Identification of manganese superoxide dismutase as a NO-regulated gene in rat glomerular mesangial cells by 2D gel electrophoresis.

The course of inflammatory glomerular diseases is accompanied by changes in the expression of matrix-associated proteins, growth factors, and mediators in renal mesangial cells. Furthermore, the production of nitric oxide (NO) by the inducible isoform of nitric oxide synthase (iNOS) is enhanced after stimulation with pro-inflammatory cytokines. NO has been demonstrated to be a potent modulator of gene expression. To identify NO-regulated genes, we compared the expression patterns of mesangial cells treated for 24h with 500 microM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) with those of un-stimulated controls by applying a proteomics approach. One protein found to be NO-modulated by 2D gel electrophoresis is the manganese superoxide dismutase (Mn-SOD). Immunoblot and Northern blot analysis demonstrated a dose- and time-dependent induction of Mn-SOD expression by S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and DETA-NO on both the protein and the mRNA levels. An upregulation of Mn-SOD expression by NO was accompanied by an increased Mn-SOD activity. Immunoblots of extracts of IL-1beta-treated cells cultivated with or without the iNOS inhibitor N(G)-monomethyl-L-arginine and the inhibitor of soluble guanylyl cyclase (sGC) 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) demonstrated that the upregulation of the Mn-SOD by NO is due to a NO-dependent activation of sGC. The upregulation of Mn-SOD mRNA expression by NO was confirmed in vivo by Northern blot analysis in kidneys from rats treated with lipopolysaccharide (LPS) either in presence or absence of the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (l-NIL).     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

Keratinocyte-derived chemotactic cytokines: expressional modulation by nitric oxide in vitro and during cutaneous wound repair in vivo

Inhibition of inducible nitric oxide-synthase (iNOS) enzymatic activity during cutaneous wound repair leads to severely impaired tissue regeneration. To assess whether disturbed leukocyte infiltration might participate in impaired repair, we determined expressional kinetics of neutrophil-attracting macrophage inflammatory protein-2 (MIP-2), and monocyte-attracting macrophage chemoattractant protein-1 (MCP-1) using an excisional wound healing model in mice. MCP-1 was induced in epithelial keratinocytes upon wounding, and our data indicate that NO serves a negative regulatory role for MCP-1 expression in vivo, as clearly reduced numbers of wound margin keratinocytes associated with NO-deficient repair compensate for high MCP-1 expression levels observed during normal healing. MIP-2 expression was restricted to hair follicles which were not reduced in number during NO-deficient repair. In vitro studies confirmed a regulatory role of NO for keratinocyte-derived chemokine expression, as NO attenuated IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression, whereas NO augmented IL-1beta-induced IL-8 (functional human homolog to murine MIP-2) mRNA expression in the human keratinocyte cell line HaCaT.     

Calpain mediates a von Hippel-Lindau protein-independent destruction of hypoxia-inducible factor-1alpha

Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1alpha requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1alpha expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2'-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1alpha protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1alpha degradation. Furthermore, chelating intracellular calcium attenuated HIF-1alpha destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1alpha even under normoxia. An active role of calpain in lowering HIF-1alpha amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1alpha that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1alpha destruction involving the calpain system.     

Differential pattern of inflammatory molecule regulation in intestinal epithelial cells stimulated with IL-1

To better predict the consequences of blocking signal transduction pathways as a means of controlling intestinal inflammation, we are characterizing the pathways up-regulated by IL-1 in intestinal epithelial cells (IEC). IL-1beta induced increased mRNA levels of MIP-2, MCP-1, RANTES, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) in the IEC-18 cell line. IL-1beta activated NF-kappaB but not ERK or p38. Infecting cells with adenovirus expressing a mutated gene for IkappaBalpha (IkappaBAA) blocked IL-1-induced mRNA increases in MIP-2, MCP-1, and iNOS but not COX-2 or RANTES. Expression of IkappaBAA attenuated the IL-1-induced increase in COX-2 protein. Unexpectedly, RANTES mRNA increased, and protein was secreted by cells expressing IkappaBAA in the absence of IL-1. Adenovirus-expressing IkappaBAA, blocking protein synthesis, and IL-1beta all resulted in activation of JNK. The JNK inhibitor SP600125 prevented the RANTES increases by all three stimuli. A human enterocyte line was similarly examined, and both NF-kappaB and JNK regulate IL-1-induced RANTES secretion. We conclude that in IEC-18, IL-1beta-induced increases in mRNA for MIP-2, MCP-1, and iNOS are NF-kappaB-dependent, whereas regulation of RANTES mRNA is independent of NF-kappaB but is positively regulated by JNK. IL-1beta-induced mRNA increases in COX-2 mRNA are both NF-kappaB- and MAPK-independent but the translation of COX-2 protein is NF-kappaB-dependent. This pattern of signaling due to a single stimulus exposed the complexities of regulating inflammatory genes in IEC.     

Cilostazol enhances IL-1beta-induced NO production and apoptosis in rat vascular smooth muscle via PKA-dependent pathway

Interleukin-1beta (IL-1beta) stimulates nitric oxide (NO) production and induces apoptosis in several tissues. Cilostazol is a Type 3 phosphodiesterase inhibitor. We investigated whether cilostazol affects IL-1beta-induced NO production and apoptosis in rat vascular smooth muscle cells. Cilostazol (100 nM-10 microM) potentiated NO production triggered by IL-1beta. The mRNA and protein expression of inducible NO synthase was also upregulated by cilostazol. KT5720, an inhibitor of protein kinase A, and N(G)-monomethyl-L-arginine, an inhibitor of NO synthase, abrogated cilostazol-enhanced IL-1beta-stimulated NO production and apoptosis. These results shows that cilostazol potentiates IL-1beta-induced NO production via PKA-pathway and thereafter augments apoptosis via NO-dependent pathway.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

Inhibitory mechanisms of highly purified vitamin B2 on the productions of proinflammatory cytokine and NO in endotoxin-induced shock in mice

Inhibitory effects of highly purified vitamin B2 (riboflavin-5'-sodium phosphate, >97%) on the interleukin (IL)-6, macrophage inflammatory protein (MIP)-2 and nitric oxide (NO) in LPS-induced shock mice were evaluated. Vitamin B2 at 20 mg/kg (protective effect on mice mortality induced by LPS), intravenously administered 6 h after LPS injection, significantly decreased the plasma elevated levels of IL-6 and MIP-2 at 9 and 12 h. In addition, vitamin B2 lowered the tissue concentration and the mRNA expression of IL-6 in lung and those of MIP-2 in liver at 9 h. Vitamin B2 also reduced concentration of MIP-2 concentration in lung, and inhibited mRNA expression in kidney, respectively. Vitamin B2 decreased the plasma elevated NO levels in accordance with a reduction in expression of inducible NO synthase (iNOS) both at 21 and 24 h. Accordingly, the reduction in elevated plasma cytokine levels and NO based on the inhibitory effect on local cytokine mRNA expression and iNOS would be responsible for the anti-septic effect of vitamin B2.     

Nitric oxide modulates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules

Although platelets have been implicated in the pathogenesis of vascular diseases, little is known about factors that regulate interactions between platelets and the vessel wall under physiological conditions. The objectives of this study were to 1) define the contribution of nitric oxide (NO) to endotoxin (lipopolysaccharide, LPS)-induced platelet-endothelial cell (P/E) adhesion in murine intestinal venules and 2) determine whether the antiadhesive action of NO is mediated by soluble guanylate cyclase (sGC). Adhesive interactions between platelets and endothelial cells were monitored by intravital microscopy. LPS administration into control wild-type mice (WT) resulted in a >15-fold increase in P/E adhesion. Similar responses were observed using endothelial NO synthase (eNOS)-deficient platelets. However, treatment with the NO donor diethylenetriamine-nitric oxide (DETA-NO) attenuated the P/E adhesion response to LPS, whereas the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester or eNOS deficiency resulted in an exacerbation. P/E adhesion response did not differ between LPS-treated WT and inducible NOS-deficient mice. Inhibition of sGC abolished the attenuating effects of DETA-NO, whereas the sGC activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) reduced LPS-induced P/E adhesion. These findings indicate that 1) eNOS-derived NO attenuates endotoxin-induced P/E adhesion and 2) sGC is responsible for the antiadhesive action of NO.     

Reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide

In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (-log EC(50) 6.55 +/- 0.12 and 5.02 +/- 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGCalpha(1) and sGCbeta(1). sGCbeta(1) protein expression was 42 +/- 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGCbeta(1) protein expression (36 +/- 3 and 31 +/- 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [(cGMP)(i)] was significantly increased, and NO-induced (cGMP)(i) was significantly decreased by 24-h DETA-NO treatment. (cGMP)(i) normalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity.     

Effect of IL-1beta on CRE-dependent gene expression in human airway smooth muscle cells

IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 microM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta-treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.     

Glycated human serum albumin enhances macrophage inflammatory protein-1beta mRNA expression through protein kinase C-delta and NADPH oxidase in macrophage-like differentiated U937 cells

BACKGROUND: In a previous report (Higai K et al., Biol Pharm Bull, 2007), glycated human serum albumin (Glc-HSA) was found to induce interleukin-8 (IL-8) mRNA expression in human monocyte-derived U937 cells through a reactive oxygen species (ROS)-dependent pathway; however, Glc-HSA signaling has not been elucidated in macrophages. METHODS: U937 cells were differentiated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days and the macrophage-like differentiated U937 (differentiated U937) cells were stimulated with Glc-HSA and glycolaldehyde dimer-modified HSA (GA-HSA) in the presence of various signaling inhibitors. Macrophage inflammatory protein-1beta (MIP-1beta) mRNA expression was determined by real-time PCR. Intracellular ROS generation was estimated by confocal laser microscopy. RESULTS: Glc-HSA and GA-HSA markedly enhanced MIP-1beta mRNA expression in differentiated U937 cells. Enhanced MIP-1beta mRNA expression was completely suppressed by the ROS scavenger N-acetyl-l-cysteine, the NADPH oxidase inhibitors diphenylene iodonium and apocynin, and the protein kinase C (PKC)-delta inhibitor rottlerin. Furthermore, ROS generation was suppressed completely by rottlerin but not by the PKC-gamma inhibitor Ro318425 or the PKC-alpha, -beta1 and -micro inhibitor Go6976. CONCLUSION: Glc-HSA and GA-HSA enhance MIP-1beta mRNA expression in differentiated U937 cells through PKC-delta-dependent activation of NADPH oxidase.     

Modulator of intracellular Ca(2+), thapsigargin, interferes with in vitro secretion of cytokines and nitric oxide

Interference of thapsigargin (TG), an inhibitor of endoplasmic reticulum Ca(2+) ATPase, with immune reactivity of murine macrophages was investigated under conditions in vitro. The activation of cells with lipopolysaccharide (LPS), interferon-(gamma) (IFN-(gamma)), and with acyclic nucleoside phosphonate N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]- 2,6-diaminopurine (N(6)-isobutyl-PMEDAP) resulted in enhanced production of cytokines TNF-alpha, IL-10, chemokines RANTES/CCL5 and MIP-1alpha/CCL3, as well as in substantially augmented production of nitric oxide (NO) triggered by IFN-(gamma). The effects were in a dual mode of action influenced by TG (1 microM). While TG upregulated secretion of TNF-alpha, it inhibited secretion of IL-10 and RANTES. The immune-stimulated secretion of MIP-1alpha remained virtually unaffected, though TG on its own activated expression of MIP-1alpha in macrophages. The high-output NO production induced by IFN-(gamma), high concentrations of LPS, or by combination of IFN-(gamma) plus LPS or N(6)-isobutyl-PMEDAP was inhibited by TG. On the other hand, production of NO which was marginally activated by low concentration of LPS was upregulated by TG.