Expression with early postnatal peak and female-dominant sexual dimorphism of phospholipase D (PLD) 2 in submandibular gland ducts in situ of mice.

In the present study, expression and localization of PLD2 were examined in mouse submandibular glands in situ in comparison with those of PLD1 previously reported by the present authors. In immunoblots, PLD2-expression was high at postnatal 0 week (P0W) and P2W, and at P4W decreased considerably with the decrease of the male gland more marked, and at P8W it was undetectable in the male gland, but remained faint in the female. In the male gland at P8W after castration at P6W, PLD2-expression reappeared faintly. In the female glands with daily injections of testosterone started at P6W, at P8W the expression was undetectable. In immunohistochemistry, PLD2 was localized throughout immature duct epithelia without distinct regional differences in its intensity at P0W and P2W. PLD2-localization was weak and diffuse in granular convoluted tubules at P4W and negligible there at P8W in the female gland, while it was at negligible levels in the tubules at P4W and at undetectable levels there at P8W in the male gland. The expression changes detected in immunoblots after castration or testosterone injection were duplicated in granular convoluted tubules in immunohistochemistry. The present findings suggest that PLD2 is dominantly involved in proliferation/differentiation/apoptosis of most immature ductal cells at early postnatal and at least perinatal stages, different from PLD1. This also indicates that the female-dominant sexual dimorphism of PLD2-expression occurs in synchrony of differentiation of granular convoluted tubules at puberty and at young adulthood, similar to PLD1 though at much lower levels.

Involvement of MCL1, c-myc, and cyclin D2 protein degradation in ponatinib-induced cytotoxicity against T315I(+) Ph+leukemia cells.

T315I mutation found in chronic myelogenous leukemia (CML) and Ph + ALL patients is the most serious one among resistance against BCR/ABL kinase inhibitors including imatinib and is only responsive to ponatinib (PNT). However, the novel strategy is required to reduce life-threatening adverse effects of PNT including ischemic cardiovascular disease. We examined the mechanism of PNT-induced cytotoxicity against a T315I(+) Ph + ALL cell line, TccY/Sr. PNT induced apoptosis (increased sub G1 cells, and cleaved caspase3 and PARP), and suppressed protein expression of MCL1, cyclin D2 and c-myc, which were reversed by a proteasome inhibitor, MG132, suggesting enhanced proteasomal degradation by PNT. Among BCL2 family inhibitors, MCL1 inhibitors (maritoclax and AZD5991) robustly induced cell death, showing the MCL1-dependent survival of TccY/Sr cells. Decreased MCL1 and c-myc expression by PNT was also observed in T315I(+) MEGA2/STIR cells. PNT suppressed PI3K activation followed by AKT inhibition and GSK3 dephosphorylation. PI3K/AKT inhibitors mimicked PNT, suggesting that PI3K/AKT signaling is important for survival of TccY/Sr cells. Moreover, GSK3 inhibitor (SB216763) reduced PNT-induced cytotoxicity and degradation of c-myc and MCL1. AZD5991 exhibited the synergistic action with PNT, anti-cancer drugs and venetoclax (BCL2 inhibitor), suggesting the utility of MCL1 inhibitor alone or in combination as a future clinical option for Ph + leukemia patients.

Expression of endogenous phospholipase D1, localized in mouse submandibular gland, is greater in females and is suppressed by testosterone.

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4 weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2 weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.

Mechanism of paclitaxel resistance in a human prostate cancer cell line, PC3-PR, and its sensitization by cabazitaxel.

Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patient's prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of ßIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells.

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment.

Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines.

Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture: The Involvement of CREB Transcription Factor.

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.

CCR1/CCL5 interaction promotes invasion of taxane-resistant PC3 prostate cancer cells by increasing secretion of MMPs 2/9 and by activating ERK and Rac signaling.

Castration-refractory prostate cancer (CRPC) is treated with taxane-based chemotherapy, but eventually becomes drug resistant. It is thus essential to identify novel therapeutic targets for taxane resistance in CRPC patients. We investigated the role of the chemokine (C-C motif) receptor 1 (CCR1) and its ligand, chemokine (C-C motif) ligand 5 (CCL5), in taxane-resistant CRPC using paclitaxel-resistant prostate cancer cells (PC3PR) established from PC3 cells. We found that the expression levels of CCR1 mRNA and protein were up-regulated in PC3PR cells compared to PC3 cells. In order to investigate the role of increased CCR1 in PC3PR cells, we stimulated cells with CCL5, one of the chemokine ligands of CCR1. In CCL5-stimulated PC3PR cells, siRNA-mediated knockdown of CCR1 expression reduced phosphorylation of ERK1/2 and Rac1/cdc42. Furthermore, CCR1 knockdown and MEK1/2 inhibition decreased CCL5-stimulated secretion of MMPs 2 and 9, which play important roles in cancer cell invasion and metastasis. In the Matrigel invasion assay, knockdown of CCR1 and inhibition of the ERK and Rac signaling pathways significantly decreased the number of invading cells. Finally, the serum CCL5 protein level as measured by ELISA was not different among the three groups of patients: those with negative prostate biopsy, those at initial diagnosis of prostate cancer, and those with taxane-resistant prostate cancer. These results demonstrated for the first time that the interaction of CCR1 with CCL5 caused by increased expression of CCR1 promotes invasion of PC3PR cells by increasing secretion of MMPs 2 and 9 and by activating ERK and Rac signaling. Our findings suggest that CCR1 could be a novel therapeutic target for taxane-resistant CRPC.

A kavalactone derivative inhibits lipopolysaccharide-stimulated iNOS induction and NO production through activation of Nrf2 signaling in BV2 microglial cells.

Neuroinflammation and oxidative stress are involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's diseases and Parkinson's disease. Naturally derived kavalactones isolated from Piper methysticum (Piperaceae) have been shown to exhibit neuroprotective effects. We have previously reported that a chemically synthesized kavalactone derivative, 2',6'-dichloro-5-methoxymethyl-5,6-dehydrokawain (compound 1) protects against oxidative stress-induced neuronal cell death through activation of Nrf2 signaling. In the present study, we examined the effect of compound 1 on neuroinflammation. In BV2 microglial cells, compound 1 strongly inhibited LPS-stimulated iNOS induction and NO production, but did not affect LPS-stimulated induction of COX2. At 6h after LPS challenge, when iNOS induction was not clearly seen, treatment with LPS or compound 1 alone increased expression of heme oxygenase 1 (HO-1) whose transcription is regulated by Nrf2. When treated with both, compound 1 enhanced LPS-stimulated HO-1 induction, which was more evident at 24h after LPS treatment. Furthermore, LPS-stimulated activation of Nrf2 signaling and nuclear translocation of Nrf2 were potentiated by compound 1. The mechanism by which compound 1 activated Nrf2 signaling was supposed to be a covalent modification of the sulfhydryl groups of Keap1 by an α,ß-unsaturated carbonyl group present in the compound 1. Treatment with hemin, a HO-1 inducer, and with [Ru(CO)3Cl2]2, a CO donor, decreased LPS-stimulated iNOS induction and NO production. In contrast, siRNA-mediated knockdown of HO-1 expression reduced the inhibitory effect of compound 1 on LPS-stimulated iNOS induction and NO production. The compound 1 inhibited LPS-stimulated ERK phosphorylation after LPS treatment. Finally, compound 1 suppressed LPS/IFN-γ-stimulated NO production in primary microglial cells. These results suggest that compound 1 is capable of inhibiting LPS-stimulated iNOS induction and NO production via activation of Nrf2 signaling and HO-1 induction in microglial cells. Taken together, compound 1 has a potential to reduce neuroinflammation as well as oxidative stress in neurodegenerative diseases through activation of Nrf2 signaling.

Heterogeneous sphingosine-1-phosphate lyase gene expression and its regulatory mechanism in human lung cancer cell lines.

The role of sphingolipid metabolic pathway has been recognized in determining cellular fate. Although sphingolipid degradation has been extensively studied, gene expression of human sphingosine 1-phosphate lyase (SPL) catalyzing sphingosine 1-phosphate (S1P) remains to be determined. Among 5 human lung cancer cell lines examined, SPL protein levels paralleled the respective mRNA and enzyme activities. Between H1155 and H1299 cells used for further experiments, higher cellular S1P was observed in H1155 with higher SPL activity compared with H1299 with low SPL activity. GATA-4 has been reported to affect SPL transcription in Dictyostelium discoideum. GATA-4 was observed in H1155 but not in other cell lines. Overexpression of GATA-4 in H1299 increased SPL expression. However, promoter analysis of human SPL revealed that the most important region was located between -136bp and -88bp from the first exon, where 2 Sp1 sites exist but no GATA site. DNA pull-down assay of H1155 showed increased DNA binding of Sp1 and GATA-4 within this promoter region compared with H1299. Electrophoresis mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, reporter assay using mutated binding motif, and mithramycin A, a specific Sp1 inhibitor, suggest the major role of Sp1 in SPL transcription and no direct binding of GATA-4 with this 5' promoter region. The collaborative role of GATA-4 was proved by showing coimmunoprecipitation of Sp1 and GATA-4 using GST-Sp1 and overexpressed GATA-4. Thus, high SPL transcription of H1155 cells was regulated by Sp1 and GATA-4/Sp1 complex formation, both of which bind to Sp1 sites of the 5'-SPL promoter.

ETS1 promotes chemoresistance and invasion of paclitaxel-resistant, hormone-refractory PC3 prostate cancer cells by up-regulating MDR1 and MMP9 expression.

ETS1, which belongs to the ETS transcription factor family, plays important roles in diverse aspects of cancer such as drug resistance and metastasis. In the present study, we examined the functional roles of ETS1 in paclitaxel resistance and invasion using human prostate cancer PC3 cells and paclitaxel-resistant PC3PR cells established from PC3 cells. Our results showed that ETS1mRNA and protein expression was markedly up-regulated in paclitaxel-resistant PC3PR cells compared with paclitaxel-sensitive PC3 cells. The mRNA levels of MDR1 as well as MMP1, MMP3, MMP9 and uPA were positively correlated with that of ETS1. In PC3PR cells, silencing of ETS1 expression by siRNAs inhibited the activity of the MDR1 promoter containing ETS binding sites, reduced the mRNA and protein levels of MDR1 and suppressed paclitaxel resistance. Furthermore, ETS1 knockdown decreased secretion of MMP9 as well as its intracellular mRNA level, and dramatically inhibited invasion of PC3PR cells. Our results suggest that ETS1 promotes paclitaxel resistance and invasion in part by up-regulating MDR1 and MMP9 expression. Taken together, a novel therapeutic strategy targeting the ETS1 gene could be designed to overcome chemoresistance and metastasis of taxane-resistant, hormone-refractory prostate cancer.

Hesperetin upregulates ABCA1 expression and promotes cholesterol efflux from THP-1 macrophages.

ABCA1, a member of the ATP-binding cassette transporter family, regulates high-density lipoprotein (HDL) metabolism and cholesterol transport. Its expression is upregulated mainly by the activation of the liver X receptor (LXR). Since ABCA1 plays a pivotal role in cholesterol and HDL metabolism, identification of a compound capable of increasing its expression may be beneficial for the prevention and therapy of atherosclerosis. Firefly luciferase reporter assays were developed for human ABCA1 promoters and LXR enhancers, and an in-house phytochemical library was screened. It was found that a citrus flavonoid, hesperetin (1), increased ABCA1 promoter and LXR enhancer activities in THP-1 macrophages. It was also found that this flavonoid promoted PPAR-enhancing activity. In accordance with these findings, 1 increased mRNA and protein expression of ABCA1 and consequently upregulated ApoA-I-mediated cholesterol efflux. These results provide evidence that 1 promotes ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression through the activation of LXRα and PPARγ.

Association of a genetic variant of BTN2A1 with metabolic syndrome in East Asian populations.

BACKGROUND: The authors previously showed that the C→T polymorphism (rs6929846) of butyrophilin, subfamily 2, member A1 gene (BTN2A1) was significantly associated with myocardial infarction in Japanese individuals. Given that metabolic syndrome (MetS) is an important risk factor for myocardial infarction, the association of the rs6929846 of BTN2A1 with myocardial infarction might be attributable, at least in part, to its effect on susceptibility to MetS. AIM: The aim of the present study was to examine the relation of the rs6929846 of BTN2A1 to MetS in East Asian populations. METHODS: The study population comprised 5210 Japanese or Korean individuals (3982 individuals with MetS, 1228 controls) from three independent subject panels. Japanese subject panels A and B comprised 1322 individuals with MetS and 654 controls, and 1909 individuals with MetS and 170 controls, respectively, whereas the Korean population samples comprised 751 individuals with MetS and 404 controls. RESULTS: Comparison of genotype distributions using the χ(2) test revealed that the genotype distributions and allele frequencies of rs6929846 were significantly (p<0.05) associated with MetS in Japanese subject panels A (T allele frequency: MetS, 0.091; controls, 0.054; p=6.1×10(-5)) and B (T allele frequency: MetS, 0.091; controls, 0.039; p=0013) but not in the Korean population samples (T allele frequency: MetS, 0.102; controls, 0.125; p=0.0997). Multivariable logistic regression analysis with adjustment for covariates revealed that the rs6929846 of BTN2A1 was significantly (p<0.017) associated with MetS in Japanese subject panel A (p=0.0055, OR 1.97) and in all individuals (p=0.0038, OR 1.38), with the T allele representing a risk factor for this condition. CONCLUSION: BTN2A1 may be a susceptible gene for MetS in Japanese individuals.

Inhibition of cortactin and SIRT1 expression attenuates migration and invasion of prostate cancer DU145 cells.

OBJECTIVES: Cortactin is overexpressed in various types of cancer and enhances cell motility. It has been recently reported that silent mating type information regulation 2 homolog 1 interacts with cortactin and promotes cell migration. Here, we examined the role of cortactin and silent mating type information regulation 2 homolog 1 in migration and invasion of prostate cancer cells. METHODS: The cortactin expression levels in DU145, LNCaP and PC3 prostate cancer cells, and in PrEC normal human prostate epithelial cells were evaluated by western blot analysis. In DU145 cells, the expression of cortactin or silent mating type information regulation 2 homolog 1 was inhibited by small interfering RNA, and the effects of their knockdown on migration and invasion were examined by cell migration and invasion assays. To determine the localization of cortactin and silent mating type information regulation 2 homolog 1, western blot and immunofluorescence microscopic analyses were carried out. The functional interaction between silent mating type information regulation 2 homolog 1 and cortactin was also studied by in vivo acetylation assay. RESULTS: The protein expression of cortactin was significantly higher in DU145 cells than in other cell lines. Knockdown of cortactin or silent mating type information regulation 2 homolog 1 expression inhibited both migration and invasion of DU145 cells. Similarly to cortactin, silent mating type information regulation 2 homolog 1 was found to be predominantly expressed in the cytoplasm. Finally, the knockdown of silent mating type information regulation 2 homolog 1 expression increased the acetylation level of cortactin. CONCLUSIONS: Our findings suggest that inhibition of cortactin or silent mating type information regulation 2 homolog 1 expression attenuates migration and invasion of DU145 cells and this could represent a promising strategy to regulate metastasis of prostate cancer.

MiR-148a attenuates paclitaxel resistance of hormone-refractory, drug-resistant prostate cancer PC3 cells by regulating MSK1 expression.

MicroRNAs are involved in cancer pathogenesis and act as tumor suppressors or oncogenes. It has been recently reported that miR-148a expression is down-regulated in several types of cancer. The functional roles and target genes of miR-148a in prostate cancer, however, remain unknown. In this report, we showed that miR-148a expression levels were lower in PC3 and DU145 hormone-refractory prostate cancer cells in comparison to PrEC normal human prostate epithelial cells and LNCaP hormone-sensitive prostate cancer cells. Transfection with miR-148a precursor inhibited cell growth, and cell migration and invasion, and increased the sensitivity to anti-cancer drug paclitaxel in PC3 cells. Computer-aided algorithms predicted mitogen- and stress-activated protein kinase, MSK1, as a potential target of miR-148a. Indeed, miR-148a overexpression decreased expression of MSK1. Using luciferase reporter assays, we identified MSK1 as a direct target of miR-148a. Suppression of MSK1 expression by siRNA, however, showed little or no effects on malignant phenotypes of PC3 cells. In PC3PR cells, a paclitaxel-resistant cell line established from PC3 cells, miR-148a inhibited cell growth, and cell migration and invasion, and also attenuated the resistance to paclitaxel. MiR-148a reduced MSK1 expression by directly targeting its 3'-UTR in PC3PR cells. Furthermore, MSK1 knockdown reduced paclitaxel-resistance of PC3PR cells, indicating that miR-148a attenuates paclitaxel-resistance of hormone-refractory, drug-resistant PC3PR cells in part by regulating MSK1 expression. Our findings suggest that miR-148a plays multiple roles as a tumor suppressor and can be a promising therapeutic target for hormone-refractory prostate cancer especially for drug-resistant prostate cancer.

Sphingosine kinase 1/S1P pathway involvement in the GDNF-induced GAP43 transcription.

Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPß transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98 bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPß to the 5'-promoter.

Molecular hydrogen inhibits lipopolysaccharide/interferon γ-induced nitric oxide production through modulation of signal transduction in macrophages.

Molecular hydrogen has been reported to be effective for a variety of disorders and its effects have been ascribed to the reduction of oxidative stress. However, we have recently demonstrated that hydrogen inhibits type I allergy through modulating intracellular signal transduction. In the present study, we examined the hydrogen effects on lipopolysaccharide/interferon γ LPS/IFNγ-induced nitric oxide (NO) production in murine macrophage RAW264 cells. Treatment with hydrogen reduced LPS/IFNγ-induced NO release, which was associated with a diminished induction of inducible isoform of nitric oxide synthase (iNOS). Hydrogen treatment inhibited LPS/IFNγ-induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream signaling molecules, p38 MAP kinase and JNK, as well as IκBα, but did not affect activation of NADPH oxidase and production of reactive oxygen species (ROS). As ROS is an upstream activator of ASK1, inhibition of ASK1 by hydrogen without suppressing ROS implies that a potential target molecule of hydrogen should be located at the receptor or immediately downstream of it. These results suggested a role for molecular hydrogen as a signal modulator. Finally, oral intake of hydrogen-rich water alleviated anti-type II collagen antibody-induced arthritis in mice, a model for human rheumatoid arthritis. Taken together, our studies indicate that hydrogen inhibits LPS/IFNγ-induced NO production through modulation of signal transduction in macrophages and ameliorates inflammatory arthritis in mice, providing the molecular basis for hydrogen effects on inflammation and a functional interaction between two gaseous signaling molecules, NO and molecular hydrogen.

Involvement of heme oxygenase-1 induction via Nrf2/ARE activation in protection against H2O2-induced PC12 cell death by a metabolite of sesamin contained in sesame seeds.

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE (antioxidant response element) signaling has been considered as a promising strategy to combat with oxidative stress-related diseases. In the present study, we tested for potential effects of sesamin, a major lignan contained in sesame seeds, its stereoisomer episesamin, and their metabolites on Nrf2/ARE activation in rat pheochromocytoma PC12 cells. Luciferase reporter assays showed that primary metabolites of sesamin and episesamin, SC-1 and EC-1 were the most potent ARE activators among all tested compounds. SC-1 {(1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane} enhanced nuclear translocation of Nrf2 and up-regulated expression of phase II antioxidant enzymes including heme oxygenase-1 (HO-1). Treatment with SC-1 resulted in increased phosphorylation of p38 MAP kinase and transient increase in intracellular ROS levels. N-acetylcysteine (NAC) treatment abolished p38 phosphorylation as well as HO-1 induction caused by SC-1, indicating that ROS are upstream signals of p38 in Nrf2/ARE activation by SC-1. Furthermore, preconditioning with SC-1 attenuated H(2)O(2)-induced cell death in a dose-dependent manner. Finally, treatment with a HO-1 inhibitor, Zn-protoporphyrin (ZnPP), and overexpression of a dominant-negative mutant of Nrf2 diminished SC-1-mediated neuroprotection. Our results demonstrate that SC-1 is capable of protecting against oxidative stress-induced neuronal cell death in part through induction of HO-1 via Nrf2/ARE activation, suggesting its potential to reduce oxidative stress and ameliorate oxidative stress-related neurodegenerative diseases.

Association of a genetic variant of BTN2A1 with chronic kidney disease in Japanese individuals.

AIM: Although recent genetic studies suggested that several genetic variants increase the risk for chronic kidney disease (CKD), the genes that underlie genetic susceptibility to this condition remain to be identified definitively. We showed that the C→T polymorphism (rs6929846) of BTN2A1 and A→G polymorphism (rs2569512) of ILF3 were significantly associated with myocardial infarction in Japanese individuals by a genome-wide association study. The purpose of the present study was to examine a possible association of these polymorphisms (rs6929846, rs2569512) with CKD in Japanese individuals. METHODS: A total of 7542 Japanese individuals from two independent populations were examined: Subject panel A comprised 971 individuals with CKD (estimated glomerular filtration rate (eGFR) <60 mL/min 1.73 m(-2)) ) and 2269 controls (eGFR ≥60 mL/min 1.73 m(-2) ); and subject panel B comprised 1318 individuals with CKD and 2984 controls. RESULTS: The χ(2) test revealed that rs6929846 of BTN2A1, but not rs2569512 of ILF3, was significantly related to the prevalence of CKD both in subject panels A (P = 0.0383) and B (P = 0.0477). Multivariable logistic regression analysis with adjustment for covariates revealed that the C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with the prevalence of CKD in subject panels A (P = 0.0422; recessive model; odds ratio, 2.36) and B (P = 0.0386; dominant model; odds ratio, 1.21) with the T allele representing a risk for this condition. CONCLUSION: Our results suggest that BTN2A1 may be a susceptibility gene for CKD in Japanese individuals.

Adaptive regulation of membrane lipids and fluidity during thermal acclimation in Tetrahymena.

The free-living eukaryotic protozoan Tetrahymena is a potentially useful model for the thermoadaptive membrane regulation because of easy growth in the axenic culture, systematic isolation of subcellular organelles, and quick response to temperature stress. Exposure of Tetrahymena cells to the cold temperature induces marked alterations in the lipid composition and the physical properties (fluidity) of various membranes. The increase in fatty acid unsaturation of membrane phospholipids is required to preserve the proper fluidity. In this homeoviscous adaptive response, acyl-CoA desaturase plays a pivotal role and its activity is regulated by induction of the enzyme via transcriptional activation.