kappaB Activation Research Articles

Bioactive constituents and medicinal importance of genus Alnus

The genus Alnus has been reviewed for its chemical constituents and biological activities including traditional importance of some common species. The plants of this genus contain terpenoids, flavonoids, diarylheptanoids, phenols, steroids, and tannins. Diarylheptanoids are the dominant constituents within the genus Alnus, few of them exhibited antioxidant effects and inhibitory activity against nuclear factor kappaB activation, nitric oxide and tumor necrosis factor-α production, human umbilical vein endothelial cells, farnesyl protein transferase, cell-mediated low-density lipoprotein oxidation, HIF-1 in AGS cells, and the HIV-1-induced cytopathic effect in MT-4 cells. Some ellagitannines showed hepatoprotective activity even in a dose of 1 mg/kg which is ten-fold smaller compared with the dose of traditional flavonoid-based drugs. The members of genus Alnus are well known for their traditional uses in the treatment of various diseases like cancer, hepatitis, inflammation of uterus, uterine cancer, rheumatism, dysentery, stomachache, diarrhea, fever, etc. The aim of the present review is to summarize the various researches related to the chemistry and pharmacology of genus Alnus.

Ghrelin promotes nuclear factor kappa-B activation in a human B-lymphocyte cell line

Ghrelin, an orexigenic hormone of gastric origin that stimulates growth hormone secretion, may modulate inflammation. This experimental study examines the effect of ghrelin on NFκB (p65 subunit), a transcriptional factor involved in inflammation on a human B-lymphocyte cell (WILCL). After confirming the expression of ghrelin receptor protein using western blotting the cells were transferred to wells maintaining a density of 1 × 10(6) cells per ml and a proportion activated with phytohaemagluttinin. Activated and resting cells were exposed to octanoyl-, desoctanoyl ghrelin and a non-peptide ghrelin agonist (Pfizer CP-464709) in increasing concentrations for 6 h. Cell protein extracts were analyzed for NFκB activation using Trans AM NFκB p65 assay. IL-6, IL-8, IL-10, IL-13 and TNFα were measured in the media using Lincoplex human cytokine assay. In octanoyl ghrelin treated resting cells, NFκB activity (Optical Density OD(450 nm)) (mean ± SEM) in control cells was 0.42 ± 0.10 and increased to 0.61 ± 0.20 (P = 0.044), 0.54 ± 0.10 (P = 0.043), 0.52 ± 0.08 at 1, 10 and 100 nM concentrations respectively. No effect was detected with desoctanoyl ghrelin or ghrelin agonist and no specific change in cytokine production. In conclusion, Octanoyl ghrelin increased NFκB activation by up to 50% in a B-lymphocyte cell line suggesting an effect on the inflammatory process.

Involvement of advanced glycation end products, oxidative stress and nuclear factor-kappaB in the development of diabetic keratopathy

The purpose of the experiment reported here was to assess the involvement of advanced glycation end products (AGEs), oxidative stress, and nuclear factor kappa-B (NF-κB) activation in the development of diabetic keratopathy. Diabetes was induced by intraperitoneal streptozotocin injection in male Sprague-Dawley rats. The thickness of the cornea was measured. Apoptosis was detected by TUNEL assay and western blot for caspase-3. The expression of AGEs and 8-hydroxydeoxyguanosine (8-OHdG) were studied by immunohistochemistry in corneal tissues of normoglycaemic and diabetic rats. NF-κB activation was evaluated by electrophoretic mobility shift assay and southwestern histochemistry. Corneal edema was observed in diabetic rats. The thickness of cornea was higher in diabetic than in control rats. AGEs were accumulated in corneal tissues. 8-OHdG and NF-κB were identified in corneal epithelium, stroma and endothelium, and its expressions were greater in diabetic than in those of control rats. Diabetes induces significant alterations in rat corneal tissue structure. The higher expression of AGE, 8-OHdG and NF-κB in corneal tissues of diabetic rats suggests that these factors are involved in apoptosis and in subsequent corneal alterations related to diabetic keratopathy.

Loss of tumor progression locus 2 (tpl2) enhances tumorigenesis and inflammation in two-stage skin carcinogenesis

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase in the mitogen-activated protein kinase signal transduction cascade known to regulate inflammatory pathways. Previously identified as an oncogene, its mutation or overexpression is reported in a variety of human cancers. To address its role in skin carcinogenesis, Tpl2(-/-) or wild-type (WT) C57BL/6 mice were subjected to a two-stage dimethylbenzanthracene/12-O-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis model. Tpl2(-/-) mice developed a significantly higher incidence of tumors (80%) than WT mice (17%), as well as a reduced tumor latency and a significantly higher number of total tumors (113 vs 6). Moreover, Tpl2(-/-) mice treated with TPA experienced significantly higher nuclear factor kappaB (NF-κB) activation, edema, infiltrating neutrophils and production of proinflammatory cytokines than did WT mice. We investigated the role of the p38, JNK, MEK and NF-κB signaling pathways both in vitro and in vivo in WT and Tpl2(-/-) mice by using inhibitors for each of these pathways. We confirmed that the proinflammatory effect in Tpl2(-/-) mice was due to heightened activity of the NF-κB pathway. These studies indicate that Tpl2 may serve more as a tumor suppressor than as an oncogene in chemically induced skin carcinogenesis, with its absence contributing to both tumorigenesis and inflammation.

Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury.

We investigated the neuroprotective effect of glucosamine (GlcN) in a rat middle cerebral artery occlusion model. At the highest dose used, intraperitoneal GlcN reduced infarct volume to 14.3% ± 7.4% that of untreated controls and afforded a reduction in motor impairment and neurological deficits. Neuroprotective effects were not reproduced by other amine sugars or acetylated-GlcN, and GlcN suppressed postischemic microglial activation. Moreover, GlcN suppressed lipopolysaccharide (LPS)-induced upregulation of proinflammatory mediators both in vivo and in culture systems using microglial or macrophage cells. The anti-inflammatory effects of GlcN were mainly attributable to its ability to inhibit nuclear factor kappaB (NF-κB) activation. GlcN inhibited LPS-induced nuclear translocation and DNA binding of p65 to both NF-κB consensus sequence and NF-κB binding sequence of inducible nitric oxide synthase promoter. In addition, we found that GlcN strongly repressed p65 transactivation in BV2 cells using Gal4-p65 chimeras system. P65 displayed increased O-GlcNAcylation in response to LPS; this effect was also reversed by GlcN. The LPS-induced increase in p65 O-GlcNAcylation was paralleled by an increase in interaction with O-GlcNAc transferase, which was reversed by GlcN. Finally, our results suggest that GlcN or its derivatives may serve as novel neuroprotective or anti-inflammatory agents.

Astrocytes contacting HIV‐1‐infected macrophages increase the release of CCL2 in response to the HIV‐1‐dependent enhancement of membrane‐associated TNFα in macrophages

The presence of human immunodeficiency virus (HIV)-infected macrophages in the parenchyma of central nervous system is an hallmark of acquired immunodeficiency syndrome-related neuroinflammation. Once penetrated the blood-brain barrier (BBB), macrophages closely interact with astrocytes, beginning with those lying beneath the BBB endothelium. By investigating the consequences of the cell-cell interaction between HIV-infected macrophages and astrocytes, we observed that the HIV-1 expression in macrophagic cells correlated with increased chemotactic activity in supernatants of astroglial cells. Gene array analysis revealed an impressive increase in the transcription of the gene for the CCL2/MCP-1 chemokine in astroglial cells isolated from HIV-1-infected co-cultures compared with cells from uninfected co-cultures. This phenomenon coupled with the increase in CCL2 release and depended on the cell-cell contact. In addition, it was a consequence of the HIV-1-induced enhancement of membrane-associated tumor necrosis factor-α in macrophagic cells, and correlated with increased levels of nuclear factor kappaB activation in astroglial cells. These observations could mirror a mechanism of recruitment of leukocytes through the BBB, likely contributing to the increase in both viral load and inflammation in central nervous system of HIV-infected patients.

A novel role of CCN3 in regulating endothelial inflammation

The vascular endothelium plays a fundamental role in the health and disease of the cardiovascular system. The molecular mechanisms regulating endothelial homeostasis, however, remain incompletely understood. CCN3, a member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators, has been shown to play an important role in numerous cell types. The function of CCN3 in endothelial cells has yet to be elucidated. Immunohistochemical analysis of CCN3 expression in mouse tissues revealed robust immunoreactivity in the endothelium of large arteries, small resistance vessels, and veins. We found that CCN3 expression in human umbilical vein endothelial cells (HUVECs) is transcriptionally induced by laminar shear stress (LSS) and HMG CoA-reductase inhibitors (statins). Promoter analyses identified the transcription factor Kruppel-like factor 2 (KLF2) as a direct regulator of CCN3 expression. In contrast to LSS, proinflammatory cytokines reduced CCN3 expression. Adenoviral overexpression of CCN3 in HUVEC markedly inhibited the cytokine-mediated induction of vascular adhesion molecule-1 (VCAM-1). Consistent with this observation, CCN3 significantly reduced monocyte adhesion. Conversely, CCN3 knockdown in HUVECs resulted in enhancement of cytokine-induced VCAM-1 expression. Concordant effects were observed on monocyte adhesion. Gain and loss-of-function mechanistic studies demonstrated that CCN3 negatively regulates nuclear factor kappaB (NF-κB) activity by reducing its translocation into the nucleus and subsequent binding to the VCAM-1 promoter, suggesting that CCN3's anti-inflammatory effects occur secondary to inhibition of NF-κB nuclear accumulation. This study identifies CCN3 as a novel regulator of endothelial proinflammatory activation.

Вплив інгібітора активації NF-kB куркуміну на оогенез і загибель фолікулярних клітин при імунному ушкодженні яєчників мишей

In experiments on CBA mice, we studied the influence of an inhibitor of nuclear transcription factor kappaB activation curcumin, obtained from Curcuma longa, on the meiotic maturation of oocytes and apoptotic and necrotic death of follicular cells at immune ovary failure induced by immunization of animals with allogenic ovarian extracts. NF-kappaB plays a pivotal role in the induction of genes encoding pro-inflammatory factors (cytokines, adhesion molecules, inducible NO-synthase and cyclooxygenase) and in regulation of cell proliferation and death. It has been shown that immunization of mice increased the death of follicular cells through anapoptotic and necrotic pathways, which led to inflammatory response (according to blood leukogram and impairment the oocyte meiotic maturation at metaphase I and II). Intragastric administration of curcumin (Sigma, USA, 2 mg of the mouse weight, four times a week during the period of immunization) reduced the number of the follicular cells died through apoptotic and especially necrotic pathway. Curcumin attenuated an inflammatory response and improved the meiotic maturation of oocytes impaired under experimental immune ovarian failure in mice.

Ubiquitin and Cancer: New Discussions for a New Journal

Ubiquitin and Cancer: New Discussions for a New Journal

Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB

The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor kappaB (NF-kappaB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-kappaB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-kappaB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells.

RETRACTED ARTICLE: A glucagon-like peptide-1 (GLP-1) analogue, liraglutide, upregulates nitric oxide production and exerts anti-inflammatory action in endothelial cells

Glucagon-like peptide-1 (GLP-1), a member of the proglucagon-derived peptide family, was seen to exert favourable actions on cardiovascular function in preclinical and clinical studies. The mechanisms through which GLP-1 modulates cardiovascular function are complex and incompletely understood. We thus investigated whether the GLP-1 analogue, liraglutide, which is an acylated GLP-1, has protective effects on vascular endothelial cells. Nitrite and nitrate were measured in medium with an automated nitric oxide detector. Endothelial nitric oxide synthase (eNOS) activation was assessed by evaluating the phosphorylation status of the enzyme and evaluating eNOS activity by citrulline synthesis. Nuclear factor kappaB (NF-kappaB) activation was assessed by reporter gene assay. Liraglutide dose-dependently increased nitric oxide production in HUVECs. It also caused eNOS phosphorylation, potentiated eNOS activity and restored the cytokine-induced downregulation of eNOS (also known as NOS3) mRNA levels, which is dependent on NF-kappaB activation. We therefore examined the effect of liraglutide on TNFalpha-induced NF-kappaB activation and NF-kappaB-dependent expression of proinflammatory genes. Liraglutide dose-dependently inhibited NF-kappaB activation and TNFalpha-induced IkappaB degradation. It also reduced TNFalpha-induced MCP-1 (also known as CCL2), VCAM1, ICAM1 and E-selectin mRNA expression. Liraglutide-induced enhancement of nitric oxide production and suppression of NF-kappaB activation were attenuated by the AMP-activated protein kinase (AMPK) inhibitor compound C or AMPK (also known as PRKAA1) small interfering RNA. Indeed, liraglutide induced phosphorylation of AMPK, which occurs through a signalling pathway independent of cyclic AMP. Liraglutide exerts an anti-inflammatory effect on vascular endothelial cells by increasing nitric oxide production and suppressing NF-kappaB activation, partly at least through AMPK activation. These effects may explain some of the observed vasoprotective properties of liraglutide, as well as its beneficial effects on the cardiovascular system.

Preserved Immune Functions and Controlled Leukocyte Oxidative Stress in Naturally Long-lived Mice: Possible Role of Nuclear Factor Kappa B

In order to verify the survival biomarker role of several immune functions, and to determine the oxidation and inflammation mechanisms underlying variability in the aging process, we have investigated a variety of immune functions and oxidative stress parameters as well as activation of the nuclear factor kappa B (NF kappaB) in peritoneal leukocytes from four different age groups of mice, including natural extreme longevity. Immune cells from naturally long-lived animals showed preservation of immune function in response to stimuli and controlled oxidative stress as well as nuclear factor kappa B activation in resting conditions. Moreover, leukocytes from extreme long-lived animals showed increased catalase activity when compared with the adults. In contrast, the old and very old animal groups showed impaired immune function and increased oxidation as well as NF kappaB activation. Our results support preserved immune function as a biomarker of extended survival and point to controlled regulation of NF kappaB activity as a key mechanism restraining oxidative stress in immune cells and contributing to reach longevity.

Phytoestrogen bavachin mediates anti-inflammation targeting IκB kinase-IκBα-NF-κB signaling pathway in chondrocytes in vitro

The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) plays critical roles in pathogenesis of osteoarthritis. Although estrogen is protective for cartilage in osteoarthritis patients, it also potentially increases the risk of stroke and cancer. Phytoestrogens acting as natural estrogen receptor modulators may serve as alternatives. This study aimed to identify medicinal phytoestrogens that preserve anti-inflammatory property and may function as potential chondro-protective compounds. Both human chondrocytes and chondrocytic cell line CHON-002 were used for this study. Protein concentrations or expressions were measured by ELISA or Western blot, respectively. The DNA-binding activity and transcriptional activity of transcription factors were evaluated by electrophoretic mobility shift assay and dual-luciferase reporter assay, respectively. Cell migration was analyzed by chemotaxis assays. We found that among screened phytoestrogens, bavachin could potently decrease IL-1 beta-induced nuclear factor-kappa B (NF-kappaB) but not activator protein-1 (AP-1) DNA-binding activity. Bavachin also inhibited I kappaB alpha degradation, increased nuclear translocation of p65 and p50 as well as decreased I kappaB alpha kinase (IKK) activity. Furthermore, bavachin inhibited IL-1 beta-induced chemokine production that resulted in reduced migration of THP-1 monocytic cells. Our results suggest that through decreasing IL-1 beta-induced activation of IKK-I kappaB alpha-NF-kappaB signaling pathway, bavachin potentially protects cartilage from inflammation-mediated damage in joints of osteoarthritis patients.

Fluoxetine Induces Apoptosis in Ovarian Carcinoma Cell Line OVCAR‐3 Through Reactive Oxygen Species‐Dependent Activation of Nuclear Factor‐κB

The apoptotic effect of fluoxetine (FLX), an antidepressant, against human epithelial ovarian cancer cell lines OVCAR-3 and SK-OV-3 was investigated in relation to the mitochondria-mediated cell death process and nuclear factor (NF)-kappaB activation. FLX-induced mitochondrial membrane permeability change and formation of reactive oxygen species, leading to cell death. FLX-induced increase in mitochondrial Bax levels, decrease in cytosolic Bid and Bcl-2 levels, loss of the mitochondrial transmembrane potential, cytochrome c release, caspase-3 activation and up-regulation of p53. Oxidant scavengers and Bay 11-7085 [an inhibitor of nuclear factor kappaB (NF-kappaB) activation] prevented the FLX-induced cell death, increase in phosphorylated inhibitory kappaB-alpha and NF-kappaB p65 levels, and binding of NF-kappaB p65 to DNA. Results from this study suggest that FLX may exhibit apoptotic effect against ovarian cancer cell lines by inducing the mitochondrial membrane permeability change, which leads to cytochrome c release and subsequent caspase-3 activation, through reactive oxygen species-dependent activation of NF-kappaB.

Mutations in modified virus Ankara protein 183 render it a non-functional counterpart of B14, an inhibitor of nuclear factor B activation

Vaccinia virus (VACV) encodes multiple proteins to evade host innate immunity, including B14, a virulence factor that binds to the inhibitor of κB kinase β (IKKβ) and blocks nuclear factor κB (NF-κB) activation. B14 shares 95 % amino acid identity with the 183 protein encoded by modified virus Ankara (MVA), an attenuated VACV strain being developed as a vaccine vector. To evaluate whether the immunogenicity of MVA might be increased by manipulation of MVA immunomodulatory proteins, the MVA counterpart of B14, protein 183, was characterized. Unlike B14, protein 183 was unstable in eukaryotic cells unless proteasome-mediated protein degradation was inhibited. Furthermore, 183 did not inhibit NF-κB activation in response to cytokine stimulation, and did not restore the virulence of VACV strain Western Reserve lacking gene B14R. The instability and non-functionality of 183 are probably explained by a deletion of 6 aa within α-helix 6 of the B14 crystal structure.

α-LIPOIC ACID AS A POTENTIAL TARGET FOR THE TREATMENT OF LUNG INJURY CAUSED BY CECAL LIGATION AND PUNCTURE-INDUCED SEPSIS MODEL IN RATS

One of the common lethal complications of septic shock, a major cause of morbidity and mortality in patients with severe trauma and so on, is acute lung injury. alpha-Lipoic acid (ALA), with antioxidant properties, is a popular agent. Thus, we investigated the potential protective effects of ALA (200 mg/kg) on sepsis-induced acute lung injury. Rats were exposed to cecal ligation and puncture (CLP) to induce sepsis. Rat groups were designed as (a) sham operated, (b) sham operated + ALA treated, (c) CLP applied, (d) CLP + ALA treated. Sixteen hours after CLP induction, serum samples and lung tissues were obtained for biochemical and histopathological examination. alpha-Lipoic acid decreased the serum levels of inflammatory cytokines such as TNF-alpha and IL-6, which increased after CLP. Increased activity of nuclear factor kappaB in septic lung tissues was decreased by ALA. alpha-Lipoic acid improved the decreased antioxidant activity and alleviated the increased oxidant activity, which occurred after CLP application. We can suggest that ALA showed beneficial effects by decreasing nuclear factor kappaB activation in lung tissues, resulting in decreased serum levels of TNF-alpha and IL-6, and also increasing the antioxidant capacity of the lungs.

M1669 Inhibition of Acid Sphingomyelinase Causes Hepatic Glucose Intolerance and Insulin Resistance

Background and Aims: Acid sphingomyelinase (ASM) regulates sphingolipids homeostasis including ceramides and sphingosine-1-phosphate. Although the sphingolipids are involved in AKT activation, little information is available for the role of ASM in glucose and lipid metabolism. To explore this issue, we investigated glucose and lipid metabolisms in ASMinhibited hepatocytes. Methods: Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed on ASM+/+ and ASM-/mice. Primary cultured hepatocytes were isolated and the effects of ASM inhibitor imipramine or ASM deficiency were examined for AKT activation, glucose up-take, glycogen deposition, and lipid accumulation. Results: Food intake decreased most of sphingomyelin species in mice liver. IPGTT and ITT showed glucose intolerance accompanied with hepatic insulin resistance in ASM-/mice. In primary cultured hepatocytes, insulin treatment alone was unable to increase glucose uptake and glycogen because the cells had high basal nuclear factor (NF)-kappaB activity due to the stimulation by the culturing on collagen-coated plastic dishes. When NF-kappaB was inhibited by either pretreatment of the IkappaB kinase inhibitor PS-1145 or introduction of an adenoviral IkappaB super repressor (Ad5IkappaB), insulin treatment increased glucose uptake and glycogen levels in the hepatocytes. Thus, Ad5IkappaB-infected hepatocytes were used in case of insulin treatment. Pretreatment with imipramine inhibited glucose uptake by insulin, and also inhibited glycogen deposition by insulin or high dose of glucose in rat hepatocytes as did the knockout of ASM in mouse hepatocytes. Moreover, either insulin or high dose of glucose treatment increased lipid droplets in primary hepatocytes, and imipramine or ASM deficiency suppressed this increase. Insulin induced phosphorylation of AKT in primary hepatocytes, and overexpression of dominant negative-AKT by adenovirus abrogated the phosphorylation of endogenous AKT by insulin. DN-AKT inhibited glucose uptake, glycogen deposition, and lipid accumulation by insulin or high dose of glucose suggesting that AKT has central roles in hepatic glucose and lipid metabolisms. However, pretreatment of imipramine or knockout of ASM did not affect AKT phosphorylation induced by insulin. Conclusion: ASM deficiency causes glucose intolerance and hepatic insulin resistance without affecting insulin-induced AKT activation in hepatocytes.

M1672 An Analysis of CD133 Positive Bone Marrow in Oval Cell Induced Model and a Role of CD133 Bone Marrow in Mice With Liver Fibrosis

Background and Aims: Acid sphingomyelinase (ASM) regulates sphingolipids homeostasis including ceramides and sphingosine-1-phosphate. Although the sphingolipids are involved in AKT activation, little information is available for the role of ASM in glucose and lipid metabolism. To explore this issue, we investigated glucose and lipid metabolisms in ASMinhibited hepatocytes. Methods: Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed on ASM+/+ and ASM-/mice. Primary cultured hepatocytes were isolated and the effects of ASM inhibitor imipramine or ASM deficiency were examined for AKT activation, glucose up-take, glycogen deposition, and lipid accumulation. Results: Food intake decreased most of sphingomyelin species in mice liver. IPGTT and ITT showed glucose intolerance accompanied with hepatic insulin resistance in ASM-/mice. In primary cultured hepatocytes, insulin treatment alone was unable to increase glucose uptake and glycogen because the cells had high basal nuclear factor (NF)-kappaB activity due to the stimulation by the culturing on collagen-coated plastic dishes. When NF-kappaB was inhibited by either pretreatment of the IkappaB kinase inhibitor PS-1145 or introduction of an adenoviral IkappaB super repressor (Ad5IkappaB), insulin treatment increased glucose uptake and glycogen levels in the hepatocytes. Thus, Ad5IkappaB-infected hepatocytes were used in case of insulin treatment. Pretreatment with imipramine inhibited glucose uptake by insulin, and also inhibited glycogen deposition by insulin or high dose of glucose in rat hepatocytes as did the knockout of ASM in mouse hepatocytes. Moreover, either insulin or high dose of glucose treatment increased lipid droplets in primary hepatocytes, and imipramine or ASM deficiency suppressed this increase. Insulin induced phosphorylation of AKT in primary hepatocytes, and overexpression of dominant negative-AKT by adenovirus abrogated the phosphorylation of endogenous AKT by insulin. DN-AKT inhibited glucose uptake, glycogen deposition, and lipid accumulation by insulin or high dose of glucose suggesting that AKT has central roles in hepatic glucose and lipid metabolisms. However, pretreatment of imipramine or knockout of ASM did not affect AKT phosphorylation induced by insulin. Conclusion: ASM deficiency causes glucose intolerance and hepatic insulin resistance without affecting insulin-induced AKT activation in hepatocytes.

Paired Immunoglobulin-Like Receptor B (PIR-B) Negatively Regulates Macrophage Activation in Experimental Colitis

Innate and adaptive immune responses are regulated by cross talk between activation and inhibitory signals. Dysregulation of the inhibitory signal can lead to aberrant chronic inflammatory diseases such as the inflammatory bowel diseases (IBD). Little is known about negative regulation of innate intestinal immune activation. We examined the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of macrophage function in innate intestinal immunity. We examined the susceptibility of Pirb-/- and wild-type (WT) mice to dextran sodium sulfate (DSS)-induced colitis. We assessed proinflammatory cytokine release and mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) activation in Pirb-/- and WT macrophages following Escherichia coli stimulation. Macrophage transfer experiments were performed to define the role of PIR-B in the negative regulation of macrophage function in DSS-induced colitis. We also assessed expression of PIR-B human homologues (immunoglobulin-like transcript [ILT]-2 and ILT-3) in colon biopsy samples from healthy individuals (controls) and patients with IBD. Pirb-/- mice had increased susceptibility to DSS-induced colitis. In vitro analysis showed increased production of proinflammatory cytokines (interleukin-6, interleukin-1beta, and tumor necrosis factor alpha) and activation of MAPK and NF-kappaB in Pirb-/- macrophages following bacterial activation. Adoptive transfer of bone marrow-derived Pirb-/- macrophages into WT mice was sufficient to increase disease susceptibility. ILT-2 and ILT-3 were expressed on CD68+ and CD68- mononuclear cells and intestinal epithelium in colon biopsy samples from patients and controls. PIR-B negatively regulates macrophage functions in response to pathogenic bacteria and chronic intestinal inflammatory responses. Inhibitory receptors such as PIR-B might be used as therapeutic targets for treatment of patients with IBD.

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes.

Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. mRNA and protein for A(1), A(2A), A(2B) and A(3) adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors. Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status. These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.