one approach to prevent and improve hyperpigmentation and wrinkling. Zn(2+) is well-known as an inducer of MT (metallothionein) and γGCS (γ-glutamyl cysteinyl synthetase: a rate-limiting enzyme of glutathione synthesis) via the up-regulation of their mRNAs through a metal transcription factor. The inductions of both MT and glutathione are expected to reduce oxidative stress due to the more effective scavenging of intracellular ROS. Several complexes of Zn(2+) and amino acids were synthesized and then evaluated for effects on MT synthesis in HaCaT keratinocytes. Among the complexes tested, we found a superior induction by a Zn(2+) glycine complex, Zn(Gly)(2). The anti-pigmentation and anti-wrinkling effects of Zn(Gly)(2) are introduced in this paper.

Pathophysiological process of ethanol physical dependence and its withdrawal syndrome is supposed to result from adaptive changes in a number of neurotransmission systems, and several reports have demonstrated functional relationship between behavioral responses and neurotransmission systems in ethanol-dependent and -withdrawn animals. However, the molecular mechanisms underlying behavioral responses observed in these animals are still controversial at present. Alterations of beta-adrenergic receptor (beta-AR) function in the brains of mice physically dependent on ethanol were examined because of few available data on functional changes of beta-ARs and its significance in ethanol withdrawal signs. The protein level of beta-ARs also significantly increased in the frontal cortex of mouse with ethanol physical dependence and conditions withdrawn from ethanol after chronic treatment with ethanol vapor for 9 days. Intracerebroventricular administration of nadolol, a non-selective antagonist for beta-ARs, immediately after discontinuation of ethanol inhalation clearly attenuated the expression of withdrawal signs. These findings suggests that ethanol physical dependence induces beta-AR up-regulation, that is their increased number and facilitation of beta-AR signaling, which may at least in part participates in expression of tonic-clonic convulsion as one of ethanol withdrawal signs. We further investigated relationship between actin of cytoskeleton and relapse of alcohol dependence after withdrawal from alcohol vapor inhalation for 9 days. After 3 days of withdrawal from alcohol vapor alcohol-induced place preference was enhanced, which was blocked by the treatment with an actin polymerization inhibitor, cytochalasin D, and anactin depolymerization inhibitor, phalloidin, during 3 days after ethanol withdrawal. These findings suggest that changes in actin turnover on withdrawal from alcohol vapor involve in sensitization of alcohol-induced place preference.

Excessive Ca(2+) elevation resulting from activation of NMDA and other Ca(2+) channels is thought to play a pivotal role in pathologic events following brain ischemia. The Ca(2+) elevation directly triggers necrotic or apoptotic cell death through activation of Ca(2+)/calmodulin (CaM)-dependent enzymes, including calcineurin (CaN). CaN, a Ca(2+)/CaM-dependent serine/threonine protein phosphatase, partly mediates apoptosis associated with neuronal death. In a mouse middle cerebral artery occlusion (MCAO) model, calpain, a Ca(2+)-dependent cysteine protease, converted CaN to the constitutively active form of 48 kDa in vivo. The calpain-induced CaN activation mediated delayed neuronal death through translocation of nuclear factor of activated T-cells (NFAT) and FKHR, a forkhead box class O family member (FOXO) into neuronal nuclei after brain ischemia. The FKHR activation occurred through decreased Akt activity with concomitant dephosphorylation by constitutively active CaN. Thereafter, FKHR formed a complex with CaN and in turn translocated into nuclei after brain ischemia. After nuclear translocation of NFAT and FKHR, the transcription factors stimulated expression of Fas-ligand by binding to its promoter regions. Taken together, constitutively active CaN mediates delayed neuronal death through Fas-ligand expression via up regulation of both NFAT and FKHR transcriptional activity in brain ischemia.

Mast cells originate from hematopoietic stem cells and undergo terminal differentiation in the tissues, in which they are ultimately resident. Heterogeneity of tissue mast cells is, therefore, one of the key concepts for a better understanding of immune modulation by mast cells. Since no appropriate culture model has been developed for tissue mature mast cells, it was difficult to investigate the tissue-specific functions of mast cells. We established a novel cutaneous mast cell model by modifying the previously reported co-culture system with fibroblastic cell line. This model shares many characteristics with cutaneous mast cells, such as staining properties, sensitivity to cationic secretagogues, and higher levels of granule histamine and proteases. We extracted the candidate genes that should regulate differentiation and functions of mast cells by analyses of the gene expression profiles during the co-culture period. We further investigated the functions of cluster of differentiation 44 (CD44), which is the primary receptor of hyaluronan in mast cells, since CD44 was up-regulated during the co-culture period. Fluorescence study revealed that mast cells expressing CD44 were bound to the extracellular matrix containing hyaluronan and lack of CD44 impaired proliferation of the co-cultured mast cells. In the CD44(-/-) mice, the number of cutaneous mast cells was significantly decreased. Reconstitution analyses with the mast cell deficient strain revealed that CD44 expressed in mast cells should be required in the proliferation in the cutaneous tissues. In the next phase of mast cell research, it might become increasingly important to focus on the heterogeneity of tissue mast cells.

Glial cells are abundant in the CNS and play diverse roles in the regulation of neuronal activity, vascular function, and gliotransmitter release, whereas pathologically activated glial cells have been reported to disturb brain function in conjunction with Ca(2+) signaling, however there is no enough explanation for a unique Ca(2+) entry. Transient receptor potential (TRP) superfamily comprises a group of non-selective cation channels that open in response to divergent stimuli in their environment. Although TRP channels are widely distributed in the mammalian brain, their roles remain to be elucidated. Here we provide an overview of the roles of TRP channels in pathophysiological processes, especially focusing on TRPC3 and TRPV4 channels in glial cells. Using rat cortical astrocytes, we found that TRPC3 was upregulated by thrombin via Ca(2+) signaling through TRPC3 itself. Thrombin also upregulated S100B, a marker of reactive astrocytes, and increased cell proliferation, both of which were inhibited by Ca(2+) signaling blockers and specific knockdown of TRPC3 using siRNA, suggesting that TRPC3 contributes to the pathological activation of astrocytes in part through a feed-forward upregulation of its own expression. Moreover, we found that TRPV4 stimulation by its agonist 4alpha-PDD suppressed LPS-induced microglial activation while TRPV4 mRNA was downregulated in LPS-treated cultured rat microglia. These results suggest that TRP channels play pivotal roles in the process of astrocytic and microglial activation.

Neuropathic pain is a debilitating pain that occurs after nerve injury and is generally resistant to currently available treatments including morphine. Such pain involves aberrant excitability in dorsal horn neurons after nerve injury. Emerging evidence indicate that the enhanced activity of dorsal horn neurons requires a communication with microglia. Results of our laboratory have shown that activating P2X4R upregulated in spinal microglia after nerve injury contributes to neuropathic pain through a release of BDNF from microglia, which is a crucial factor to signal to dorsal horn neurons to cause neuronal hyperexcitability. Activated spinal microglia also express P2Y12R, and P2Y12R-KO mice display impaired neuropathic pain. The mechanisms of microglia activation are unknown, but our recent study shows that interferon-gamma (IFN-gamma) can be an important factor that causes spinal microglia activation after nerve injury. IFN-beta upregulates P2X4R in microglia and causes P2X4R-dependent allodynia. These findings suggest that purinoceptors in spinal microglia is crucial for pathological intractable pain.

Insulin and insulin-like growth factor-I (IGF-I) are potent bone anabolic factors through the balance of distinct signals of the two adaptor molecules, insulin receptor substrate (IRS) -1 and IRS-2 : IRS-1 for maintenance of bone turnover by up-regulating anabolic and catabolic functions of osteoblasts, while IRS-2 for retaining the predominance of the anabolic function over the catabolic function.

Osteocytes form a network through processes and canaliculi throughout bone. Osteocytes have been reported to stimulate or inhibit bone formation and to inhibit bone resorption. As these functions have been estimated from the bone changes after osteocyte death, however, an inflammatory reaction in the microenvironment after necrosis has to be considered. The osteocyte network is thought to be a mechanosensor and mechanotransduction system due to its ideal anatomical feature. Further, osteocytes play an important role in phosphate metabolism, because osteocytes are important producers of FGF23, and FGF23 is upregulated in the osteocytes of Hyp mice and DMP1 knockout mice.

microRNAs (miRNAs) are non-coding RNAs, and are negatively regulating the gene functions through translational repression or digesting mRNAs by targeting 3'-UTR in messenger RNAs (mRNAs). To clarify roles of miRNAs in normal and pathological erythropoiesis, we analyzed the expression of miR-451, miR-155, miR-221 in normal human erythroid colony forming cells (ECFCs) and circulating red blood cells using quantitative real-time polymerase chain reaction. Remarkable down-regulation of miR-155 (about 200-fold) and up-regulation of miR-451 (270-fold) were observed during 12 days of ECFC cultures. miR-221 was down-regulated moderately (20-fold). Since mature erythroid cells expressed miR-451 at high levels, we analyzed the content of miR-451 in fractionated peripheral blood cells obtained from normal subjects (n= 3), and found that the expression level of miR-451 in red blood cells was about 10(4)-fold more than in granulocytes and mononuclear cells. Analysis of packed red cells from normal subjects (n=3) and patients with hematological disorders (n=16), showed that there was no significant difference of the expression levels of miR-451 between normal and patients. In conclusion, expression profiles of miR-155 and miR-451 are stage-specific during normal erythropoiesis in which miR-155 is down-regulated and miR-451 is up-regulated. A high expression of miR-451 is specific to red blood cells among circulating blood cells. These observations suggest that an analysis of microRNAs is a new relevant approach to understand underlying mechanisms of hematological disorders.

We have identified the vaspin gene(serpina12), which is up-regulated in visceral white adipose tissues (WATs) of Otsuka Long-Evans Tokushima fatty (OLETF) rat, an animal model of abdominal obesity and type 2 diabetes. Vaspin mRNA was barely detectable at 6 weeks of age, but was abundantly and exclusively expressed in visceral WATs at 30 weeks of age, when OLETF rats reach their peak body weight. However, vaspin mRNA decreased with worsening of diabetes and body weight loss. Vaspin mRNA increased with administration of thiazolidinediones, i.e. pioglitazone. Administration of recombinant vaspin into high fat high sucrose (HFHS) chow-induced obese ICR mice improved glucose tolerance and insulin sensitivity. Vaspin may be the compensatory molecule in the pathogenesis of metabolic syndrome and vaspin recombinant protein or vaspin-mimicking agents such as vaspin analogues, antibodies or small molecule agents would link to drug discovery and development.

Shortly after encountering the muscle surface, the motor axon releases agrin to the postsynaptic muscle membrane to regulate postsynaptic differentiation. Neural agrin activates postsynaptic muscle-specific kinase to induce aggregation of muscle nicotinic acetylcholine receptors (nAChR) expressed throughout the muscle surface into the subsynaptic area. Agrin also regulates the distribution of other synaptic proteins, including rapsyn, ErbB receptor. Rapsyn, 43-kd cytoplasmic protein, attaches to the beta subunit of synaptic nAChR and anchors it at the neuromuscular junction. Neureglin binds to ErbB receptors to activate a pathway that leads to enhance mature nAChR gene transcription in synaptic nuclei. While, a voltage change evoked by binding of acetylcholine to nAChR leads to down-regulate fetal nAChR expression in extrasynaptic nuclei. Denervation causes destruction of normal neuromuscular synapse and induces up-regulation of immature nAChR at extrajunctional sites. Importantly in clinical settings, the immature nAChR is resistant to non-depolarizing neuromuscular blocking agents and more sensitive to depolarizing muscle relaxants.

Epstein-Barr virus (EBV) is associated with the development of a variety of highly metastatic carcinomas, including nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1) is essential for B-cell transformation. In this study, we used two-dimensional differential gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) to study the mechanism behind tumor invasion and metastasis. Eight proteins, including Vimentin and Ezrin, were identified from the alteration of expressed proteins in HEK-293 cells responding to LMP1 gene transfection. Vimentin is a major protein of the mesenchymal intermediate filament, which maintains the cytoskeleton conformation. Ezrin is also an essential protein that links the cell membrane to the actin cytoskeleton. The up-regulation of Vimentin and Ezrin in the LMP1 gene-transfected cells suggests that EBV LMP1 is involved in the progression and metastasis of NPC.

Osteoprotegerine (OPG) acts as a decoy receptor for receptor activator of NF-kappaB (RANK) ligand (RANKL) to compete against RANK. OPG-deficient mice exhibit severe osteoporosis with accelerated bone remodeling. In human, several homozygous mutations in OPG gene were found in patients with juvenile Paget's disease. Missense mutations in cysteine residues in ligand binding domain, as well as complete deletion mutation, were associated with a severe phenotype characterized by enhanced bone remodeling and deformity. Serum RANKL level was elevated in OPG-deficient mice and a patient with complete deletion mutation. These results suggest that OPG represses the production of soluble form of RANKL. The elevated RANKL level in serum of OPG-deficient mice was not reduced by treatment with anti-resorbing agent, risedronate, but was upregulated by 1,25 (OH) (2)D(3) administration or ovariectomy.

Nonalcoholic steatohepatitis (NASH), which is considered the hepatic manifestation of the metabolic syndrome is an increasingly cause of chronic liver disease in Japan. NASH is finally lead to liver cirrhosis and hepatocellular carcinoma as viral hepatitis, therefore, medical treatment should be considered, when NASH occurs. Treatment of patients with metabolic syndrome has been focused on the management of associated conditions such as obesity, hyperlipidemia, hypertension and hyperinsulinemia. Insulin resistance, that could accelerate liver inflammation and fibrosis by up-regulation of TNFa seems to be most important factor in many cases of NASH. The insulin-sensitizing drugs, which were biguanides (metformin) and thiazolidinediones (pioglitazone) have been shown to correct not only insulin resistance but also steatosis and inflammation in the liver. Metformin and pioglitazone might be useful drugs against NASH, however further investigations were needed.

Runt-related transcription factor 2 (Runx2) is an essential transcription factor for osteoblast differentiation, and the expression is regulated by many factors including bone morphogenetic protein (BMP). Core binding factorbeta (Cbf beta) is required for DNA binding of Runx2 and Runx2-dependent transcription, and Cbf beta regulates Runx2 function isoform-dependently. Further, ability of Runx2 for transcriptional activation is regulated by many molecules. Phosphoinositide 3-kinase (PI3K)-Akt signaling enhances DNA binding of Runx2 and Runx2-dependent transcription, and Runx2 upregulates PI3K subunits (p85 and p110 beta) and Akt. Therefore, Runx2 and PI3K-Akt signaling are mutually dependent each other in the regulation of osteoblast and chondrocyte differentiation and their migration.

Indoleamine 2,3-dioxygenase (IDO), one of the enzymes of tryptophan catabolism, has been shown to play an essential role for successful pregnancy through the inhibition of allogenic fetus-induced T-cell proliferation, and interferon-gamma (IFN-gamma) induces the expression of IDO in CD14-positive (CD14(+)) cells. On the other hand, prolactin (PRL) is the hormone whose serum levels drastically elevate during pregnant period and is shown to play an important role in the early stages of pregnancy including implantation. However little is known about the physiological significance of the elevation of PRL from second trimester except for its fundamental role in lactation. Since receptors of PRL and IFN-gamma share their structure and the signal transduction pathway, we hypothesized the potential crosstalk between two substances. To test this idea, we examined the effect of PRL on IFN-gamma-induced IDO expression in CD14(+) cells. CD14(+) cells were prepared from peripheral blood of 12 healthy controls who had informed consent, and IDO expression and transcriptions were analyzed by flow cytometry and RT-PCR. The results showed that although PRL by itself had little effect on IDO expression, PRL significantly enhanced the IFN-gamma-induced IDO expression at comparable to those seen in a pregnant period. In contrast, no such effect was observed with PRL at lower concentrations comparable to those seen in a non-pregnant period. These findings suggest that PRL may contribute to the maintenance of pregnancy by augmenting IFN-gamma induction of IDO expression.

SUMO conjugating enzyme Ubc9 has been shown to upregulate GLUT4 in muscle cells although the mechanism of action is unknown. We investigated the physiological significance of Ubc9 in GLUT4 turnover and subcellular targeting by adenovirus vector-mediated overexpression and by siRNA-mediated gene silencing of Ubc9 in 3T3-L1 adipocytes. Overexpression of Ubc9 resulted in inhibition of GLUT4 degradation and promotion of its targeting to the non-endosomal, non-TGN GLUT4 storage vesicles (GSV), leading to an up-regulation of GLUT4 expression level and insulin-responsive glucose transport. While long-term insulin stimulation caused GLUT4 down-regulation by 40-50%, which was inhibited with lysosomal inhibitors and was associated with a selective reduction in GLUT4 in GSV, overexpression of Ubc9 antagonized these long-term effects of insulin. By contrast, Ubc9 gene silencing with siRNA caused a marked decrease in the GLUT4 level, whereas overexpression of the catalytically inactive mutant of Ubc9 increased GLUT4 and insulin-stimulated glucose transport to the level comparable to that with wild-type Ubc9. These results suggest that Ubc9 up-regulates GLUT4 by inhibition of lysosomal sorting and promotes GLUT4 targeting to GSV by a mechanism unrelated to its catalytic activity. Thus, Ubc9 plays an indispensable role in the expression and maintenance of the insulin-sensitive glucose transport system in adipocytes.

The Corticotropin-releasing hormone (CRH) is a key mediator in the stress response. Two CRH-receptor subtypes have been identified in the brain, CRH-receptor 1 (CRH-R1) and CRH-receptor 2, and stress responses are mediated by the CRH-R1. In this study we have examined the effect of chronic variable stress (CVS) on the CRH-R1 immunoreactivity in the hypothalamic and limbic brain regions of the rat. For CVS we selected the six stressors described in previous reports. Male Wistar rats were randomly exposed to one stressor per a day during 13 days and finally exposed to a novel stressor (footshock). There was a significant increase of the CRH-R1 immunoreactivity 24 hours after CVS in the bed nucleus of the stria terminalis (BNST) and basolateral amygdala (BLA), whereas a single footshock had no influence on the immunoreactivity in any of the regions. A novel stressor after CVS suppressed within 24 hours the CVS-induced increase in the CRH-R1 immuoreactivity in the BNST and BLA. Our results suggest that an up-regulation of the limbic CRH-R1 may contribute to stress sensitization (vulnerability) such as an anxiety, arousal and hypersympathetic autonomic symptoms under stressful situations.

In dialysis patients, deficiency of calcitriol down regulates vitamin D receptor (VDR) and decreases density of Ca sensing receptor, resulting right shift of set point of extra cellular Ca concentrations to release of parathyroid hormone. And then, renal hyperparathyroidism occurs and progresses. The purpose of pulse therapy with calcitriol or its analogue is elevating extra cellular calcitriol level up to supra physiological level, which up regulates VDR and shift the set point to left. The extra cellular calcitriol level after injection of calcitriol cannot compare with the level after oral administration of calcitriol at the same dose. The left shift of set point is investigated 4 weeks after intravenous pulse therapy with calcitriol or maxacalcitol, and degree of shift to the left depends on the single dose of these medicines. There is another advantage of intravenous pulse therapy, which takes no account of drug compliance or existence of malabsorption of lipid soluble vitamins.

The aim of this study was to define transcriptional changes that occur during dexamethasone-induced in vitro osteoblastic differentiation using human mesenchymal stem cells (hMSCs). Bone marrow derived hMSCs from three individual donors were grown in DMEM containing 10% fetal bovine serum and antibiotics (basal media, BM). At confluence (Day 0), cells were grown in BM or BM plus ascorbic acid, beta-glycerophosphate and dexamethasone (OS). At 0, 3, 7 and 14 days, total RNA was isolated from hMSCs, and 32ρ-labelled probes were synthesized. Atlas 1.2 gene arrays (Clontech) were hybridized with donor-specific cDNA probes. Acquired data were analyzed using GeneSpring Software (Silicon Genetics) enabling normalization and averaging of donor-specific data sets at each time point. Only 8, 31, and 27 genes were upregulated more than 3-fold at 3, 7 and 14 days, respectively. Over 50 genes were downregulated at each time point. Interestingly, the results of this study did not reflect commonly identified genes that are frequently considered in cellular differentiation along the osteoblastic lineage. Instead, some interesting genes were upregulated. Seven days exposure to OS media resulted in the relatively high induction of several genes, including growth factors and receptors which affect adipocyte, chondrocyte and osteoblast differentiation. IGF II and FGFR 3, FGFR 2 were upregulated in the middle of 14 days' induction. Examination of gene expression at day 14 also revealed a number of receptors (LEPR) and transcription factors (ROR alpha) were upregulated during 14 days. These could be of importance to the process of early stage differentiation.

CD80 and CD86, which are costimulatory molecules in T-cell activation, play important roles in the differentiation of Th1- or Th2-phenotypes. The results of blocking studies using neutralizing antibodies have suggested that CD80 and CD86 also play important roles in sensitization to cedar pollen antigen, but very few studies have examined the kinetics of CD80 and CD86 expression on antigen-presenting cells (APC). We studied the kinetics of CD80 and CD86 expression on APC after allergen-stimulation in cedar pollinosis subjects. A skin-prick test was performed in nine subjects with pollinosis and seven control subjects. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with Japanese cedar pollen extract. Zero to 8 days following in vitro stimulation, the expression of CD80 and CD86 in either CD14+ or CD19+ cells was analyzed by two-color flow cytometry. The expression of CD28, CTLA-4 and CD40L on CD4+ cells was analyzed by two-color flow cytometry after eliminating either CD14+ or CD19+ cells. After in vitro stimulation, the expression of both CD80 and CD86 was significantly upregulated in pollinosis subjects compared to control subjects. However, the difference was observed in the kinetics of CD80 and CD86 expression following allergen stimulation. The expression of CD86 was upregulated earlier than that of CD80 after in vitro stimulation. In the absence of CD19+ cells, the expression of CD28, CTLA-4, and CD40L in CD4+ cells was significantly lower than that in the absence of CD14+ cells. These results indicate that CD19+ cells of pollinosis subjects expressed higher CD80 and CD86 than that of control subjects, and that the kinetics of CD80 and CD86 expression following stimulation differed. In pollinosis subjects, CD19+ cells may thus function as APC in allergen-induced activation of PBMC.

Findings of chemokines and chemokine receptors in multiple sclerosis(MS) are reviewed. MS is a T-helper type 1 (Th1) dominant condition, and Th1-associated chemokine receptors(CCR5 and CXCR3) on CD4- and CD8-positive T cells and their ligands are upregulated in the CNS of the patients with active disease. Meanwhile, Th2-associated chemokine receptors(CCR3 and CCR4) on CD4- and CD8-positive T cells are suppressed during relapse. Their expressions are useful immunological measures of disease activity, clinical subtypes and therapy. CCR7 and the ligands are expressed in the CNS of MS and may be important for the recruitment of immune cells committed to immunological memory and antigen presentation.

The deposition of amyloid-beta peptide (Abeta) causes the long-term pathological cascade of Alzheimer's disease (AD). Neprilysin is a rate-limiting peptidase, which participates in Abeta degradation in brain. As demonstrated by reverse genetics, the disruption of neprilysin gene causes an elevation in endogenous Abeta levels in the mouse brain in a gene-dose-dependent manner. Therefore, a reduction of neprilysin activity will contribute to Abeta deposition and thus to AD development. Neprilysin is localized at presynapses and on axons, and its expression levels are decreased at the terminal zones and on axons of the lateral perforant pathway and the mossy fibers with aging in mice, suggesting that local concentrations of Abeta are likely to be elevated at the sites, which play crucial roles on certain forms of learning and memory and are highly vulnerable to AD. Overexpression of neprilysin decreased both extracellular and intracellular Abeta levels in primary cortical neurons. These results indicate that up-regulation of neprilysin activity would be a relevant strategy for therapy and prevention through reduction of the Abeta levels. Recently, we have found that a certain neuropeptide regulates the expression of neprilysin in primary neurons. Since a number of receptors for neuropeptides are G-protein-coupled receptors, we would control brain Abetalevels pharmacologically by the manipulation of neprilysin activity.

Antidepressants are divided into several groups such as monoamine oxidase inhibitors, serotonin reuptake inhibitors, noradrenaline reuptake inhibitors and alpha-adrenoceptor antagonists. Antidepressants show, however, a common clinical profile: their clinical actions occur after a chronic treatment. It is, therefore, suggested that antidepressants have a common pharmacological profile. We reviewed possible involvement of gamma-aminobutyric acid (GABA)B receptors in action of antidepressants. Our data in combination with previous ones from other laboratories suggest that antidepressants suppress the activity of presynaptic 5-hydroxytryptamine3 receptors on GABA neurons, resulting in the reduction of GABA release. It is likely that the decrease in GABA release attenuates GABAB receptor-mediated neurotransmission, thereby upregulating GABAB receptors.

Through disruption of the hsp47 gene in mice, we found that HSP47, a collagen-specific molecular chaperone residing in the endoplasmic reticulum, is essential for mouse development. Improper triple helix formation was observed in hsp47-null embryos, and no collagen fibrils in the mesenchyme or basement membranes between the mesenchyme and epithelial cell layers were seen in those mice, which resulted in embryonic lethality. Interestingly, constitutive expression of HSP47 is always correlated with that of collagens in various cells or tissues. HSP47 is markedly up-regulated during the progression of fibrosis in the liver, kidney, lung, and so on. A preliminary experiment showed that down-regulation of HSP47 caused the reduction in the progression of fibrosis by down-regulating the accumulation of collagens in the tissues, which suggests a novel strategy for the therapy of fibrotic diseases including liver cirrhosis.

In this study, we performed a global analysis of the differential gene expression of a gastric cancer cell line established from a primary main tumor (SNU-1) and that of other cell lines established from the metastasis to the peritoneal cavity (KATO-III, SNU-5, SNU-719, MKN45P, HS39). The application of a high-density cDNA microarray method made it possible to analyze the expression of approximately 21,168 genes. Our examinations of KATO-III, SNU-5, SNU-719, MKN45P, and HS39 showed that several genes were up-regulated in addition to expression of sequence tags (ESTs). The analysis revealed altered up-regulation of CD44 (cell adhesion), CEA, 14-3-3, Ubiquitin A and several kinds of ESTs in gastric cancer cells from malignant ascites. We then analyzed these gastric cancer cell lines with Northern blots and observed preferential up-regulation of these selected genes in cells prone to peritoneal dissemination. RT-PCR confirmed that several genes selected by DNA microarray were also overexpressed in clinical samples of malignant ascites. It is therefore considered that these genes may be related to the peritoneal dissemination of gastric cancers. The results of this global gene expression analysis of gastric cancer cells with peritoneal dissemination promise to provide new insights into the study of human gastric cancer peritoneal dissemination.

Shiga toxins(Stxs), which are produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae serotype I, induce proinflammatory cytokines including tumor necrosis factor-alpha, interleukin(IL)-1 beta, IL-6, interferon-gamma, and chemokines such as IL-8 in intestinal epithelial cells, vascular endothelial cells, and monocytes/macrophages in vitro and in kidneys and spleen in vivo. Cytokines induced by Stxs and lipopolysaccharide enhance the toxicity of Stxs via up-regulation of the expression of Gb3, a Stx receptor, and infiltration of neutrophils. Stxs bind to neutrophils and transmigrate across intestinal mucosa and are transported to the target organs through bloodstreams. Stxs induce cytokines in vascular endothelial cells and peripheral blood monocytes and may injure organ tissues, finally resulting in hemolytic uremic syndrome and encephalopathia.