Zinc-histidine complex protects cultured cortical neurons against oxidative stress-induced damage

The levels of zinc in the brain are directly affected by dietary zinc and deficiency has been associated with alcohol withdrawal seizures, excitotoxicity, impaired learning and memory and an accelerated rate of dysfunction in aged brain. Although zinc is essential for a healthy nervous system, high concentrations of zinc are neurotoxic, thus it is important to identify the most effective forms of zinc for treatment of conditions of the central nervous system. Accumulating evidence suggests that zinc-histidine complex (Zn(HiS)(2)) has greater biological potency and enhanced bioavailability compared with other zinc salts and also has antioxidant potential. Therefore, in this study we investigated the ability of zinc-histidine to protect cultured cortical neurons against hydrogen peroxide-induced damage. Pre-treating neurons for 18h with subtoxic concentrations of zinc-histidine (5-25 muM) improved neuronal viability and strongly inhibited hydrogen peroxide-induced (75 muM, 30 min) cell damage as assessed by MTT turnover and morphological analysis 24 It later. Low concentrations of zinc-histidine were more neuroprotective than zinc chloride. There was evidence of an anti-apoptotic mechanism of action as zinc-histidine inhibited hydrogen peroxide-induced caspase-3 activation and c-jun-N-terminal kinase phosphorylation. In summary, zinc supplementation with zinc-histidine protects cultured neurons against oxidative insults and inhibits apoptosis which suggests that zinc-histidine may be beneficial in the treatment of diseases of the CNS associated with zinc deficiency. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Thyroid hormone can increase estrogen-mediated transcription from a consensus estrogen response element in neuroblastoma cells

Thyroid hormones (T) and estrogens (E) are nuclear receptor ligands with at least two molecular mechanisms of action: (i) relatively slow genomic effects, such as the regulation of transcription by cognate T receptors (TR) and E receptors (ER); and (ii) relatively rapid nongenomic effects, such as kinase activation and calcium release initiated at the membrane by putative membrane receptors. Genomic and nongenomic effects were thought to be disparate and independent. However, in a previous study using a two-pulse paradigm in neuroblastoma cells, we showed that E acting at the membrane could potentiate transcription from an E-driven reporter gene in the nucleus. Because both T and E can have important effects on mood and cognition, it is possible that the two hormones can act synergistically. In this study, we demonstrate that early actions of T via TRalpha1 and TRbeta1 can potentiate E-mediated transcription (genomic effects) from a consensus E response element (ERE)-driven reporter gene in transiently transfected neuroblastoma cells. Such potentiation was reduced by inhibition of mitogen-activated protein kinase. Using phosphomutants of ERalpha, we also show that probable mitogen-activated protein kinase phosphorylation sites on the ERalpha, the serines at position 167 and 118, are important in TRbeta1-mediated potentiation of ERalpha-induced transactivation. We suggest that crosstalk between T and E includes potential interactions through both nuclear and membrane-initiated molecular mechanisms of hormone signaling.

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na, K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (gamma C-33) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K+, ATP and N-4(+)center dot K-0.5 for Na+ was unaffected. Exogenous pig FXYD2 increased the V-max for stimulation of gill Na,K-ATPase activity by Na+, K+ and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na, K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity. (C) 2012 Elsevier B.V. All rights reserved.

Endo-PDI is required for TNFα-induced angiogenesis

Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function

Xanthohumol ameliorates atherosclerotic plaque formation, hypercholesterolemia, and hepatic steatosis in ApoE-deficient mice

SCOPE: Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits positive effects on lipid and glucose metabolism. Based on its favorable biological properties, we investigated whether XN attenuates atherosclerosis in western-type diet-fed apolipoprotein-E-deficient (ApoE(-/-) ) mice. METHODS AND RESULTS: XN supplementation markedly reduced plasma cholesterol concentrations, decreased atherosclerotic lesion area, and attenuated plasma concentrations of the proinflammatory cytokine monocyte chemoattractant protein 1. Decreased hepatic triglyceride and cholesterol content, activation of AMP-activated protein kinase, phosphorylation and inactivation of acetyl-CoA carboxylase, and reduced expression levels of mature sterol regulatory element-binding protein (SREBP)-2 and SREBP-1c mRNA indicate reduced lipogenesis in the liver of XN-fed ApoE(-/-) mice. Concomitant induction of hepatic mRNA expression of carnitine palmitoyltransferase-1a in ApoE(-/-) mice-administered XN suggests increased fatty acid beta-oxidation. Fecal cholesterol concentrations were also markedly increased in XN-fed ApoE(-/-) mice compared with mice fed western-type diet alone. CONCLUSION: The atheroprotective effects of XN might be attributed to combined beneficial effects on plasma cholesterol and monocyte chemoattractant protein 1 concentrations and hepatic lipid metabolism via activation of AMP-activated protein kinase.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding a4b1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic a4b1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by a4b1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 Tcell survival. Conclusions: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to a4b1 expressing proinflammatory T lymphocytes in CLD.

Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCKA receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCKA and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.

Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness

Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner, in vitro. Because NCSCs are likely exposed to multiple signals in vivo, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor β1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48–96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor β1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons, in vivo.

Fetuin (α2-HS-glycoprotein) opsonizes cationic macrophagedeactivating molecules

Macrophages become activated by bacterial endotoxin (lipopolysaccharide) and other stimuli to release proinflammatory cytokines and NO. To prevent release of toxic or potentially lethal quantities of these factors, the state of macrophage activation is counter-regulated by anti-inflammatory mediators (e.g., glucocorticoid hormones, interleukin 10, and transforming growth factor type β). Fetuin, a negative acute-phase protein, recently was implicated as an anti-inflammatory mediator, because it is required for macrophage deactivation by spermine. In the present studies, we found that fetuin is necessary for macrophages to respond to CNI-1493, a tetravalent guanylhydrazone inhibitor of p38 mitogen-activated protein kinase phosphorylation. Fetuin dose-dependently increases macrophage uptake of CNI-1493, which can be specifically inhibited by anti-human fetuin antibodies. Anti-human fetuin antibodies render primary human peripheral blood mononuclear cells insensitive to deactivation by CNI-1493. Thus, macrophages use fetuin as an opsonin for cationic-deactivating molecules, both endogenous (e.g., spermine) and pharmacologic (e.g., CNI-1493). This role of fetuin as an opsonic participant in macrophage-deactivating mechanisms has implications for understanding and manipulating the innate immune response.

Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell spreading on fibronectin but not on plastic. Cell adhesion on anti-integrin antibodies is also able to specifically induce PECAM-1 dephosphorylation while concurrently inducing pp125 focal adhesion kinase phosphorylation. Inhibition of dephosphorylation with sodium orthovanadate suggests that this effect is at least partially mediated by phosphatase activity. Tyr-663 and Tyr-686 are identified as potential phosphorylation sites and mutated to phenylalanine. When expressed, both mutants show reduced PECAM-1 phosphorylation but Phe-686 mutants also show significant reversal of PECAM-1-mediated inhibition of cell migration and do not localize PECAM-1 to cell borders. Our results suggest that beta 1-integrin engagement can signal to dephosphorylate PECAM-1 and that this signaling pathway may play a role during endothelial cell migration.

The cis-acting phorbol ester "12-O-tetradecanoylphorbol 13-acetate"-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression.

Vascular endothelial cells, serving as a barrier between vessel and blood, are exposed to shear stress in the body. Although endothelial responses to shear stress are important in physiological adaption to the hemodynamic environments, they can also contribute to pathological conditions--e.g., in atherosclerosis and reperfusion injury. We have previously shown that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells and that the regulation is at the transcriptional level. These observations led us to functionally analyze the 550-bp promoter region of the MCP-1-encoding gene to define the cis element responding to shear stress. The shear stress/luciferase assay on the deletion constructs revealed that a 38-bp segment (-53 to -90 bp relative to the transcription initiation site) containing two divergent phorbol ester "12-O-tetradecanoylphorbol 13-acetate" (TPA)-responsive elements (TRE) is critical for shear inducibility. Site-specific mutations on these two sites further demonstrated that the proximal one (TGACTCC) but not the distal one (TCACTCA) was shear-responsive. Shear inducibility was lost after the mutation or deletion of the proximal site. This molecular mechanism of shear inducibility of the MCP-1 gene was functional in both the epithelial-like HeLa cells and bovine aortic endothelial cells (BAEC). In a construct with four copies of the TRE consensus sequences TGACTACA followed by the rat prolactin minimal promoter and luciferase gene, shear stress induced the reporter activities by 35-fold and 7-fold in HeLa cells and BAEC, respectively. The application of shear stress on BAEC also induced a rapid and transient phosphorylation of mitogen-activated protein kinases. Pretreatment of BAEC with TPA attenuated the shear-induced mitogen-activated protein kinase phosphorylation, suggesting that shear stress and TPA share a similar signal transduction pathway in activating cells. The present study provides a molecular basis for the transient induction of MCP-1 gene by shear stress.

Oleic acid modulates mRNA expression of liver X receptor (LXR) and its target genes ABCA1 and SREBP1c in human neutrophils

Purpose: Regulation of liver X receptors (LXRs) is essential for cholesterol homeostasis and inflammation. The present study was conducted to determine whether oleic acid (OA) could regulate mRNA expression of LXRα and LXRα-regulated genes and to assess the potential promotion of oxidative stress by OA in neutrophils. Methods: Human neutrophils were treated with OA at different doses and LXR target gene expression, oxidative stress production, lipid efflux and inflammation state were analyzed. Results: We describe that mRNA synthesis of both LXRα and ABCA1 (a reverse cholesterol transporter) was induced by OA in human neutrophils. This fatty acid enhanced the effects of LXR ligands on ABCA1 and LXR expression, but it decreased the mRNA levels of sterol regulatory element-binding protein 1c (a transcription factor that regulates the synthesis of triglycerides). Although OA elicited a slight oxidative stress in the short term (15–30 min) in neutrophils, it is unlikely that this is relevant for the modulation of transcription in our experimental conditions, which involve longer incubation time (i.e., 6 h). Of physiological importance is our finding that OA depresses intracellular lipid levels and that markers of inflammation, such as ERK1/2 and p38 mitogen-activated protein kinase phosphorylation, were decreased by OA treatment. In addition, 200 μM OA reduced the migration of human neutrophils, another marker of the inflammatory state. However, OA did not affect lipid peroxidation induced by pro-oxidant agents. Conclusions: This work presents for the first time evidence that human neutrophils are highly sensitive to OA and provides novel data in support of a protective role of this monounsaturated acid against the activation of neutrophils during inflammation.

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.

Mécanismes moléculaires de régulation de l'activité du récepteur A des peptides natriurétiques

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.