Ubiquinone binding site of yeast NADH dehydrogenase revealed by structures binding novel competitive- and mixed-type inhibitors.

Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.

Fermented Brown Rice Extract Causes Apoptotic Death of Human Acute Lymphoblastic Leukemia Cells via Death Receptor Pathway.

Mixture of brown rice and rice bran fermented with Aspergillus oryzae, designated as FBRA, has been reported to reveal anti-carcinogenic and anti-inflammatory effects in rodents. Then, to test its potential anti-cancer activity, the aqueous extract was prepared from FBRA powder, and the effect of this extract on human acute lymphoblastic leukemia Jurkat cells was directly examined. The exposure to FBRA extract reduced the cell viability in a concentration- and time-dependent manner. The reduction of the cell viability was accompanied by the DNA fragmentation, and partially restored by treatment with pan-caspase inhibitor. Further studies showed that FBRA extract induced the cleavage of caspase-8, -9, and -3, and decreased Bcl-2 protein expression. Moreover, the expression of tBid, DR5, and Fas proteins was enhanced by FBRA extract, and the pretreatment with caspase-8 inhibitor, but not caspase-9 inhibitor, restored the reduction of the cell viability induced by FBRA extract. These findings suggested that FBRA extract could induce the apoptotic death of human acute lymphoblastic leukemia cells probably through mainly the death receptor-mediated pathway and supplementarily through the tBid-mediated mitochondrial pathway, proposing the possibility that FBRA was a potential functional food beneficial to patients with hematological cancer.

Involvement of S1P1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells.

COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca(2+) concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [(3)H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis.

Mifepristone promotes adiponectin production and improves insulin sensitivity in a mouse model of diet-induced-obesity.

The steroid receptor antagonist mifepristone is used as an anti-cancer agent, eliciting both cytostatic and cytotoxic effects on malignant cells. However, the metabolic effects of long-term treatment with mifepristone have remained unclear. The effects of mifepristone on insulin sensitivity and adiponectin secretion were evaluated both in in vivo and in vitro. First, we explored the effects of mifepristone, on metabolic functions in obese mice receiving a high-fat diet. When these mice were fed mifepristone, they exhibited a marked improvement in insulin sensitivity, attenuated hepatic injury, and decreased adipocyte size, compared with mice that received only the high-fat diet. Intriguingly, mifepristone-treated mice showed significantly elevated plasma adiponectin levels. Second, we tested the effects of mifepristone on differentiated 3T3-L1 adipocytes in vitro. When differentiated adipocytes were treated with mifepristone for 48 h, adiponectin was upregulated at both mRNA and protein levels. Collectively, these results reveal novel actions of mifepristone on metabolic functions, in vivo and in vitro, in which the drug exerts antidiabetic effects associated with an upregulation in adiponectin-secretion.

Deranged epidermal differentiation in kl/kl mouse and the effects of ßKlotho siRNA on the differentiation of HaCaT cells.

Mice deficient in the klotho gene (kl/kl mice) display the phenotypes of human ageing. We found that the expression of epidermal differentiation-associated factors (keratin 1, keratin 10, filaggrin and loricrin) was lower in the skin of kl/kl mice than that of wild-type mice. In vitro experiments showed that the expression of ßKlotho, a family of klotho gene-encoded protein, was induced concomitantly with the differentiation of an immortalized human epidermal keratinocyte cell line (HaCaT cells) when they were cultured in an air-liquid interface. ßKlotho knockdown by small interfering ribonucleic acid suppressed the expression of the above differentiation-associated factors in HaCaT cells. ßKlotho small interfering ribonucleic acid increased the expression of keratin 14, which is expressed in mitotically active basal layer cells, and activated p44/p42 mitogen-activated protein kinase in the HaCaT cells grown in the air-liquid interface. These findings suggest that the epidermal differentiation is deranged in kl/kl mice, and ßKlotho is required for the differentiation of human epidermal keratinocytes.

Dexamethasone induces caveolin-1 in vascular endothelial cells: implications for attenuated responses to VEGF.

Steroids exert direct actions on cardiovascular cells, although underlying molecular mechanisms remain incompletely understood. We examined if steroids modulate abundance of caveolin-1, a regulatory protein of cell-surface receptor pathways that regulates the magnitudes of endothelial response to vascular endothelial growth factor (VEGF). Dexamethasone, a synthetic glucocorticoid, induces caveolin-1 at both levels of protein and mRNA in a time- and dose-dependent manner in pharmacologically relevant concentrations in cultured bovine aortic endothelial cells. Aldosterone, a mineralocorticoid, but not the sex steroids 17ß-estradiol, testosterone, or progesterone, elicits similar caveolin-1 induction. Caveolin-1 induction by dexamethasone and that by aldosterone were abrogated by RU-486, an inhibitor of glucocorticoid receptor, and by spironolactone, a mineralocorticoid receptor inhibitor, respectively. Dexamethasone attenuates VEGF-induced responses at the levels of protein kinases Akt and ERK1/2, small-G protein Rac1, nitric oxide production, and migration. When induction of caveolin-1 by dexamethasone is attenuated either by genetically by transient transfection with small interfering RNA or pharmacologically by RU-486, kinase responses to VEGF are rescued. Dexamethasone also increases expression of caveolin-1 protein in cultured human umbilical vein endothelial cells, associated with attenuated tube formation responses of these cells when cocultured with normal fibroblasts. Immunohistochemical analyses revealed that intraperitoneal injection of dexamethasone induces endothelial caveolin-1 protein in thoracic aorta and in lung artery in healthy male rats. Thus steroids functionally attenuate endothelial responses to VEGF via caveolin-1 induction at the levels of signal transduction, migration, and tube formation, identifying a novel point of cross talk between nuclear and cell-surface receptor signaling pathways.

Reduced expression of epidermal growth factor receptor, E-cadherin, and occludin in the skin of flaky tail mice is due to filaggrin and loricrin deficiencies.

Disruption of skin barrier function leads to increases in the percutaneous transfer of allergens and the incidence of atopic dermatitis. Flaky tail (Flg(ft)) mice have been used as a model of atopic dermatitis with skin barrier dysfunction. Although Flg(ft) mice are known to have filaggrin mutation, the mechanism responsible for the skin barrier dysfunction that they display needs to be determined, especially for the roles of epidermal adhesion and junction proteins. Herein, we report the decreased expression of epidermal growth factor receptor (EGFR), E-cadherin, occludin, and SIRT1 in the skin of Flg(ft) mice, compared with those in C57BL/6J mice. Administration of N-acetyl-L-cysteine, an antioxidant, in the drinking water improved these protein expressions in the skin of Flg(ft) mice. Notably, we discovered that loricrin expression was suppressed in Flg(ft) mice. In vitro experiments showed that filaggrin small interfering RNA, loricrin small interfering RNA, or SIRT1 inhibitor sirtinol suppressed the expression levels of EGFR, E-cadherin, and occludin in a human immortalized keratinocyte cell line (HaCaT cells). Our findings suggest that the observed reductions in EGFR, E-cadherin, and occludin expression were due to filaggrin deficiency accompanied with subsequent loricrin deficiency and disruption of the SIRT1 pathway in the skin of Flg(ft) mice.

Pioglitazone promotes preadipocyte proliferation by downregulating p16(Ink4a).

Pioglitazone, a synthetic ligand of peroxisome proliferator-activated receptor (PPAR)γ, causes preadipocyte proliferation through a mechanism which still remains elusive. Here, to address the mechanism, we investigated the effects of PPARγ and pioglitazone on the kinetics of cyclin-dependent kinase inhibitors, especially with p16(Ink4a) (p16) centered, by employing 3T3-L1 preadipocytes. Pioglitazone promoted preadipocyte proliferation by increasing S and G(2)/M cell-cycle entry, which was accompanied by decreased p16 mRNA expression. PPARγ overexpression along with the luciferase reporter assay confirmed that PPARγ was crucial for the downregulation of p16 mRNA transcription, and that the action was augmented by pioglitazone. Thus, pioglitazone exerted cell-cycle dependent promoting effect on preadipocyte proliferation, of which mechanisms include p16-downregulation through PPARγ.

Lipid peroxidation-induced VEGF expression in the skin of KKAy obese mice.

Obesity is known to be associated with a number of effects on skin physiology. KKA(y) obese mouse is a model of type 2 diabetes characterized by systemic oxidative stress because of severe obesity, hypertriglyceridaemia, hyperglycaemia and hyperinsulinaemia. We investigated lipid peroxidation and vascular endothelial growth factor (VEGF) expression in the skin of KKA(y) obese mice. We also investigated the effect of lipid peroxidation derivatives on VEGF production and proliferation in human epidermal keratinocyte cell line (HaCaT). The lipid peroxidation level in the mouse skin tissue was determined by measuring the levels of thiobarbituric acid-reactive substances. The levels of VEGF expression, p44/p42 mitogen-activated protein kinase (MAPK) activation and CD36 expression were analysed by Western blot. Their localization was examined by immunofluorescence. For the in vitro experiments, an enzyme-linked immunosorbent assay was utilized to measure VEGF secretion in the medium. In vitro experiments demonstrated that lipid peroxidation derivatives increased VEGF production in HaCaT cells, which was blocked by a p44/p42 MAPK inhibitor and anti-CD36 antibody. We observed increased levels of lipid peroxidation derivatives, p44/p42 MAPK activation and VEGF expression in the skin of KKA(y) obese mice. Notably, pitavastatin, an inhibitor of competitive 3-hydroxy-3-methylglutaryl coenzyme A reductase, suppressed all of these processes. Our results suggest that lipid peroxidation induces VEGF expression via CD36 and p44/p42 MAPK pathway in the skin of obese mice.

Reaction mechanism of single subunit NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae: evidence for a ternary complex mechanism.

The flavoprotein rotenone-insensitive internal NADH-ubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin.

Expression of wild-type, but not mutant, loricrin causes programmed cell death in HaCaT keratinocytes.

The epidermal cornified cell envelope is a complex protein-lipid composite that replaces the plasma membrane of corneocytes and is crucial for epidermal barrier function. Loricrin is a major constituent of the epidermal cornified cell envelope, contributing approximately 70% by mass. In order to explore novel function of wild-type (WT) loricrin other than the major component of the epidermal cornified cell envelope, we transiently expressed construct encoding human WT and mutant loricrin (730insG) in HaCaT keratinocytes. HaCaT cells transfected with WT or mutant loricrin were at differentiation level. WT loricrin in the transfected cells was seen diffusely in the cytoplasm and nuclei. Positive transferase deoxytidyl uridine end labeling staining was observed in the nuclei of WT loricrin-transfected HaCaT keratinocytes. Data from the DNA fragmentation assay showed that only WT loricrin induced DNA ladders compared with that of mutant loricrin. WT loricrin-transfected HaCaT keratinocytes were susceptible to programmed cell death (PCD). Activation of caspase-14 was also seen. In contrast, PCD or activation of caspase-14 did not occur in mutant loricrin-transfected HaCaT cells. These results suggest that the expression of WT loricrin facilitates induction of PCD in HaCaT keratinocytes.

A novel nucleic acid analogue shows strong angiogenic activity.

A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100muM was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

Activation of vascular endothelial growth factor receptor 2 in a cellular model of loricrin keratoderma.

Loricrin is a major constituent of the epidermal cornified cell envelope. Recently, heterozygous loricrin gene mutations have been identified in two dominantly inherited skin diseases, Vohwinkel syndrome with ichthyosis and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. We generated stable HaCaT cell lines that express wild-type (WT) loricrin and a mutant form found in Vohwinkel syndrome with ichthyosis, using an ecdysone-inducible promoter system. The cells expressing the mutant loricrin grew more rapidly than those expressing WT loricrin after induction for 5 days. Confocal immunofluorescence microscopy revealed that phospho-Akt occurred in the nucleolus where the mutant loricrin was also located. The level of activity of Akt kinase was about nine times higher in cells with the mutant than in those with WT loricrin. ERK1/2, the epidermal growth factor receptor, vascular endothelial growth factor (VEGF) receptor 2 and Stat3 were all phosphorylated in cells with the mutant loricrin. The docking proteins, Gab1 and c-Cbl, were also tyrosine-phosphorylated in these cells. Furthermore, chromatin immunoprecipitation assays showed that Stat3 protein bound to the VEGF promoter in cells with the mutant. Thus, this study suggests that VEGF release and the subsequent activation of VEGF receptor 2 link loricrin gene mutations to rapid cell proliferation in a cellular model of loricrin keratoderma.

Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling.

The Ndi1 enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone oxidoreductase. As Ndi1 is expected to be a possible remedy for complex I defects of mammalian mitochondria, a detailed biochemical characterization of the enzyme is needed. To identify the ubiquinone (UQ) binding site in Ndi1, we conducted photoaffinity labeling using a photoreactive biotinylated UQ mimic (compound 2) synthesized following a concept of the least possible modification of the substituents on the quinone ring. Cleavage with CNBr of Ndi1 cross-linked by 2 revealed the UQ ring of 2 to be specifically cross-linked to the Phe281-Met410 region (130 amino acids). Digestion of the CNBr fragment with V8 protease and lysylendopeptidase (Lys-C) gave approximately 8 and approximately 4 kDa peptides, respectively. The approximately 8 kDa V8 digest was identified as the Thr329-Glu399 region (71 amino acids) by an N-terminal sequence analysis. Although the approximately 4 kDa Lys-C digest could not be identified by N-terminal sequence analysis, the band was thought to cover the Gly374-Lys405 region (32 amino acids). Taken together, the binding site of the Q ring of 2 must be located in a common region of the V8 protease, and Lys-C digests Gly374-Glu399 (26 amino acids). Superimposition of the Ndi1 sequence onto a three-dimensional structural model of NDH-2 from Escherichia coli suggested that the C-terminal portion of this region is close to the isoalloxazine ring of FAD.

Transforming growth factor-beta1 downregulates caveolin-1 expression and enhances sphingosine 1-phosphate signaling in cultured vascular endothelial cells.

In vascular endothelial cells, specialized microdomains of plasma membrane termed caveolae modulate various receptor signal transduction pathways regulated by caveolin-1, a resident protein of caveolae. We examined whether transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine, alters expression levels of caveolin-1 and influences heterologous receptor signaling. Treatment of cultured bovine aortic endothelial cells (BAEC) with TGF-beta1 induces marked decreases in caveolin-1 expression in a time- and dose-dependent fashion at both levels of protein and mRNA. A pharmacological inhibitor of activin receptor-like kinase 5 (ALK-5) counteracts caveolin-1 downregulation by TGF-beta1, indicating the involvement of ALK-5 receptor subtype for TGF-beta1. Sphingosine 1-phosphate (S1P) is a serum-borne angiogenic lipid growth factor that exerts a wide variety of biological actions. S1P modulates G protein-coupled S1P receptors, activating downstream molecules kinases AMP-activated protein kinase (AMPK), and Akt as well as a small G protein Rac1, ultimately to promote migration. Because S1P receptor signaling is associated with caveolae/caveolin-1, we examined whether pretreatment with TGF-beta1 enhances effects of S1P on BAEC. Whereas S1P alone evokes robust BAEC responses to S1P, pretreatment with TGF-beta1 leads to even higher magnitudes of S1P-elicited signaling responses and cell migration. Conversely, genetic knockdown of caveolin-1 using small interfering RNA mimics TGF-beta1-induced promotion of BAEC responses to S1P. Collectively, these data demonstrate that TGF-beta1 downregulates caveolin-1 of cultured endothelial cells, involving ALK-5 receptor subtype. Because downregulation of caveolin-1 by TGF-beta1 promotes subsequent heterologous receptor signaling by S1P, these results may also identify novel point of cross-talk between cytokines and sphingolipids within endothelial signal transduction machineries.

HB-EGF-induced VEGF production and eNOS activation depend on both PI3 kinase and MAP kinase in HaCaT cells.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of growth factors that have been implicated in skin patho-physiology. Although endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) appear to be involved in mitogenesis and chemotaxis in epidermal keratinocytes, the activation of eNOS and VEGF production induced by HB-EGF and its signaling mechanism remains undefined. OBJECTIVE: We examined possible signal transduction pathways by which HB-EGF leads to eNOS activation and VEGF production in human epidermal keratinocyte cell line (HaCaT cells). METHODS: The phosphorylation of epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK; p42/p44 MAPK), Akt and eNOS were examined by Western blotting analysis. VEGF production was determined by enzyme-linked immunosorbent assay. Various inhibitors were utilized to investigate the signaling mechanisms of eNOS activation and VEGF production. RESULTS: HB-EGF-induced phosphorylation of EGFR with maximum phosphorylation at 1h. HB-EGF-induced phosphorylation of p42/p44 MAPK in a few minutes. It activated Akt with maximum phosphorylation at 1h and eNOS with maximum phosphorylation at 3h. The HB-EGF-induced eNOS activation was significantly blocked by the p42/p44 MAPK inhibitor U0126 and the phosphatidylinositol 3-kinase (P13K) inhibitor LY294002. HB-EGF increased VEGF production. The HB-EGF-induced VEGF production was blocked by U0126 and LY294002. Finally, the HB-EGF-induced activation of Akt and eNOS was suppressed by VEGF competitive antagonist, CBO-P11. CONCLUSION: These results demonstrate that HB-EGF-induced eNOS activation depends on p42/p44 MAPK, PI3K/Akt pathways and endogenous VEGF in HaCaT cells.

Effects of combined olmesartan and pravastatin on glucose intolerance and cardiovascular remodeling in a metabolic-syndrome model.

Hypertension and dyslipidemia frequently coexist in patients with progressive insulin resistance and thus constitute metabolic syndrome. We sought to determine the merits of combining an angiotensin II receptor blocker and a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor in treating this pathological condition. Five-week-old Otsuka Long-Evans Tokushima Fatty rats, a model of metabolic syndrome, were untreated or treated with olmesartan 3 mg kg(-1) per day, pravastatin 30 mg kg(-1) per day or their combination for 25 weeks. Long-Evans Tokushima Otsuka rats served as normal controls. The antihypertensive effect of olmesartan and the lipid-lowering properties of pravastatin were both augmented by the combination. The oral glucose tolerance test revealed that only the combined treatment significantly reduced the area under the time-glucose curve, which was accompanied by augmented adiponectin messenger RNA expression in epididymal adipose tissue. Although the total cardiac endothelial nitric oxide synthetase (eNOS) content did not significantly differ among the groups, the combined treatment significantly increased the content of dihydrofolate reductase, a key eNOS coupler. Dihydroethidium staining of the aorta showed that the combination most significantly attenuated superoxide production. Moreover, Azan-Mallory staining revealed that the combination most significantly limited the perivascular fibrosis and wall thickening of intramyocardial coronary arteries. In conclusion, the combination of olmesartan and pravastatin augmented adiponectin expression in white adipose tissue and improved glucose tolerance in a rat model of metabolic syndrome, which was associated with more significant ameliorations of cardiovascular redox state and remodeling than those by treatments with either agent alone.

Sphingosine kinase is induced in mouse 3T3-L1 cells and promotes adipogenesis.

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that exerts numerous biological activities both as a receptor ligand and as an intracellular second messenger. In the present study, we explored roles of sphingosine kinase (SphK), an S1P-producing enzyme, in adipose tissue. We utilized mouse 3T3-L1 cells as an in vitro model of adipogenesis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. Real-time quantitative PCR (qRT-PCR) assays revealed that the expression levels of transcripts encoding both isoforms of SphK-1 and SphK-2 are up-regulated during adipogenesis (37.6- and 6.6-fold vs. basal, P < 0.05, respectively). Concomitantly, SphK-1/SphK-2 protein abundance and S1P contents of these cells increased at 3 days after hormonal stimulation. Loss-of-function approaches by pharmacological inhibition of SphK activity as well as by transfection with small interfering RNA (siRNA) against SphK-1 led to significant attenuation of lipid droplet accumulation and adipocyte marker gene expression. We detected marked elevation of SphK-1 mRNA in adipose tissue derived from 13-week-old ob/ob mice with obese phenotype than their lean littermates. These results suggest that increased expression of SphK, an S1P-producing enzyme, plays a significant role during adipogenesis, potentially providing a novel point of control in adipose tissue.

Angiotensin II enhances EGF receptor expression levels via ROS formation in HaCaT cells.

BACKGROUND: Recent work has shown a novel function of angiotensin II (Ang II) in skin wound healing in which reactive oxygen species might be involved. As Ang II is known to increase superoxide production by activating NADPH oxidase in some non-phagocytic cells, we hypothesized that the produced superoxide by NADPH activation could contribute to the regulation of epidermal growth factor receptor (EGFR) in keratinocytes. OBJECTIVE: We examined whether Ang II could generate superoxide and enhance EGFR expression levels in HaCaT cells. METHODS: Superoxide formation was assessed by using hydroethidine. EGFR expression levels were examined by Western blotting. RESULTS: Ang II (1-100 microM) increased the superoxide formation. Ang II (1-100 microM) resulted in a dose-dependent increase in cell proliferation in HaCaT cells. Heparin-binding epidermal growth factor activated the EGFR at 5-10 min. Although Ang II did not activate the EGFR, the expression levels of EGFR protein were increased in HaCaT cells treated with Ang II (1 microM) at 6h. Apocynin, a NADPH oxidase inhibitor, decreased the expression levels of EGFR. Xanthine/xanthine oxidase system, an exogenous superoxide generating system, enhanced the EGFR protein expression. Although Ang II did not affect the nitric oxide (NO) production, a NO synthase inhibitor N(omega)-nitro-l-arginine methyl ester suppressed the Ang II-induced EGFR expression levels in HaCaT cells. Thus, constitutive NO is required for the Ang II-induced EGFR expression in HaCaT cells. CONCLUSION: These results suggest that Ang II enhances the cell proliferation and EGFR expression via superoxide production under the regulation of NO in HaCaT cells, implying that Ang II may regulate the proliferation, differentiation and tumorigenesis of the epidermis by harmonizing the superoxide and NO production.

Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells.

Reactive oxygen species including H(2)O(2) lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H(2)O(2). Treatment of cultured bovine aortic EC (BAEC) with H(2)O(2) (750 microM for 6h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H(2)O(2) were alike markedly attenuated by pretreatment with S1P (1 microM, 30 min). H(2)O(2) induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H(2)O(2)-induced caspase-3 activation. Thus S1P attenuates H(2)O(2)-induced apoptosis of cultured BAEC, involving p38 MAP kinase.