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BACKGROUND Patients with major depressive disorder (MDD) frequently retain cognitive disturbances after recovery from mood symptoms. We investigated the relationship between early response of mood symptoms and/or remission, and residual cognitive disturbances after 6 months of antidepressant treatment. METHODS 518 patients with MDD were followed up for 6 months after antidepressant treatment initiation (first-line or switch from a previous drug). Subjective and objective cognitive disturbances were assessed by the Perceived Deficits Questionnaire - Depression (PDQ-D) and digit symbol substitution test (DSST), respectively. Depressive symptoms, as well as remission and early response to treatment, were assessed using the Montgomery-Asberg Depression Rating Scale (MADRS). Multivariable linear and logistic regression models were used to adjust for confounders. RESULTS Early response of depressive mood (≥50% reduction in MADRS score at month 1) was related with fewer residual subjective cognitive symptoms, as evaluated by the PDQ-D at month 6 (p

Amyloid-β peptide (Aβ), which is generated by the β- and γ-secretase-mediated proteolysis of β-amyloid precursor protein (APP), plays an important role in the pathogenesis of Alzheimer's disease (AD). We recently reported that prostaglandin E(2) (PGE(2) ) stimulates the production of Aβ through both EP(2) and EP(4) receptors and that activation of the EP(4) receptor stimulates Aβ production through endocytosis and activation of γ-secretase. We here found that transgenic mice expressing mutant APP (APP23) mice showed a greater or lesser apparent cognitive deficit when they were crossed with mice lacking EP(2) or EP(4) receptors, respectively. Mice lacking the EP(4) receptor also displayed lower levels of Aβ plaque deposition and less neuronal and synaptic loss than control mice. Oral administration of a specific EP(4) receptor antagonist, AE3-208 to APP23 mice, improved their cognitive performance, as well as decreasing brain levels of Aβ and suppressing endocytosis and activation of γ-secretase. Taken together, these results suggest that inhibition of the EP(4) receptor improves the cognitive function of APP23 mice by suppressing Aβ production and reducing neuronal and synaptic loss. We therefore propose that EP(4) receptor antagonists, such as AE3-208, could be therapeutically beneficial for the prevention and treatment of AD.

Measurement of multichannel continuous-wave near-infrared spectroscopy (CW-NIRS) is dependent on the modified Beer-Lambert law, which includes optical path length (PL) as an essential parameter. PLs are known to differ across different head regions and between individuals, but the distribution of PLs for the whole head has not been evaluated so far. Thus, using time-resolved near-infrared spectroscopy (TR-NIRS), we measured the optical characteristics, including PL, scattering coefficients (μls), and absorption coefficients (μa) at three wavelengths (760, 800, and 830 nm). We then constructed maps of these parameters on the surface of the subject′s head. While the PLs in nearby channels are similar, they differ depending on the regions of the head. The PLs in the region above the Sylvian fissure tended to be shorter than those in the other regions at all of the wavelengths. The difference in the distribution of PLs may be attributed to differences in tissue absorption and scattering properties. The current study suggests the importance of considering PL differences in interpreting functional data obtained by CW-NIRS.

Skin hyperpigmentation disorders due to abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and cosmetic problem. UV irradiation stimulates melanin production in melanocytes by increasing intracellular cAMP. Expression of heat shock proteins (HSPs), especially HSP70, is induced by various stressors, including UV irradiation, to provide cellular resistance to such stressors. In this study we examined the effect of expression of HSP70 on melanin production both in vitro and in vivo. 3-Isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, stimulated melanin production in cultured mouse melanoma cells, and this stimulation was suppressed in cells overexpressing HSP70. IBMX-dependent transcriptional activation of the tyrosinase gene was also suppressed in HSP70-overexpressing cells. Expression of microphthalmia-associated transcription factor (MITF), which positively regulates transcription of the tyrosinase gene, was up-regulated by IBMX; however, this up-regulation was not suppressed in HSP70-overexpressing cells. On the other hand, immunoprecipitation and immunostaining analyses revealed a physical interaction between and co-localization of MITF and HSP70, respectively. Furthermore, the transcription of tyrosinase gene in nuclear extract was inhibited by HSP70. In vivo, UV irradiation of wild-type mice increased the amount of melanin in the basal layer of the epidermis, and this increase was suppressed in transgenic mice expressing HSP70. This study provides the first evidence of an inhibitory effect of HSP70 on melanin production both in vitro and in vivo. This effect seems to be mediated by modulation of MITF activity through a direct interaction between HSP70 and MITF.

Irradiation with UV light, especially UVB, causes epidermal damage via the induction of apoptosis, inflammatory responses, and DNA damage. Various stressors, including UV light, induce heat shock proteins (HSPs) and the induction, particularly that of HSP70, provides cellular resistance to such stressors. The anti-inflammatory activity of HSP70, such as its inhibition of nuclear factor kappa B (NF-kappaB), was recently revealed. These in vitro results suggest that HSP70 protects against UVB-induced epidermal damage. Here we tested this idea by using transgenic mice expressing HSP70 and cultured keratinocytes. Irradiation of wild-type mice with UVB caused epidermal damage such as induction of apoptosis, which was suppressed in transgenic mice expressing HSP70. UVB-induced apoptosis in cultured keratinocytes was suppressed by overexpression of HSP70. Irradiation of wild-type mice with UVB decreased the cutaneous level of IkappaB-alpha (an inhibitor of NF-kappaB) and increased the infiltration of leukocytes and levels of pro-inflammatory cytokines and chemokines in the epidermis. These inflammatory responses were suppressed in transgenic mice expressing HSP70. In vitro, the overexpression of HSP70 suppressed the expression of pro-inflammatory cytokines and chemokines and increased the level of IkappaB-alpha in keratinocytes irradiated with UVB. UVB induced an increase in cutaneous levels of cyclobutane pyrimidine dimers and 8-hydroxy-2'-deoxyguanosine, both of which were suppressed in transgenic mice expressing HSP70. This study provides genetic evidence that HSP70 protects the epidermis from UVB-induced radiation damage. The findings here also suggest that the protective action of HSP70 is mediated by anti-apoptotic, anti-inflammatory, and anti-DNA damage effects.

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

Alteration in the expression of claudins, consisting of tight junctions (TJs), has been reported in various clinically isolated tumors. Claudins play an important role not only in the intercellular barrier function of TJs but also in migration and invasiveness of cancer cells. However, the use of different types of cells and different claudins in these studies has complicated the picture. In this study, we systematically examined the effect of claudin (claudin-1, -2, -3, -4 and -15) overexpression on the paracellular permeability, migration and invasiveness of Caco-2 colonic cancer cells. Overexpression of claudin-4 or claudin-2 increased or decreased, respectively, paracellular permeability. Overexpression of claudin-4 specifically stimulated the invasive activity of the Caco-2 cells. Furthermore, activation of matrix metalloproteinase (MMP)-2 and MMP-9 were observed in the claudin-4-overexpressing cells, suggesting that the invasive activity was stimulated through an increase in MMP activity. Overexpression of claudin-2 or claudin-3 and -4 stimulated or inhibited, respectively, the migration activity of the Caco-2 cells. Immunostaining analysis revealed that each of the overexpressed claudins localized at TJs under the conditions used to evaluate paracellular permeability. In contrast, they localized mainly in intracellular compartments under experimental conditions designed to assess cell invasion and migration. Overall, the results of this study show that the effect exerted by the claudins on the intercellular barrier function of TJs, as well as on cell migration and invasive activity, differs depending on the particular claudin species. Furthermore, the subcellular localization of the claudins varies according to the culture conditions.

Amyloid-beta peptides (Abeta), generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE(2) receptors (EP(1-4)), EP(4) receptors alone or EP(2) and EP(4) receptors together are responsible for this PGE(2)-stimulated production of Abeta in HEK293 or SH-SY5Y cells, respectively. An EP(4) receptor antagonist suppressed the PGE(2)-stimulated production of Abeta in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Abeta, demonstrating that increases in the cellular level of cAMP are responsible for the PGE(2)-stimulated production of Abeta. Immunoblotting experiments and direct measurement of gamma-secretase activity suggested that PGE(2)-stimulated production of Abeta is mediated by activation ofgamma-secretase but not of beta-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Abeta in the brain, when they were crossed with mice lacking either EP(2) or EP(4) receptors, suggesting that PGE(2)-mediated activation of EP(2) and EP(4) receptors is involved in the production of Abeta in vivo and in the pathogenesis of AD.

Abeta (amyloid-beta peptides) generated by proteolysis of APP (beta-amyloid precursor protein), play an important role in the pathogenesis of AD (Alzheimer's disease). ER (endoplasmic reticulum) chaperones, such as GRP78 (glucose-regulated protein 78), make a major contribution to protein quality control in the ER. In the present study, we examined the effect of overexpression of various ER chaperones on the production of Abeta in cultured cells, which produce a mutant type of APP (APPsw). Overexpression of GRP78 or inhibition of its basal expression, decreased and increased respectively the level of Abeta40 and Abeta42 in conditioned medium. Co-expression of GRP78's co-chaperones ERdj3 or ERdj4 stimulated this inhibitory effect of GRP78. In the case of the other ER chaperones, overexpression of some (150 kDa oxygen-regulated protein and calnexin) but not others (GRP94 and calreticulin) suppressed the production of Abeta. These results indicate that certain ER chaperones are effective suppressors of Abeta production and that non-toxic inducers of ER chaperones may be therapeutically beneficial for AD treatment. GRP78 was co-immunoprecipitated with APP and overexpression of GRP78 inhibited the maturation of APP, suggesting that GRP78 binds directly to APP and inhibits its maturation, resulting in suppression of the proteolysis of APP. On the other hand, overproduction of APPsw or addition of synthetic Abeta42 caused up-regulation of the mRNA of various ER chaperones in cells. Furthermore, in the cortex and hippocampus of transgenic mice expressing APPsw, the mRNA of some ER chaperones was up-regulated in comparison with wild-type mice. We consider that this up-regulation is a cellular protective response against Abeta.

Pancreatic cancer is one of the most difficult malignancies to treat owing to the rapid acquisition of resistance to chemotherapy. Gemcitabine, a first-line treatment for pancreatic cancer, prolongs patient survival by several months, and combination treatment with gemcitabine and other anti-cancer drugs in the clinic do not show any significant effects on overall survival. Thus, identification of a drug that resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine and a better understanding of the molecular mechanisms of gemcitabine resistance are critical to develop new therapeutic options for pancreatic cancer. Here, we report that zidovudine resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine as shown by screening a compound library, including clinical medicine, using gemcitabine-resistant cells. In analyzing the molecular mechanisms of zidovudine effects, we found that the epithelial-to-mesenchymal transition (EMT)-like phenotype and downregulation of human equilibrative nucleoside transporter 1 (hENT1) are essential for the acquisition of gemcitabine resistance, and zidovudine restored these changes. The chemical biology investigations also revealed that activation of the Akt-GSK3β-Snail1 pathway in resistant cells is a key signaling event for gemcitabine resistance, and zidovudine resensitized resistant cells to gemcitabine by inhibiting this activated pathway. Moreover, our in vivo study demonstrated that co-administration of zidovudine and gemcitabine strongly suppressed the formation of tumors by gemcitabine-resistant pancreatic cancer and prevented gemcitabine-sensitive pancreatic tumors from acquiring gemcitabine-resistant properties, inducing an EMT-like phenotype and downregulating hENT1 expression. These results suggested that co-treatment with zidovudine and gemcitabine may become a novel therapeutic strategy for pancreatic cancer by inhibiting chemoresistance-specific signaling.

Gastric mucosal cell death by non-steroidal anti-inflammatory drugs (NSAIDs) is suggested to be involved in NSAID-induced gastric lesions. Therefore, cellular factors that suppress this cell death are important for protection of the gastric mucosa from NSAIDs. Heme oxygenase-1 (HO-1) is up-regulated by various stressors and protects cells against stressors. Here, we have examined up-regulation of HO-1 by NSAIDs and the contribution of HO-1 to the protection of gastric mucosal cells against NSAIDs both in vitro and in vivo. In cultured gastric mucosal cells, all NSAIDs tested up-regulated HO-1. In rats, orally administered indomethacin up-regulated HO-1, induced apoptosis, and produced lesions at gastric mucosa. An inhibitor of HO-stimulated NSAID-induced apoptosis in vitro and in vivo and also stimulated NSAID-produced gastric lesions, suggesting that NSAID-induced up-regulation of HO-1 protects the gastric mucosa from NSAID-induced gastric lesions by inhibiting NSAID-induced apoptosis. Indomethacin activated the HO-1 promoter and caused nuclear accumulation of NF-E2-related factor 2 (Nrf2), a transcription factor for the HO-1 gene. Examination of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and experiments with its inhibitor strongly suggest that the nuclear accumulation of Nrf2 and resulting up-regulation of HO-1 by NSAIDs is mediated through NSAID-dependent activation (phosphorylation) of p38 MAPK. This is the first report showing the protective role of HO-1 against irritant-induced gastric lesions.

Background and Purpose— To evaluate the mechanisms of failure of blood oxygenation level–dependent (BOLD) imaging in stroke, we compared the evoked cerebral blood oxygenation (CBO) responses and activation volumes (AVs) of BOLD functional MRI (fMRI) in chronic stroke patients with moderate and severe cerebral ischemia. Methods— We measured the evoked CBO responses in the primary sensorimotor cortex (PSMC) by means of near-infrared spectroscopy during contralateral motor tasks. We compared the AV of BOLD-functional MRI in the PSMC on the nonlesion and lesion sides. Single-photon emission computed tomography was used to classify ischemic status as moderate (slight reduction of regional cerebral blood flow and cerebrovascular reserve capacity [CVRC]) or severe (marked reduction of regional cerebral blood flow and CVRC; ie, misery perfusion). Results— In age-matched controls, deoxyhemoglobin concentration decreased with concomitant increases in oxyhemoglobin and total hemoglobin concentrations during activation. The PSMC on the nonlesion side exhibited a normal CBO response pattern. On the lesion side, moderate cerebral ischemia did not affect the CBO response pattern, but severe cerebral ischemia caused an increase of deoxyhemoglobin during the task, associated with increases of oxyhemoglobin and total hemoglobin. Moderate cerebral ischemia induced only a slight reduction of the AV on the lesion side; however, severe cerebral ischemia markedly reduced the AV on the lesion side. The BOLD signal did not change in some areas of the PSMC on the lesion side in severe cerebral ischemia, whereas it tended to decrease in other areas during the tasks. Conclusions— Misery perfusion caused a marked reduction of the AV on BOLD imaging, associated with an increase of deoxyhemoglobin concentration during activation. BOLD-fMRI investigations of stroke patients should be performed while giving consideration to baseline circulatory status. Functional near-infrared spectroscopy could be an alternative means to assess the CVRC.

We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells.

Prostaglandin E(2) (PGE(2)) has a strong protective effect on the gastric mucosa in vivo; however, the molecular mechanism of a direct cytoprotective effect of PGE(2) on gastric mucosal cells has yet to be elucidated. Although we reported previously that PGE(2) inhibited gastric irritant-induced apoptotic DNA fragmentation in primary cultures of guinea pig gastric mucosal cells, we show here that PGE(2) inhibits the ethanol-dependent release of cytochrome c from mitochondria. Of the four main subtypes of PGE(2) receptors, we also demonstrated, using subtype-specific agonists, that EP(2) and EP(4) receptors are involved in the PGE(2)-mediated protection of gastric mucosal cells from ethanol-induced apoptosis. Activation of EP(2) and EP(4) receptors is coupled with an increase in cAMP, for which a cAMP analogue was found here to inhibit the ethanol-induced apoptosis. The increase in cAMP is known to activate both protein kinase A (PKA) and phosphatidylinositol 3-kinase pathways. An inhibitor of PKA but not of phosphatidylinositol 3-kinase blocked the PGE(2)-mediated protection of cells from ethanol-induced apoptosis, suggesting that a PKA pathway is mainly responsible for the PGE(2)-mediated inhibition of apoptosis. Based on these results, we considered that PGE(2) inhibited gastric irritant-induced apoptosis in gastric mucosal cells via induction of an increase in cAMP and activation of PKA, and that this effect was involved in the PGE(2)-mediated protection of the gastric mucosa from gastric irritants in vivo.

A novel fabrication approach for electrochemical sensing of nicotinamide adenine dinucleotide (NADH) using neutral red (NR) functinalized carbon nanotube/plasma-polymerized film (PPF) composite electrode is reported. The configuration of sensing electrode was NR-functionalized CNTs sandwiched between two acetonitrile PPFs on sputtered gold thin film. The NR as an electron transfer mediator shuttles the electron from CNT to gold electrode. Due to the synergistic effect between NR and CNT, the resulting electrode showed the lower detection potential and the larger sensitivity (current) than that with NR or CNT alone. As a result, the electrode showed the selectivity against the interference ascorbic acid.

Amyloid-β peptide (Aβ), especially its oligomeric form, is believed to play an important role in the pathogenesis of Alzheimer's disease (AD). To this end, the binding of Aβ oligomer to cellular prion protein (PrP(C)) plays an important role in synaptic dysfunction in a mouse model of AD. Here, we have screened for compounds that inhibit Aβ oligomer binding to PrP(C) from medicines already used clinically (Mizushima Approved Medicine Library 1), and identified dextran sulfate sodium (DSS) as a candidate. In a cell-free assay, DSS inhibited Aβ oligomer binding to PrP(C) but not to ephrin receptor B2, another endogenous receptor for Aβ oligomers, suggesting that the drug's action is specific to the binding of Aβ oligomer to PrP(C) . Dextran on the other hand did not affect this binding. DSS also suppressed Aβ oligomer binding to cells expressing PrP(C) but not to control cells. Furthermore, while incubation of mouse hippocampal slices with Aβ oligomers inhibited the induction of long-term potentiation, simultaneous treatment with DSS restored the long-term potentiation. As DSS has already been approved for use in patients with hypertriglyceridemia, and its safety in humans has been confirmed, we propose further analysis of this drug as a candidate for AD treatment. Amyloid-β peptide (Aβ) oligomer-binding to cellular prion protein (PrP(C) ) is important in synaptic dysfunction in Alzheimer's disease (AD). We found here that dextran sulfate sodium (DSS) inhibits Aβ oligomer binding to PrP(C) . Simultaneous treatment of hippocampal slices with DSS restored long-term potentiation (LTP) in the presence of Aβ oligomers. Since DSS has already been approved for clinical use, we propose this drug is a candidate drug for AD treatment.

Background The expression of heat shock proteins (HSPs), particularly HSP70, is receiving considerable attention in the field of cosmetics, particularly given our recent report that ultraviolet-induced melanin production, skin damage and wrinkle formation were all suppressed in transgenic mice expressing HSP70. Objective In the present study, we searched for HSP70-inducers from a library of herbal extracts that have already been approved as quasi-pharmaceutical products in Japan. We selected an ethanol extract of Arnica montana ( A. montana ), based on its high HSP70-inducing activity and low cytotoxicity. Methods Cell viability was determined by MTT method and expression of HPS70 was monitored by immunoblotting analysis. Results From the extract, we purified and identified eight sesquiterpene lactones (AM1–8) as HSP70-inducers, among which AM-2 (helenalin 2-methylbutyrate) was selected due to its good HSP70-inducing properties and low cytotoxicity. Treatment of cultured mouse melanoma cells with AM-2 or A. montana extract up-regulated the expression of HSP70 in a dose-dependent manner. This treatment also activated heat shock factor-1, a transcription factor for hsp genes. Furthermore, pre-treatment of cells with AM-2 or A. montana extract decreased melanin production and expression and activity of tyrosinase. Conclusion These results suggest that AM-2 and A. montana extract could be beneficial for use in hypopigmenting cosmetics as a consequence of their stimulatory effects on HSP70 expression.

Skin hyperpigmentation disorders as a result of abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and a cosmetic problem. This melanin production is mediated by tyrosinase whose expression is positively regulated by microphthalmia-associated transcription factor (MITF). We recently found that expression of heat shock protein 70 (HSP70) inhibits melanin production. In this study, we searched for HSP70 inducers from Chinese herbs and selected an ethanol extract of Eupatorium lindleyanum (E. lindleyanum). Not only melanin production but also the activity and expression of tyrosinase were significantly suppressed in cells treated with E. lindleyanum extract as well as in HSP70-overexpressing cells. The expression of MITF was clearly suppressed in cells treated with E. lindleyanum extract but not in HSP70-overexpressing cells. These results suggest that E. lindleyanum extract suppresses the expression of tyrosinase and melanin production through both HSP70-dependent and HSP70-independent mechanisms.

UV-induced wrinkle formation owing to the degeneration of the extracellular matrix (ECM) is a major dermatological problem in which abnormal activation of matrix metalloproteinases (MMPs) and elastases have important roles. Heat shock protein 70 (HSP70) has cytoprotective and anti-inflammatory activities. In this study, we examined the effect of HSP70 expression on UV-induced wrinkle formation. Mild heat treatment (exposure to heated water at 42 °C) of the dorsal skin of hairless mice induced the expression of HSP70. The long-term repeated exposure to UV induced epidermal hyperplasia, decreased skin elasticity, degeneration of ECM, and wrinkle formation, which could be suppressed in mice concomitantly subjected to this heat treatment. The UV-induced epidermal hyperplasia, decreased skin elasticity, and degeneration of ECM were less apparent in transgenic mice expressing HSP70 than in wild-type mice. UV-induced fibroblast cell death, infiltration of inflammatory cells, and activation of MMPs and elastase in the skin were also suppressed in the transgenic mice. This study provides evidence for an inhibitory effect of HSP70 on UV-induced wrinkle formation. The results suggest that this effect is mediated by various properties of HSP70, including its cytoprotective and anti-inflammatory activities. We propose that HSP70 inducers used in a clinical context could prove beneficial for the prevention of UV-induced wrinkle formation.

Amyloid-β peptide (Aβ) plays an important role in the pathogenesis of Alzheimer's disease (AD). Aβ is generated by proteolysis of β-amyloid precursor protein (APP) and is cleared by enzyme-mediated degradation and phagocytosis by microglia and astrocytes. Some cytokines, such as TGF-β1, stimulate this phagocytosis. In contrast, cellular upregulation of HSP70 expression provides cytoprotection against Aβ. HSP70 activity in relation to inhibition of Aβ oligomerization and stimulation of Aβ phagocytosis has also been reported. Although thesein vitroresults suggest that stimulating the expression of HSP70 could prove effective in the treatment of AD, there is a lack ofin vivoevidence supporting this notion. In this study, we address this issue, using transgenic mice expressing HSP70 and/or a mutant form of APP (APPsw). Transgenic mice expressing APPsw showed less of an apparent cognitive deficit when they were crossed with transgenic mice expressing HSP70. Transgenic mice expressing HSP70 also displayed lower levels of Aβ, Aβ plaque deposition, and neuronal and synaptic loss than control mice. Immunoblotting experiments and direct measurement of β- and γ-secretase activity suggested that overexpression of HSP70 does not affect the production Aβ. In contrast, HSP70 overexpression did lead to upregulation of the expression of Aβ-degrading enzyme and TGF-β1 bothin vivoandin vitro. These results suggest that overexpression of HSP70 in mice suppresses not only the pathological but also the functional phenotypes of AD. This study provides the firstin vivoevidence confirming the potential therapeutic benefit of HSP70 for the prevention or treatment of AD.

Both the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, and infection with Helicobacter pylori are major causes of gastric ulcers. Although some clinical studies suggest that infection with H. pylori increases the risk of developing NSAID-induced gastric lesions, the molecular mechanism governing this effect is unknown. We recently found that in cultured gastric cells, expression of endoplasmic reticulum (ER) chaperones (such as 150-kDa oxygen-regulated protein (ORP150) and glucose-regulated protein 78 (GRP78)) is induced by NSAIDs and confers protection against NSAID-induced apoptosis, which is important in the development of NSAID-induced gastric lesions. In this study we have found that co-culture of gastric cells with H. pylori suppresses the expression of ER chaperones. This suppression was regulated at the level of transcription and accompanied by a reduction in the level of activating transcription factor 6 (ATF6), one of the transcription factors for ER chaperone genes. In vivo, inoculation of mice with H. pylori suppressed the expression of ER chaperones at gastric mucosa both with and without administration of indomethacin. Inoculation with H. pylori also stimulated formation of indomethacin-induced gastric lesions and mucosal cell death. In addition, we found that heterozygous ORP150-deficient mice are sensitive to the development of indomethacin-induced gastric lesions and mucosal cell death. The results of this study suggest that H. pylori exacerbates NSAID-induced gastric lesions through suppression of expression of ER chaperones, which stimulates NSAID-induced mucosal cell death.

Drug-induced interstitial lung disease (ILD), particularly pulmonary fibrosis, is a serious clinical concern and myofibroblasts have been suggested to have a major role, with it recently being revealed that some of these myofibroblasts are derived from lung epithelial cells through epithelial-mesenchymal transition (EMT). In this study, we examined the EMT-inducing abilities of drugs known to induce ILD clinically. EMT-like phenotypes were induced by A771726, an active metabolite of leflunomide having an inhibitory effect on dihydroorotate dehydrogenase (DHODH). Smad-interacting protein 1 (a transcription factor regulating EMT) and the Notch-signaling pathway but not transforming growth factor-β was shown to be involved in A771726-induced EMT-like phenotypes. When the cultures were supplemented with exogenous uridine, the A771726-induced EMT-like phenotypes and activation of the Notch-signaling pathway disappeared. Similarly, an A771726 analog without inhibitory activity on DHODH produced no induction, suggesting that this process is mediated through the inhibition of DHODH. In vivo, administration of leflunomide stimulated bleomycin-induced EMT-like phenomenon in pulmonary tissue, and exacerbated bleomycin-induced pulmonary fibrosis, both of which were suppressed by coadministration of uridine. Taken together, these findings suggest that leflunomide-dependent exacerbation of bleomycin-induced pulmonary fibrosis is mediated by stimulation of EMT of lung epithelial cells, providing the first evidence that drug-induced pulmonary fibrosis involves EMT of these cells.

Amyloid-beta (Abeta) peptides, generated by the proteolysis of beta-amyloid precursor protein by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta through EP(2) and EP(4) receptors, and here we have examined the molecular mechanism. Activation of EP(2) and EP(4) receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP(2), but not EP(4), receptor-mediated stimulation of the Abeta production. In contrast, inhibitors of endocytosis suppressed EP(4), but not EP(2), receptor-mediated stimulation. Activation of gamma-secretase was observed with the activation of EP(4) receptors but not EP(2) receptors. PGE(2)-dependent internalization of the EP(4) receptor was observed, and cells expressing a mutant EP(4) receptor lacking the internalization activity did not exhibit PGE(2)-stimulated production of Abeta. A physical interaction between the EP(4) receptor and PS-1, a catalytic subunit of gamma-secretases, was revealed by immunoprecipitation assays. PGE(2)-induced internalization of PS-1 and co-localization of EP(4), PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP(4) receptor null mice. These results suggest that PGE(2)-stimulated production of Abeta involves EP(4) receptor-mediated endocytosis of PS-1 followed by activation of the gamma-secretase, as well as EP(2) receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.

Epidemiological studies have revealed that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer. Various mechanisms, including induction of apoptosis and inhibition of the growth and invasion of cancer cells, have been implicated in this anti-tumorigenic activity. In this study we focused on S100P, which is known to be overexpressed in clinically isolated tumors and which functions through both intracellular and extracellular mechanisms. We showed the up-regulation of S100P expression in human gastric carcinoma cells treated with various NSAIDs, including celecoxib. The celecoxib-mediated up-regulation of S100P was suppressed by the transfection of cells with small interfering RNA for activating transcription factor 4 (ATF4), a transcription factor involved in the endoplasmic reticulum stress response. Furthermore, deletion of ATF4 binding consensus sequence located in the promoter of the S100P gene resulted in inhibition of celecoxibmediated transcriptional activation of the gene. These results suggest that celecoxib up-regulates the expression of S100P through an ATF4-mediated endoplasmic reticulum stress response. Celecoxib inhibited the growth and induced apoptosis, and these actions could be either suppressed or stimulated by transfection of cells with S100P overexpression plasmid or small interfering RNA, respectively. Celecoxib also inhibited the invasive activity of the cells. Cromolyn, which inhibits the binding of S100P to its receptor, enhanced the celecoxib-mediated inhibition of cell invasion and growth but did not affect apoptosis. These results suggest that S100P affects apoptosis, cell growth, and invasion through either an intracellular or an extracellular mechanism and that the up-regulation of S100P expression by NSAIDs reduces their anti-tumorigenic activity.

Non-steroidal anti-inflammatory drugs (NSAIDs) show chemopreventive effects; however, the precise molecular mechanism of these effects is still unclear. On the other hand, the expression of proteins that form tight junctions (TJs) (such as claudins) in clinically isolated tumors is frequently altered relative to normal tissue. We previously reported that NSAIDs upregulate the expression of claudin-4 and that this upregulation contributes to NSAID-dependent inhibition of both migration activity and anchorage-independent growth of cancer cells. In the current study, we have systematically examined the effects of various NSAIDs on the expression of various TJ proteins and have found that NSAIDs specifically and drastically inhibit the expression of claudin-2. Overexpression or suppression of claudin-2 expression caused stimulation or inhibition, respectively, of the invasion and migration activity of cancer cells. Furthermore, NSAIDs inhibited the invasion and migration activity of cancer cells and this inhibition was suppressed by overexpression of claudin-2. In contrast, neither cell growth nor apoptosis induced by lack of anchorage of cancer cells was affected by overexpression or suppression of expression of claudin-2. These results suggest that inhibition of claudin-2 expression by NSAIDs contributes to NSAID-dependent inhibition of invasion of cancer cells in vitro and that it may be involved in the chemopreventive effects of NSAIDs by inhibiting metastasis in vivo.

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in cancer cells and this effect is involved in their antitumor activity. We recently demonstrated that NSAIDs upregulate GRP78, an endoplasmic reticulum (ER) chaperone, in gastric mucosal cells in primary culture. In the present study, induction of ER chaperones by NSAIDs and the effect of those chaperones on NSAID-induced apoptosis were examined in human gastric carcinoma cells. Celecoxib, an NSAID, upregulated ER chaperones (GRP78 and its cochaperones ERdj3 and ERdj4) but also C/EBP homologous transcription factor (CHOP), a transcription factor involved in apoptosis. Celecoxib also upregulated GRP78 in xenograft tumors, accompanying with the suppression of tumor growth in nude mice. Celecoxib caused phosphorylation of eukaryotic translation initiation factor 2 kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha) and production of activating transcription factor (ATF)4 mRNA. Suppression of ATF4 expression by small interfering RNA (siRNA) partially inhibited the celecoxib-dependent upregulation of GRP78. Celecoxib increased the intracellular Ca2+ concentration, while 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, an intracellular Ca2+ chelator, inhibited the upregulation of GRP78 and ATF4. These results suggest that the Ca2+-dependent activation of the PERK-eIF2alpha-ATF4 pathway is involved in the upregulation of ER chaperones by celecoxib. Overexpression of GRP78 partially suppressed the apoptosis and induction of CHOP in the presence of celecoxib and this suppression was stimulated by coexpression of either ERdj3 or ERdj4. On the other hand, suppression of GRP78 expression by siRNA drastically stimulated cellular apoptosis and production of CHOP in the presence of celecoxib. These results show that upregulation of ER chaperones by celecoxib protects cancer cells from celecoxib-induced apoptosis, thus may decrease the potential antitumor activity of celecoxib.

Orally ingested non-steroidal anti-inflammatory drugs (NSAIDs) and acid in gastric secretions are gastric irritants that co-exist at the surface of the gastric mucosa. Here, we examined the individual and combined effects of indomethacin, a typical NSAID, and hydrochloric acid on cell death in primary cultures of guinea pig gastric mucosal cells. Indomethacin alone (at concentrations less than 200 microM) did not induce apoptosis; however, hydrochloric acid-induced apoptosis was stimulated in the presence of indomethacin (50-200 microM). Isobologram analysis confirmed the presence of a cytotoxic synergy between indomethacin and hydrochloric acid. The synergistic response between the two gastric irritants was also observed for necrosis. Given that the IC50 value of indomethacin for inhibition of prostaglandin synthesis is about 5 nM, the synergistic response between indomethacin and hydrochloric acid appears to be independent of the inhibition of cyclooxygenase activity by indomethacin.

Apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved not only in the production of NSAID-induced gastric lesions but also in the antitumor activity of these drugs. The endoplasmic reticulum (ER) stress response is a cellular mechanism that aids in protecting the ER against ER stressors and is involved in ER stressor-induced apoptosis. Here, we examine the relationship between this response and NSAID-induced apoptosis in cultured guinea-pig gastric mucosal cells. Exposure of cells to indomethacin, a commonly used NSAID, induced GRP78 as well as CHOP, a transcription factor involved in apoptosis. Three factors that positively regulate CHOP expression (ATF6, ATF4 and XBP-1) were activated and/or induced by indomethacin. NSAIDs other than indomethacin (diclofenac, ibuprofen and celecoxib) also induced CHOP. Monitoring of the transcriptional activities of ATF6 and CHOP by luciferase assay revealed that both were stimulated in the presence of indomethacin. Furthermore, indomethacin-induced apoptosis was suppressed in cultured guinea-pig gastric mucosal cells by expression of the dominant-negative form of CHOP, or in peritoneal macrophages from CHOP-deficient mice. These results suggest that ER stress response-related proteins, particularly CHOP, are involved in NSAID-induced apoptosis.

Purpose Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. Methods Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. Results Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. Conclusions Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.

Amyloid-beta peptide (A beta) plays an important role in the pathogenesis of Alzheimer's disease (AD). A beta is generated by the secretase-mediated proteolysis of beta-amyloid precursor protein (APP), and cleared by enzyme-mediated degradation and phagocytosis. Transforming growth factor (TGF)-beta 1 stimulates this phagocytosis. We recently reported that the APP23 mouse model for AD showed fewer AD-related phenotypes when these animals were crossed with transgenic mice expressing heat shock protein (HSP) 70. We here examined the effect of geranylgeranylacetone, an inducer of HSP70 expression, on the AD-related phenotypes. Repeated oral administration of geranylgeranylacetone to APP23 mice for 9 months not only improved cognitive function but also decreased levels of A beta, A beta plaque deposition and synaptic loss. The treatment also up-regulated the expression of an A beta-degrading enzyme and TGF-beta 1 but did not affect the maturation of APP and secretase activities. These outcomes were similar to those observed in APP23 mice genetically modified to overexpress HSP70. Although the repeated oral administration of geranylgeranylacetone did not increase the level of HSP70 in the brain, a single oral administration of geranylgeranylacetone significantly increased the level of HSP70 when A beta was concomitantly injected directly into the hippocampus. Since geranylgeranylacetone has already been approved for use as an anti-ulcer drug and its safety in humans has been confirmed, we propose that this drug be considered as a candidate drug for the prevention of AD.

Drug-induced interstitial lung disease (ILD), particularly pulmonary fibrosis, is of serious clinical concern. Gefitinib, a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR), is beneficial as a drug for treating non-small cell lung cancer; however, this drug induces ILD and the molecular mechanisms underpinning this condition remain unclear. We recently reported that expression of heat shock protein 70 (HSP70) protects against bleomycin-induced pulmonary fibrosis, an animal model of pulmonary fibrosis. In this study, we have examined the effects of drugs known to induce ILD clinically on the expression of HSP70 in cultured lung epithelial cells and have found that gefitinib has a suppressive effect. Results of a luciferase reporter assay, pulse-labelling analysis of protein and experiments using an inhibitor of translation or transcription suggest that gefitinib suppresses the expression of HSP70 at the level of translation. Furthermore, the results of experiments with siRNA for Dicer1, an enzyme responsible for synthesis of microRNA, and real-time RT-PCR analysis suggest that some microRNAs are involved in the gefitinib-induced translational inhibition of HSP70. Mutations in the EGFR affect the concentration of gefitinib required for suppressing the expression of HSP70. These results suggest that gefitinib suppresses the translation of HSP70 through an EGFR- and microRNA-mediated mechanism. In vivo, while oral administration of gefitinib suppressed the pulmonary expression of HSP70 and exacerbated bleomycin-induced pulmonary fibrosis in wild-type mice, these effects were not as distinct in transgenic mice expressing HSP70. Furthermore, oral co-administration of geranylgeranylacetone (GGA), an inducer of HSP70, suppressed gefitinib-induced exacerbation of bleomycin-induced pulmonary fibrosis. Taken together, these findings suggest that gefitinib-induced exacerbation of bleomycin-induced pulmonary fibrosis is mediated by suppression of pulmonary expression of HSP70 and that an inducer of HSP70 expression, such as GGA, may be therapeutically beneficial for the treatment of gefitinib-induced pulmonary fibrosis.

Blood-oxygen-level-dependent contrast functional magnetic resonance imaging (BOLD-fMRI) has been used to perform functional imaging in brain disorders such as stroke and brain tumors. However, recent studies have revealed that BOLD-fMRI does not image activation areas correctly in such patients. To clarify the characteristics of the evoked cerebral blood oxygenation (CBO) changes occurring in stroke and brain tumors, we have been comparing near-infrared spectroscopy (NIRS) and BOLD-fMRI recording during functional brain activation in these patients. We review our recent studies and related functional imaging studies on the brain disorders. In the primary sensorimotor cortex (PSMC) on the nonlesion side, the motor task consistently caused a decrease of deoxyhemoglobin (deoxy-Hb) with increases of oxyhemoglobin (oxy-Hb) and total hemoglobin (t-Hb), which is consistent with the evoked CBO response observed in normal adults. BOLD-fMRI demonstrated robust activation areas on the nonlesion side. In stroke patients, severe cerebral ischemia (i.e., misery perfusion) caused an increase of deoxy-Hb during the task, associated with increases of oxy-Hb and t-Hb, in the PSMC on the lesion side. In addition, the activation volume of BOLD-fMRI was significantly reduced on the lesion side. The BOLD signal did not change in some areas of the PSMC on the lesion side, but it tended to decrease in other areas during the tasks. In brain tumors, BOLD-fMRI clearly demonstrated activation areas in the PSMC on the lesion side in patients who displayed a normal evoked CBO response. However, the activation volume on the lesion side was significantly reduced in patients who exhibited an increase of deoxy-Hb during the task. In both stroke and brain tumors, false-negative activations (i.e., marked reductions of activation volumes) in BOLD imaging were associated with increases of deoxy-Hb, which could cause a reduction in BOLD signal. BOLD-fMRI investigations of patients with brain disorders should be performed while giving consideration to atypical evoked CBO changes.