Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nuclear factor kappa-B (NF-kappa B) has been shown to play an important role in LPS-mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP-dependent protein kinase in the regulation of NF-kappa B activity. In this study we have investigated the mechanism of NF-kappa B induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF-alpha NF-kappa B element was transfected into the RAW264 macrophage-like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8-bromo cAMP. To further study NF-kappa B induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF-kappa B binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA-binding activity confirmed these results and indicated that LPS induced a 55 KD DNA-binding protein. Induction of this NF-kappa B binding activity was not inhibited by pretreatment with the PKC inhibitor H-7. H-7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down-regulates PKC, had no effect on LPS induction of NF-kappa B activity in these cells. These results suggest that the induction of NF-kappa B in macrophages by LPS is independent of PKC.
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PMID:Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester. 173 Jul 83

Asthma is a disease of airway inflammation and hyper-reactivity associated with lymphocytic infiltration in the bronchial submucosa. We recently demonstrated that human airway smooth muscle (ASM) cells express the cell adhesion molecules ICAM-1 and VCAM-1, which are up-regulated by cytokines such as TNF-alpha, and which mediate binding of activated T lymphocytes. In this study, we examined whether an increase in [cAMP]i, presumably via activation of cAMP-dependent protein kinase, modulates TNF-alpha-induced ICAM-1 and VCAM-1 on ASM. We found that treatment of ASM with either forskolin, which directly activates adenylyl cyclase, or with cholera toxin, which activates the heterotrimeric GTP-binding protein, Gs, inhibited TNF-alpha-induced cell adhesion molecule expression. In addition, treatment with either isoproterenol or prostaglandin E2, which activates receptors coupled to Gs and increases [cAMP]i, also inhibited TNF-alpha-induced expression of ICAM-1 and VCAM-1 on ASM. Furthermore, adhesion of activated T cells to TNF-alpha-stimulated ASM was inhibited by treating the ASM cells with either forskolin or PGE2. These data suggest that cAMP-dependent protein kinase activation decreases cytokine-induced expression of cell adhesion molecules on ASM cells, modulates T cell binding to airway myocytes and, thus, suggests novel therapeutic approaches to airway inflammation.
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PMID:Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF-alpha-induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion. 753 67

Increased synthesis of insulin-like growth factor I (IGF-I), a fibroblast growth factor, is induced in murine macrophages by TNF-alpha. TNF-alpha also induces macrophages to express cytocidal activity, but only during costimulation with IFNs. Since prostaglandin E2 (PGE2) is known to inhibit macrophage cytocidal activity, its possible reciprocal enhancement of IGF-I synthesis was examined. PGE2 or dibutyryl cyclic AMP (dbcAMP) stimulated the synthesis of IGF-I similarly to TNF-alpha in magnitude and time course. TNF-alpha did not increase IGF-I synthesis by first inducing PGE2 synthesis, because indomethacin was unable to block the effect of TNF-alpha. PGE2 did not stimulate IGF-I synthesis by first inducing TNF-alpha production, because 1) anti-TNF-alpha Ab did not block PGE2-induced IGF-I synthesis, and 2) PGE2 down-regulated TNF-alpha mRNA levels and did not affect levels of the cytokine in supernatants. Moreover, the difference in the induction of IGF-I was observed at the level of signal transduction, in that PGE2 and dbcAMP increased cAMP-dependent protein kinase (PKA) activity, whereas TNF-alpha stimulated the mitogen-activated protein (MAP) kinase pathway. Divergence between the two pathways was also noted in the regulation of IGF-I at the mRNA level, and an additive effect on IGF-I synthesis was observed when cells were incubated with the combination of TNF-alpha plus PGE2 or dbcAMP. Collectively, these data suggest that TNF-alpha and PGE2 stimulate IGF-I synthesis in macrophages by two separate pathways, and that PGE2 acts as a positive stimulus for IGF-I synthesis through a cyclic AMP/PKA pathway.
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PMID:Divergence in macrophage insulin-like growth factor-I (IGF-I) synthesis induced by TNF-alpha and prostaglandin E2. 763 60

Macrophages treated with IFN-gamma alone are stimulated to produce nitric oxide. The level of nitric oxide production can be enhanced significantly when IFN-gamma treatment is combined with other agents (e.g., LPS, TNF-alpha, IL-2, etc.). We tested the hypothesis that cAMP plays a role in the IFN-gamma-induced activation of macrophages. Our experiments indicate that factors that increase the concentration of cAMP in the murine macrophage cell line ANA-1 can also enhance IFN-gamma-induced production of nitric oxide. PGE2 and cholera toxin increased the production of nitrite (an indicator of nitric oxide production) in IFN-gamma-treated ANA-1 macrophages by at least twofold. These factors produced no increase in nitric oxide production in the absence of IFN-gamma treatment. The increase in nitric oxide production corresponded to an increase in the accumulation of nitric oxide synthase mRNA without a change in stability of mRNA. Dibutyryl cAMP and Sp-cAMPs (a selective activator of cAMP-dependent protein kinase I and II) also increased nitric oxide production in IFN-gamma-treated macrophages. However, at very high concentrations (i.e., >100 microM), the stimulatory effect was decreased. These studies indicate that elevation of intracellular cAMP causes a dose-dependent, biphasic alteration of IFN-gamma-induced nitric oxide production in murine macrophages. Moreover, they suggest that agents that affect nitric oxide synthesis may do so via modulation of the cAMP second messenger system.
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PMID:An increase in intracellular cyclic AMP modulates nitric oxide production in IFN-gamma-treated macrophages. 899 9

The purpose of this study was to examine whether rCGRP has effects on TNF-alpha produced by mouse resident peritoneal macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5x10(5) cells per well and allowed to adhere for 2 hr. Pretreatment with rCGRP (10 nM-1 microM) for 24 hr, the macrophages were cultured with LPS 1 microg/ml for another 24 h. The medium was harvested for measuring TNF-alpha by ELISA kits. The results showed that rCGRP had no direct effects on TNF-alpha production, but it inhibited LPS-induced TNF-alpha production in a concentration-dependent manner. When rCGRP was at a concentration of 1 microM, the LPS-induced TNF-alpha production was inhibited by 39%. The effect of rCGRP was reversed by hCGRP(8-37) (10 microM), an antagonist of CGRP1 receptor. The LPS-induced TNF-alpha production from macrophages was also inhibited by forskolin 3 microM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 microM or Rp-cAMPS 100 microM, the inhibitors of cAMP-dependent protein kinase, the effect of rCGRP was abolished. These data suggest that the LPS-induced TNF-alpha production is inhibited by rCGRP via activation of cAMP responses in mouse resident peritoneal macrophages.
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PMID:Inhibition of LPS-induced TNF-alpha production by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages. 936 15

Excessive proinflammatory cytokine and NO production by activated microglia play a role in neurodegenerative disorders. To investigate whether the neuroprotectant N-acetyl-O-methyldopamine (NAMDA) downregulates genes associated with microglial activation, we measured gene expression of TNF-alpha, IL-1beta, inducible nitric oxide synthase (NOS2), and an associated cofactor synthesis gene, GTP cyclohydrolase I (GTPCH) in LPS-stimulated microglia cells in the presence or absence of NAMDA. The temporal pattern of cytokine gene expression showed that LPS (0.2 microg/ml) increased TNF-alpha and IL-1beta gene expression at 1 and 3 h, which was repressed by cotreatment of NAMDA. Similarly, LPS also induced GTPCH and NOS2 gene expression at 3 and 6 h, and cotreatment of NAMDA repressed the induction with parallel reduction of nitrite, an oxidative metabolite of nitric oxide. Since transcription factor NF-kappaB is involved in regulating expression of these genes, the effects of NAMDA on NF-kappaB nuclear translocation and DNA binding in immunostimulated microglia were investigated. We found that neither LPS-induced NF-kappaB translocation nor DNA binding activity was affected by cotreatment with NAMDA in BV-2 microglia. On the other hand, NAMDA increased intracellular cAMP levels and potentiated LPS-induced phosphorylated cAMP-responsive element binding protein (pCREB) expression. Treatment with adenosine 3'5'-cyclic monophosphothioate, a specific inhibitor of cAMP-dependent protein kinase (PKA), reversed not only NAMDA-induced pCREB upregulation but also NAMDA-induced repression of TNF-alpha and IL-1beta gene transcription. The data demonstrate that NAMDA represses LPS-induced proinflammatory cytokines gene expression via a cAMP-dependent protein kinase pathway. Thus, repressing proinflammatory cytokines and NOS2 gene expression in activated microglia by NAMDA may provide new therapeutic strategies for ischemic cerebral disease as well as other neurodegenerative diseases.
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PMID:Repression of proinflammatory cytokine and inducible nitric oxide synthase (NOS2) gene expression in activated microglia by N-acetyl-O-methyldopamine: protein kinase A-dependent mechanism. 1124 31

Insulin is a potent regulator of Sp1 transcription factor. To examine if glucagon, which usually antagonizes insulin, regulates Sp1, we assessed the levels of Sp1 by Western blotting from H-411E cells exposed to glucagon with or without insulin. Glucagon alone (1.5 x 10(-9) to 1.5 x 10(-5) M) stimulated Sp1 accumulation but inhibited insulin's (10,000 microU/ml) stimulatory effect on Sp1. We also assessed the effect of TNF-alpha, wortmannin, a PI3K inhibitor, and cAMP-dependent protein kinase inhibitor on Sp1 accumulation. While TNF-alpha (5 ng/ml) blocked insulin-stimulated Sp1, it failed to block stimulation of Sp1 by glucagon (1.5 x 10(-5) M). Similarly, wortmannin inhibited insulin- but not glucagon-stimulated Sp1, whereas protein kinase inhibitor had an opposite effect. Thus, insulin acts primarily via PI3K, and glucagon apparently stimulates through a cAMP-dependent pathway. Insulin increased the staining intensity of Sp1 seen exclusively in the nuclei of H-411E cells. Sp1 was demonstrable in both nucleus and cytoplasm after glucagon treatment. Finally, as judged by immunoblotting to specific antibody, insulin but not glucagon, stimulated O-glycosylation of Sp1. Thus, unique signaling mechanisms mediate the response of Sp1 to glucagon in the presence or absence of insulin.
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PMID:Paradoxical regulation of Sp1 transcription factor by glucagon. 1189 10

Hormone-sensitive lipase (HSL) is presumed to be essential for lipolysis, which is defined as the mobilization of free fatty acids from adipocytes. In the present study, we investigated the effects of various lipolytic hormones on the lipolysis in adipocytes derived from mouse embryonic fibroblasts (MEF adipocytes) prepared from HSL-deficient mice (HSL-/-). HSL-/- MEF differentiated into mature adipocytes in a manner indistinguishable from that of wild-type mice. Both isoproterenol (ISO) and tumor necrosis factor (TNF)-alpha stimulated the rate of lipolysis in HSL-/- MEF adipocytes, although to a lesser extent than in wild-type cells, and these lipolytic activities were inhibited by H-89, a cAMP-dependent protein kinase inhibitor, and troglitazone, respectively. Thus, the responses of the residual lipolytic activity to lipolytic hormones and TNF-alpha were well conserved in the absence of HSL. Extracts from HSL-/- MEF adipocytes hydrolyzed triacylglycerol (TG) but not cholesterol ester, indicating that the residual lipolytic activity was mediated by another TG-specific lipase. The TG lipase activity, which was decreased in cytosolic fraction in response to ISO, was increased in fat cake fraction. Therefore, translocation of the TG lipase may explain, at least partially, the ISO-stimulated lipolysis in HSL-/- adipocytes. In conclusion, lipolysis is mediated not only by HSL but also by the non-HSL TG lipase, whose responses to lipolytic hormones are similar to those of HSL. We propose that both lipases are regulated by common mechanism of lipolysis.
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PMID:Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases. 1245 88

(1) Fractalkine is a CX(3)C chemokine for mononuclear leukocytes that is expressed mainly by vascular cells, and regulated by pro-inflammatory cytokines. This study investigated signal transduction mechanisms by which tumor necrosis factor (TNF)-alpha stimulated fractalkine expression in cultured rat vascular smooth muscle cells (VSMCs), and the modulatory effect of a haemorrheologic agent, pentoxifylline, on its production. (2) TNF-alpha (1-50 ng ml(-1)) stimulated fractalkine mRNA and protein expression in concentration- and time-dependent manners. Pretreatment with calphostin C (0.4 micro M, a selective inhibitor of protein kinase C (PKC), and PD98059 (40 micro M), a specific inhibitor of p42/44 mitogen-activated protein kinase (MAPK) kinase, attenuated TNF-alpha-stimulated fractalkine mRNA and protein expression. In contrast, H-89 (2 micro M), a selective inhibitor of cAMP-dependent protein kinase, wortmannin (0.5 micro M), a selective inhibitor of phosphatidylinositol 3-kinase, and SB203580 (40 micro M), a specific inhibitor of p38 MAPK, had no discernible effect. (3) The ubiquitin/proteosome inhibitors, MG132 (10 micro M) and pyrrolidine dithiocarbamate (200 micro M), suppressed activation of NF-kappaB as well as stimulation of fractalkine mRNA and protein expression by TNF-alpha. (4) TNF-alpha-activated phosphorylation of PKC was blocked by calphostin C, whereas TNF-alpha-augmented phospho-p42/44 MAPK and phospho-c-Jun levels were reduced by PD98059. Neither calphostin C nor PD98059 affected TNF-alpha-induced degradation of I-kappaBalpha or p65 nuclear translocation. (5) Pretreatment with pentoxifylline (0.1-1 mg ml(-1)) decreased TNF-alpha-stimulated fractalkine mRNA and protein expression, which was preceded by a reduction in TNF-alpha-activated phosphorylation of PKC, p42/44 MAPK and c-Jun as well as degradation of I-kappaBalpha and p65/NF-kappaB nuclear translocation. (6) These data indicate that activation of PKC, p42/44 MAPK kinase, and NF-kappaB are involved in TNF-alpha-stimulated fractalkine production in VSMCs. Down-regulation of the PKC, p42/44 MAPK, and p65/NF-kappaB signals by PTX may be therapeutically relevant and provide an explanation for the anti-fractalkine effect of this drug.
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PMID:Inhibition by pentoxifylline of TNF-alpha-stimulated fractalkine production in vascular smooth muscle cells: evidence for mediation by NF-kappa B down-regulation. 1264 97

Atherosclerosis involves cellular immune responses and altered vascular smooth muscle cell (VSMC) function. Nitric oxide (NO)/cGMP is uniquely capable of inhibiting key processes in atherosclerosis. In this study, we determined the effects of NO/cGMP and their molecular mechanisms in the regulation of NF-kappaB-dependent gene expression in VSMCs. We found that cGMP-elevating agents such as the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and C-type natriuretic peptide (CNP), reduced TNF-alpha-induced NF-kappaB-dependent reporter gene expression in rat aortic VSMCs in a cGMP-dependent manner. The effects of SNAP and CNP on NF-kappaB are mediated by cAMP-dependent protein kinase (PKA) but not cGMP-dependent protein kinase (PKG) based on the findings that the selective PKA inhibitor, PKI, abolished the effects of SNAP and CNP on NF-kappaB, whereas the PKG inhibitor Rp-8-Br-PET-cGMP had no effect. Inhibition of cGMP-inhibited cAMP-hydrolyzing phosphodiesterase 3 (PDE3) blocked SNAP- and CNP-elicited effects on NF-kappaB-dependent transcription. Furthermore, cGMP analogues such as 8-pCPT-cGMP, which selectively activates PKG but does not inhibit PDE3, had no effect on NF-kappaB-mediated transcription. Activation of PKA by SNAP or cAMP-elevating agents not only inhibited TNF-alpha-induced NF-kappaB-dependent reporter gene expression but also reduced endogenous NF-kappaB-dependent adhesion molecule and chemokine expression. These results suggest that SNAP and CNP exert inhibitory effects on NF-kappaB-dependent transcription by activation of PKA via cGMP-dependent inhibition of PDE3 activity. Therefore, PDE3 is a novel mediator of inflammation in VSMCs.
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PMID:Role of phosphodiesterase 3 in NO/cGMP-mediated antiinflammatory effects in vascular smooth muscle cells. 1291 48


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