Effects of diallyl disulfide administration on insulin resistance in high-fat diet-fed mice.

OBJECTIVES: Diallyl disulfide (DADS) is a natural organosulfur compound found in garlic and related plants with various pharmacologic effects. However, whether DADS improves obesity-induced insulin resistance (IR) and its underlying mechanisms remain unclear. The aim of this study was to investigate the effects of DADS on systemic IR in high-fat diet-induced obese mice. METHODS: To induce obesity, 8-wk-old male C57BL/6J mice were fed a high-fat diet (60% fat/kcal). The mice were assigned to three weight-matched groups: control (CON, n = 8), low-dose DADS (DADS-L, n = 8), and high-dose DADS (DADS-H, n = 9). The treated mice were orally administered DADS (25 or 100 mg/kg) 5 d/wk for 8 wk. At 15 wk of age, an intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed. Twenty-four hours after the final administration of DADS, epididymal fat and the liver were sampled after a 5-h fast. RESULTS: DADS administration significantly attenuated body and fat weight gains during the experimental period. Additionally, systemic IR, as evaluated by ITT, was significantly improved by DADS administration in a dose-dependent manner. High-dose DADS administration significantly decreased liver triacylglycerol levels. Moreover, high-dose DADS administration decreased the c-Jun N-terminal kinase (JNK) phosphorylation and significantly increased heat shock protein 72 expression in the liver. CONCLUSIONS: The results of this study suggested that DADS administration alleviated systemic IR in obese mice. This may be associated with decreased hepatic fat accumulation and a heat shock protein 72-mediated decrease in JNK activity in the liver.

Methylmercury-induced reactive oxygen species-dependent and independent dysregulation of MAP kinase-related signaling pathway in cultured normal rat cerebellar astrocytes.

Methylmercury (MeHg), a global environmental pollutant, could seriously damage the central nervous system (CNS) and cause neurological disorders such as cerebellar symptoms. Although numerous studies have revealed detailed toxicity mechanisms of MeHg in neurons, toxicity in astrocytes is barely known. Here, we tried to shed light on the toxicity mechanisms of MeHg exposure in cultured normal rat cerebellar astrocytes (NRA), focusing on the involvement of reactive oxygen species (ROS) in MeHg toxicity by assessing the effects of major antioxidants Trolox, a free-radical scavenger, N-acetyl-L-cysteine (NAC), a potent thiol-containing antioxidant, and glutathione (GSH), an endogenous thiol-containing antioxidant. Exposure to MeHg at just approximately 2 µM for 96 h increased cell viability, which was accompanied by the increase in intracellular ROS level and at ≥ 5 µM induced significant cell death and lowered ROS level. Trolox and NAC suppressed 2 µM MeHg-induced increases in cell viability and ROS level corresponding to control, although GSH with 2 µM MeHg induced significant cell death and ROS increase. On the contrary, against 4 µM MeHg-induced cell loss and ROS decrease, NAC inhibited both cell loss and ROS decrease, Trolox inhibited cell loss and further enhanced ROS decrease, and GSH moderately inhibited cell loss and increased ROS level above the control level. MeHg-induced oxidative stress was suggested by increases in the protein expression levels of heme oxygenase-1 (HO-1), Hsp70, and Nrf2, except for the decrease in SOD-1 and no change in catalase. Furthermore, MeHg exposure dose-dependently induced increases in the phosphorylation of MAP kinases (ERK1/2, p38MAPK, and SAPK/JNK) and phosphorylation and/or expression levels of transcription factors (CREB, c-Jun, and c-Fos) in NRA. NAC successfully suppressed 2 µM MeHg-induced alterations in all of the above-mentioned MeHg-responsive factors, whereas Trolox suppressed some MeHg-responsive factors but failed to suppress MeHg-induced increases in the protein expression levels of HO-1 and Hsp70 and increase in p38MAPK phosphorylation. Protein expression analyses in NRA exposed to 2 µM MeHg and GSH were excluded because of devastating cell death. These results suggested that MeHg could induce aberrant NRA activation, and ROS must be substantially involved in the toxicity mechanism of MeHg in NRA; however, other factors should be assumed.

PlexinA1-deficient mice exhibit decreased cell density and augmented oxidative stress in parvalbumin-expressing interneurons in the medial prefrontal cortex.

PlexinA1 (PlxnA1) is a transmembrane receptor for semaphorins (Semas), a large family of axonal guidance cues vital during neural development. PlxnA1 is expressed in embryonic interneurons, and PlxnA1 deletion in mice leads to less interneurons in the developing cortex. In addition, PlxnA1 has been identified as a schizophrenia susceptibility gene. In our previous study, PlxnA1 knockout (KO) mice under a BALB/cAJ genetic background exhibited significantly increased self-grooming and reduced prepulse inhibition, a reliable phenotype for investigating the neurobiology of schizophrenia. However, the mechanism underlying the abnormal behavior of PlxnA1 KO mice remains unclear. We first confirmed PlxnA1 mRNA expression in parvalbumin-expressing interneurons (PV cells) in the medial prefrontal cortex (mPFC) of adult mice. Immunohistochemical analysis (IHC) showed significantly decreased densities of both GABAergic neurons and PV cells in the mPFC of PlxnA1 KO mice compared with wild type mice (WT). PV cells were found to express molecule interacting with CasL 1 (MICAL1), an effector involved in Sema-Plxn signaling for axon guidance, suggesting MICAL1 and PlxnA1 co-expression in PV cells. Furthermore, IHC analysis of 8-oxo-dG, an oxidative stress marker, revealed significantly increased oxidative stress in PlxnA1-deficient PV cells compared with WT. Thus, increased oxidative stress and decreased PV cell density in the mPFC may determine the onset of PlxnA1 KO mice's abnormal behavior. Accordingly, deficient PlxnA1-mediated signaling may increase oxidative stress in PV cells, thereby disrupting PV-cell networks in the mPFC and causing abnormal behavior related to neuropsychiatric diseases.

Exercise training improves obesity-induced inflammatory signaling in rat brown adipose tissue.

Chronic inflammation is considered as an etiology of obesity and type 2 diabetes. Brown adipose tissue (BAT) of obese animals shows increased inflammation. Regular exercise has anti-inflammatory effects; however, the effects of exercise training on BAT inflammation in obese animals remain unclear. Thus, this study aimed to investigate the effects of exercise training on inflammation-related signaling in the BAT of obese and diabetic rats. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an obese/diabetic rodent model, were randomly divided into either sedentary (n = 11) or exercise training (n = 8) groups. Long-Evans Tokushima Otsuka (LETO; n = 9) rats were used as the nondiabetic sedentary controls. Exercise training using a treadmill was conducted 4 days per week for 20 weeks, starting at 5 weeks old. As a result, exercise training attenuated the phosphorylation levels of p65 and mitogen-activated protein kinases in the BAT of OLETF rats, concurrently with the improvement of obesity and systemic glucose tolerance. Moreover, exercise training decreased oxidative stress and increased the antioxidant and anti-inflammatory protein levels in the BAT. Conversely, exercise training did not alter the expression levels of uncoupling protein-1 and oxidative phosphorylation-related proteins in the BAT, which were lower in the OLETF rats than the LETO rats. In conclusion, our data suggest that exercise training prevents the activation of inflammatory signaling in the BAT of obese/diabetic rats.

Combined effects of exercise training and D-allulose intake on endurance capacity in mice.

This study investigated the combined effects of exercise training and D-allulose intake on endurance capacity in mice. Male C57BL/6J mice were fed either a control diet (Con) or a 3% D-allulose diet (Allu) and further divided into the sedentary (Sed) or exercise training (Ex) groups (Con-Sed, Con-Ex, Allu-Sed, Allu-Ex, respectively; n = 6-7/group). The mice in the Ex groups were trained on a motor-driven treadmill 5 days/week for 4 weeks (15-18 m/min, 60 min). After the exercise training period, all mice underwent an exhaustive running test to assess their endurance capacity. At 48 h after the running test, the mice in the Ex groups were subjected to run at 18 m/min for 60 min again. Then the gastrocnemius muscle and liver were sampled immediately after the exercise bout. The running time until exhaustion tended to be higher in the Allu-Ex than in the Con-Ex group (p = 0.08). The muscle glycogen content was significantly lower in the Con-Ex than in the Con-Sed group and was significantly higher in the Allu-Ex than in the Con-Ex group (p < 0.05). Moreover, exercise training increased the phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) in the muscle and liver. The phosphorylation levels of acetyl coenzyme A carboxylase (ACC), a downstream of AMPK, in the muscle and liver were significantly higher in the Allu-Ex than in the Con-Sed group (p < 0.05), suggesting that the combination of exercise training and D-allulose might have activated the AMPK-ACC signaling pathway, which is associated with fatty acid oxidation in the muscle and liver. Taken together, our data suggested the combination of exercise training and D-allulose intake as an effective strategy to upregulate endurance capacity in mice. This may be associated with sparing glycogen content and enhancing activation of AMPK-ACC signaling in the skeletal muscle.

Diphenylarsinic acid induced activation of MAP kinases, transcription factors, and oxidative stress-responsive factors and hypersecretion of cytokines in cultured normal human cerebellar astrocytes.

Diphenylarsinic acid (DPAA) is a non-natural pentavalent organic arsenic and was detected in well water in Kamisu, Ibaraki, Japan in 2003. Individuals that had consumed this arsenic-contaminated water developed cerebellar symptoms such as myoclonus. We previously revealed that DPAA exposure in rats in vitro and in vivo specifically affected astrocytes rather than neurons among cerebellar cells. Here, we evaluated adverse effects of DPAA in cultured normal human cerebellar astrocytes (NHA), which were compared with those in normal rat cerebellar astrocytes (NRA) exposed to DPAA at 10 µM for 96 h, focusing on aberrant activation of astrocytes; increase in cell viability, activation of MAP kinases (ERK1/2, p38MAPK, and SAPK/JNK) and transcription factors (CREB, c-Jun, and c-Fos), upregulation of oxidative stress-responsive factors (Nrf2, HO-1, and Hsp70), and also hypersecretion of brain cytokines (MCP-1, adrenomedullin, FGF-2, CXCL1, and IL-6) as reported in NRA. While DPAA exposure at 10 µM for 96 h had little effect on NHA, a higher concentration (50 µM for 96 h) and longer exposure (10 µM for 288 h) induced similar aberrant activation. Moreover, exposure to DPAA at 50 µM for 96 h or 10 µM for 288 h in NHA induced hypersecretion of cytokines induced in DPAA-exposed NRA (MCP-1, adrenomedullin, FGF-2, CXCL1, and IL-6), and IL-8 besides into culture medium. These results suggested that aberrantly activated human astrocytes by DPAA exposure might play a pivotal role in the pathogenesis of cerebellar symptoms, affecting adjacent neurons, microglia, brain blood vessels, or astrocyte itself through these brain cytokines in human.

PlexinA1 deficiency in BALB/cAJ mice leads to excessive self-grooming and reduced prepulse inhibition.

PlexinA1 (PlxnA1) is a transmembrane receptor for semaphorins, a large family of proteins that act as axonal guidance cues during nervous system development. However, there are limited studies on PlxnA1 function in neurobehavior. The present study examined if PlxnA1 deficiency leads to behavioral abnormalities in BALB/cAJ mice. PlxnA1 knockout (KO) mice were generated by homologous recombination and compared to wild type (WT) littermates on a comprehensive battery of behavioral tests, including open field assessment of spontaneous ambulation, state anxiety, and grooming, home cage grooming, the wire hang test of muscle strength, motor coordination on the rotarod task, working memory on the Y maze alternation task, cued and contextual fear conditioning, anxiety on the elevated plus maze, sociability to intruders, and sensory processing as measured by prepulse inhibition (PPI). Measures of motor performance, working memory, fear memory, and sociability did not differ significantly between genotypes, while PlxnA1 KO mice displayed excessive self-grooming, impaired PPI, and slightly lower anxiety. These results suggest a crucial role for PlxnA1 in the development and function of brain regions controlling self-grooming and sensory gating. PlxnA1 KO mice may be a valuable model to investigate the repetitive behaviors and information processing deficits characteristic of many neurodevelopmental and psychiatric disorders.

PlexinA1 is crucial for the midline crossing of callosal axons during corpus callosum development in BALB/cAJ mice.

The corpus callosum (CC) is the biggest commissure that links cerebral hemispheres. Guidepost structures develop in the cortical midline during CC development and express axon guidance molecules that instruct neurons regarding the proper direction of axonal elongation toward and across the cortical midline. Neuropilin-1 (Npn1), a high affinity receptor for class 3 semaphorins (Sema3s) localized on cingulate pioneering axons, plays a crucial role in axon guidance to the midline through interactions with Sema3s. However, it remains unclear which type of Plexin is a component of Sema3 holoreceptors with Npn1 during the guidance of cingulate pioneering axons. To address the role of PlexinA1 in CC development, we examined with immunohistochemistry the localization of PlexinA1, Npn1, and Sema3s using embryonic brains from wild-type (WT) and PlexinA1-deficient (PlexinA1 knock-out (KO)) mice with a BALB/cAJ background. The immunohistochemistry confirmed the expression of PlexinA1 in callosal axons derived from the cingulate and neocortex of the WT mice on embryonic day 17.5 (E17.5) but not in the PlexinA1 KO mice. To examine the role of PlexinA1 in the navigation of callosal axons, the extension of callosal axons toward and across the midline was traced in brains of WT and PlexinA1 KO mice at E17.5. As a result, callosal axons in the PlexinA1 KO brains had a significantly lower incidence of midline crossing at E17.5 compared with the WT brains. To further examine the role of PlexinA1 in CC development, the CC phenotype was examined in PlexinA1 KO mice at postnatal day 0.5 (P0.5). Most of the PlexinA1 KO mice at P0.5 showed agenesis of the CC. These results indicate the crucial involvement of PlexinA1 in the midline crossing of callosal axons during CC development in BALB/cAJ mice.

Dysregulation of MAP Kinase Signaling Pathways Including p38MAPK, SAPK/JNK, and ERK1/2 in Cultured Rat Cerebellar Astrocytes Exposed to Diphenylarsinic Acid.

Diphenylarsinic acid (DPAA) was a major compound found in the arsenic poisoning incident that occurred in Kamisu, Ibaraki, Japan in 2003. People exposed to DPAA via contaminated well water suffered from several neurological disorders, including cerebellar symptoms. We previously reported that DPAA induces cellular activation in cultured rat cerebellar astrocytes, dose-dependent promotion of cell growth (low DPAA), cell death (high DPAA), and increased phosphorylation of mitogen-activated protein (MAP) kinases (p38MAPK, SAPK/JNK, and ERK1/2). Moreover, DPAA induces up-regulation of oxidative stress-counteracting proteins, activation of CREB phosphorylation, increased protein expression of c-Jun and c-Fos, and aberrant secretion of brain-active cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Here, we explored the role of MAP kinases in DPAA-induced activation of astrocytes using specific MAP kinase signaling inhibitors [SB203580 (p38MAPK), SP600125 (SAPK/JNK), SCH772984 (ERK1/2), and U0126 (MEK1/2, a kinase for ERK1/2)]. DPAA-induced activation of MAP kinases had little contribution to DPAA-induced cell growth and death. On the other hand, a power relationship among MAP kinases was also observed, in which p38MAPK suppressed DPAA-induced SAPK/JNK and ERK1/2 activation, whereas ERK1/2 and MEK1/2 facilitated p38MAPK and SAPK/JNK activation. In addition, SAPK/JNK had minimal effects on the activation of other MAP kinases. DPAA-induced activation of transcription factors and secretion of brain-active cytokines were submissively but intricately dominated by MAP kinases. Collectively, our results indicate that DPAA-induced activation of MAP kinases is neither a cell growth-promoting response nor a cytoprotective one but leads to transcriptional disruption and aberrant secretion of brain-active cytokines in cerebellar astrocytes.

Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow.

Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild­type (WT) mice. Administration of ß­estradiol to infant Sema4D­deficient (Sema4D­/­) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same ß­estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin­B1, was examined as well as the level of apoptosis in the vaginal epithelia of five­week­old WT and Sema4D­/­ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin­B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase­3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five­week­old Sema4D­/­ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D­/­ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis­inducing activity of Sema4D. The experimental reduction of plexin­B1 expression in vaginal epithelial cells demonstrated the integral role of plexin­B1 in Sema4D­induced apoptotic cell death. These results suggest a non­redundant role of Sema4D in the postnatal tissue remodeling process in five­week­old BALB/c mice, which involves the induction of vaginal epithelial cell apoptosis through Sema4D binding to plexin­B1.

Plasma dehydroepiandrosterone sulfate levels in patients with major depressive disorder correlate with remission during treatment with antidepressants.

OBJECTIVE: We attempted to investigate whether dehydroepiandrosterone sulfate (DHEA-S) levels are associated with remission of major depressive disorder by assessing scores on the 17-Item Structured Interview Guide for the Hamilton Depression before and after antidepressant treatment. METHODS: Plasma DHEA-S levels in 24 patients diagnosed with major depressive disorder on the basis of Diagnostic and Statistical Manual of Mental Disorders, fourth edition (text revision) before and after antidepressant treatment, and 24 healthy, gender-matched, and age-matched controls were measured using a radioimmunoassay kit. RESULTS: Plasma DHEA-S levels in patients were significantly higher than those in healthy controls. In patients who achieved remission after antidepressant treatment, plasma DHEA-S levels significantly declined compared with the levels before treatment. A significant correlation was observed between changes in DHEA-S levels and Absence of Depressive and Anxious Mood scores, which are calculated from the 2-Item Structured Interview Guide for the Hamilton Depression rating as follows: severity of depressive mood and anxiety in patients before and after antidepressant treatment. CONCLUSIONS: These findings suggest that plasma DHEA-S levels can be used as a putative indicator of the state of remission in patients with major depressive disorder. Copyright © 2014 John Wiley & Sons, Ltd.

Estrogen-dependent proteolytic cleavage of semaphorin 4D and plexin-B1 enhances semaphorin 4D-induced apoptosis during postnatal vaginal remodeling in pubescent mice.

Around the fifth week after birth, the vaginal cavity in female mouse pups opens to the overlaying skin. This postnatal tissue remodeling of the genital tract occurs during puberty, and it largely depends upon hormonally induced apoptosis that mainly occurs in the epithelium at the lower part of the mouse vaginal cavity. Previously, we showed that most BALB/c mice lacking the class IV Semaphorin (Sema4D) develop imperforate vagina and hydrometrocolpos; therefore, we reasoned that the absence of Sema4D-induced apoptosis in vaginal epithelial cells may cause the imperforate vagina. Sema4D signals via the Plexin-B1 receptor; nevertheless detailed mechanisms mediating this hormonally triggered apoptosis are not fully documented. To investigate the estrogen-dependent control of Sema4D signaling during the apoptosis responsible for mouse vaginal opening, we examined structural and functional modulation of Sema4D, Plexin-B1, and signaling molecules by analyzing both wild-type and Sema4D-/- mice with or without ovariectomy. Both the release of soluble Sema4D and the conversion of Plexin-B1 by proteolytic processing in vaginal tissue peaked 5 weeks after birth of wild-type BALB/c mice at the time of vaginal opening. Estrogen supplementation of ovariectomized wild-type mice revealed that both the release of soluble Sema4D and the conversion of Plexin-B1 into an active form were estrogen-dependent and concordant with apoptosis. Estrogen supplementation of ovariectomized Sema4D-/- mice did not induce massive vaginal apoptosis in 5-week-old mice; therefore, Sema4D may be an essential apoptosis-inducing ligand that acts downstream of estrogen action in vaginal epithelium during this postnatal tissue remodeling. Analysis of ovariectomized mice also indicated that Sema4D contributed to estrogen-dependent dephosphorylation of Akt and ERK at the time of vaginal opening. Based on our results, we propose that apoptosis in vaginal epithelium during postnatal vaginal opening is induced by enhanced Sema4D signaling that is caused by estrogen-dependent structural changes of Sema4D and Plexin-B1.

Semaphorin 3A-Plexin-A1 signaling through ERK activation is crucial for Toll-like receptor-induced NO production in BV-2 microglial cells.

Semaphorin family members have been identified as axonal guidance molecules that mediate the directional determination for axonal elongation during neuronal development. Several semaphorins have been shown to play crucial roles for various immune response phases. In a previous study using knockout mice, we suggested that Plexin-A1, a Semaphorin 3A (Sema3A) receptor, is involved in the increased production of inflammatory factors such as interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the murine microglial response to lipopolysaccharide (LPS). In that study, Sema3A-Plexin-A1 signaling was also shown to have crosstalk with Toll-like receptor 4 (TLR4) signaling to increase nitric oxide production, although the specific intracellular signaling molecule involved in the NO increase was not identified. By investigating the role of Plexin-A1 in the response of the BV-2 microglial cell line to LPS, in the present study novel findings regarding the influence of Plexin-A1 activation on TLR4 signaling in microglial cells were investigated. First, the production of inflammatory markers such as inducible nitric oxide synthase (iNOS), IL-1ß and TNF-α in the response to TLR4 stimulation was significantly decreased in BV-2 cells with the knockdown of Plexin-A1. Accordingly, Plexin-A1 was required for the enhanced production of inflammatory factors induced by LPS in BV-2 microglial cells. Second, Plexin-A1 signaling in BV-2 cells showed crosstalk with the LPS-induced TLR4 pathway through activation of nuclear factor-κB (NF-κB) and extracellular signal­regulated kinase (ERK). Third, LPS-induced NO production in BV-2 cells was intensified by Sema3A-Plexin-A1 signaling in an ERK1/2 activation-dependent manner. This finding suggested the crucial role of Plexin-A1 signaling through ERK activation in TLR4 activation-induced NO production in BV-2 microglial cells. These results therefore suggest that Plexin-A1 and Sema3A are possible new targets for treating LPS-induced encephalopathy and neuroinflammation-related mental disorders.

Plexin-A1 is required for Toll-like receptor-mediated microglial activation in the development of lipopolysaccharide-induced encephalopathy.

Recent investigations have suggested that semaphorins, which are known repulsive axon guidance molecules, may play a crucial role in maintaining brain homeostasis by regulating microglial activity. Sema3A, secreted in higher amounts from injured neurons, is considered to suppress excessive inflammatory responses by inducing microglial apoptosis through its binding to Plexin-A1 receptors on activated microglia. To clarify the in vivo role of Plexin-A1-mediated signaling in lipopolysaccharide (LPS)-induced injury in mouse brain, we examined the neuroinflammatory changes initiated by LPS administration to the cerebral ventricles of wild-type (WT) and Plexin-A1-deficient (-/-) mice. WT mice administered LPS exhibited a significantly higher expression of COX-2, iNOS, IL-1ß and TNF-α in the hippocampus, and a significantly greater ventricular enlargement and intracerebral infiltration of leukocytes, as compared with the saline-treated group. By contrast, Plexin-A1-/- mice administered LPS did not exhibit a significantly increased expression of COX-2, iNOS, IL-1ß or TNF-α in the hippocampus as compared with the saline-treated group. Plexin-A1-/- mice administered LPS did not show significant increases in ventricle size or infiltration of leukocytes into the brain, as compared with the saline-treated group. In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control. These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia. Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

Impaired death-associated protein kinase-mediated survival signals in 5-fluorouracil-resistant human endometrial adenocarcinoma cells.

A recent study showed that both 5-fluorouracil (5FU)-stimulated apoptosis and Fas-mediated apoptosis in human endometrial adenocarcinoma cells are enhanced by targeted knockdown of endogenous death-associated protein kinase (DAPK) with DAPK small-interfering RNAs. Therefore, we investigated the DAPK survival signals in three 5FU-resistant subclones. DAPK knockdown did not enhance 5FU-stimulated or Fas-mediated apoptosis in any of the three 5FU-resistant subclones, but the subclones acquired resistance to VP16-stimulated cell death that was DAPK-independent. Semi-quantitative flow cytometric analyses showed that there was no differential expression in nine cell surface antigens, including Fas, and six intracellular molecules, including DAPK, that may regulate cell death or survival between the parent cells and 5FU-resistant cells. DAPK mRNA and protein were expressed in the 5FU-resistant subclones at similar levels to the parent cells. These results indicate that acquisition of 5FU-resistance may be accompanied by impairment of common apoptotic signals regulating both DAPK-dependent and DAPK-independent pathways.

Demethylation restores SN38 sensitivity in cells with acquired resistance to SN38 derived from human cervical squamous cancer cells.

Using seven monoclonal SN38-resistant subclones established from ME180 human cervical squamous cell carcinoma cells, we examined the demethylation effects of 5-aza-2'-deoxycytidine (5-aza-CdR) on the SN38-sensitivity of the cells as well as the expression of death-associated protein kinase (DAPK) in the SN38-resistant cells. The DAPK expression levels were evaluated among parent ME180 cells, SN38-resistant ME180 cells and cisplatin-resistant ME180 cells by methylation-specific DAPK-PCR, quantitative RT-PCR and western blot analysis. The SN38-resistant cells co-treated with SN38 and 5-aza-CdR strongly exhibited enhanced SN38-sensitivities resembling those found in the parent cells. In the SN38-resistant subclones, no relationships were found between the restored SN38 sensitivity and hypermethylation of the DAPK promoter, DAPK mRNA expression, DAPK protein expression and induction of DAPK protein after 5-aza-CdR treatment, unlike the strong suppression of 5-aza-CdR-induced DAPK protein expression in the cisplatin-resistant subclones. These findings indicate that reversibly methylated molecules, but not DAPK, may regulate SN38 resistance, and that demethylating agents can be strong sensitizing anticancer chemotherapeutic drugs for SN38-resistant cancers.

STAT3 enhances intracellular Fas-mediated apoptotic signals in HHUA human endometrial epithelial cells.

Several endometrial signal transducer and activator of transcription 3 (STAT3)-activating cytokines are reported to be essential for blastocyst implantation, with inhibition of STAT3 activation in the endometrium also reported to prevent implantation. To investigate STAT3 signals in endometrial epithelial cells, the activation and inactivation effects of STAT3 signals were examined in the human endometrial epithelial cell line HHUA, which is thought to retain many of the intracellular signaling pathways found in normal human endometrial epithelial cells. Five STAT3-activating cytokines, IL-11, IL-10, LIF, oncostatin M and leptin, enhanced the Fas-mediated apoptosis of the HHUA cells without any increase in cell surface Fas antigen expression. STAT3 siRNA transfection suppressed STAT3 expression in HHUA cells and significantly inhibited Fas-mediated cell death. These results indicate that intracellular apoptotic signals in HHUA cells are constitutively activated and regulated by STAT3-mediated signals. This apoptosis-promoting effect of STAT3 in HHUA cells is completely different from many previous reports demonstrating anti-apoptotic effects by STAT3 activation. The STAT3 signals in HHUA cells may be specific to the human endometrial epithelial cell lineage in the regulation of blastocyst implantation.

Death-associated protein kinase is essential for the survival of various types of uterine cancer cells.

We recently showed that targeted knockdown of death-associated protein kinase (DAPK) expression induces apoptosis in the human endometrial adenocarcinoma cell line HHUA. To investigate the possibility that DAPK may represent a molecular target for anticancer therapies for advanced uterine cancers, we examined the effects of DAPK siRNA transfections on the viability of five different human uterine cancer cell lines. The five uterine cell lines comprised three differentiated endometrial adenocarcinomas, one leiomyosarcoma and one carcinosarcoma. Cell death assays showed that the DAPK siRNA transfection significantly increased the cell death in all five uterine cancer cells examined. Ribonuclease protection assays did not show any remarkable changes in the bcl-2 family gene expressions after the DAPK siRNA transfection in HHUA cells. Since DAPK-mutant mice were reported to be fertile and do not show lethality, DAPK may play a central role in the immortalization and carcinogenesis of uterine cancer cells, possibly without bcl-2 family-related apoptotic regulation. These results indicate that DAPK can be a convincing candidate for molecularly targeted anticancer therapies for patients with various types of advanced uterine cancers, including carcinosarcoma and leiomyosarcoma.

Suppressed protein expression of the death-associated protein kinase enhances 5-fluorouracil-sensitivity but not etoposide-sensitivity in human endometrial adenocarcinoma cells.

Targeted knockdown of the death-associated protein kinase (DAPK) expression in the endometrial adenocarcinoma HHUA cells reportedly induces cell death by enhancing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in an autocrine/paracrine manner. This suggests that endogenous DAPK is a potential candidate for a molecularly targeted anticancer therapy for patients with endometrial adenocarcinoma. To investigate the role of endogenous DAPK in anticancer drug sensitivity, we examined effects on cellular anticancer drug sensitivities of transfections with 5 different specific DAPK small-interfering RNAs (siRNAs) into HHUA cells. DAPK siRNA transfections strongly enhanced 5-fluorouracil (5FU)-sensitivity, but not etoposide-sensitivity, of HHUA cells compared with control siRNA-transfected cells. These results indicate that etoposide-stimulated cell death signals may share or include TRAIL-mediated apoptotic signals, and that 5FU-stimulated cell death signals may be independent from TRAIL-mediated apoptotic signals induced by DAPK siRNA transfections. Moreover, 5FU-combined chemotherapy with DAPK siRNA transfection may show stronger anticancer effects on patients with endometrial adenocarcinoma than does chemotherapy alone.