Nitric oxide activates diverse signaling pathways to regulate gene expression

Nitric oxide signaling is crucial for effecting long lasting changes in cells, including gene expression, cell cycle arrest, apoptosis, and differentiation. We have determined the temporal order of gene activation induced by NO in mammalian cells and have examined the signaling pathways that mediate the action of NO. Using microarrays to study the kinetics of gene activation by NO, we have determined that NO induces three distinct waves of gene activity. The first wave is induced within 30 min of exposure to NO and represents the primary gene targets of NO. It is followed by subsequent waves of gene activity that may reflect further cascades of NO-induced gene expression. We verified our results using quantitative real time PCR and further validated our conclusions about the effects of NO by using cytokines to induce endogenous NO production. We next applied pharmacological and genetic approaches to determine the signaling pathways that are used by NO to regulate gene expression. We used inhibitors of particular signaling pathways, as well as cells from animals with a deleted p53 gene, to define groups of genes that require phosphatidylinositol 3-kinase, protein kinase C, NF-kappaB, p53, or combinations thereof for activation by NO. Our results demonstrate that NO utilizes several independent signaling pathways to induce gene expression.     

Inhibitory effect of curcumin on nitric oxide production from lipopolysaccharide-activated primary microglia

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the modulatory effect of curcumin on the functional activation of primary microglial cells, brain mononuclear phagocytes causing the neuronal damage, largely remains unknown. The current study examined whether curcumin influenced NO production in rat primary microglia and investigated its underlying signaling pathways. Curcumin decreased NO production in LPS-stimulated microglial cells in a dose-dependent manner, with an IC(50) value of 3.7 microM. It also suppressed both mRNA and protein levels of inducible nitric oxide synthase (iNOS), indicating that this drug may affect iNOS gene expression process. Indeed, curcumin altered biochemical patterns induced by LPS such as phosphorylation of all mitogen-activated protein kinases (MAPKs), and DNA binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein (AP)-1, assessed by reporter gene assay. By analysis of inhibitory features of specific MAPK inhibitors, a series of signaling cascades including c-Jun N-terminal kinase (JNK), p38 and NF-kappaB was found to play a critical role in curcumin-mediated NO inhibition in microglial cells. The current results suggest that curcumin is a promising agent for the prevention and treatment of both NO and microglial cell-mediated neurodegenerative disorders.     

CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogen-activated protein kinase pathways

Clinical and basic science data support an integral role of calcitonin gene-related peptide (CGRP) in the pathophysiology of temporomandibular joint disorders. Recently, we have shown that CGRP can stimulate the synthesis and release of nitric oxide (NO) from trigeminal ganglion glial cells. The goal of this study was to determine the role of mitogen-activated protein kinase (MAPK) signaling pathways in CGRP regulation of iNOS expression and NO release from cultured trigeminal ganglion glial cells from Sprague–Dawley rats. CGRP treatment for 2 h significantly increased activity of the MAPK reporter genes, Elk, ATF-2, and CHOP. In addition, CGRP increased nuclear staining for the active forms of the MAPKs: extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38. This stimulatory event was not observed in cultures pre-treated with the CGRP receptor antagonist peptide CGRP_8–37. Similarly, pre-treatment with selective MAPK inhibitors repressed increases in reporter gene activity as well as CGRP-induced increases in iNOS expression and NO release mediated by MAPKs. In addition, over-expression of MAPK kinase 1 (MEK1), MEK3, MEK6, and MEK kinase significantly increased iNOS expression and NO production in glial cells. Results from our study provide evidence that CGRP binding to its receptor can stimulate iNOS gene expression via activation of MAPK pathways in trigeminal ganglion glial cells.     

Mechanism of macrophage activation induced by β-glucan produced from Paenibacillus polymyxa JB115

β-Glucans are heterogeneous groups of glucose polymers found in the cell walls of fungi, plants and some bacteria. Our previous report showed that a novel β-1,3/1,6-glucan produced from Paenibacillus (P.) polymyxa JB115 can induce nitric oxide (NO) production in RAW264.7 cells. In the present study, the β-glucan significantly increased luciferase activity in cells transfected with NFκB or AP1, but not STAT1, reporter vector DNA, which contain their binding promoter site. All specific NFκB and MAPKs pathway inhibitors (pyrrolidine dithiocarbamate, AG490, U0126, SB203580 and SP600125) remarkably attenuated NO production induced by the β-glucan. Furthermore, Western blot analysis revealed that the stimulation of Raw264.7 cells by β-glucan induced phosphorylation of IκB and the consequent translocation of NFκB into the nucleus. Meanwhile, phosphorylation of ERK1/2, JNK/SAPK and p38 MAPKs in cytoplasm were also confirmed. All these results indicated that β-glucan from P. polymyxa JB115 activates macrophages through MAPKs and NFκB signaling pathway.     

Glucocorticoid-induced TNF receptor functions as a costimulatory receptor that promotes survival in early phases of T cell activation

Glucocorticoid-induced TNFR (GITR) is a member of the TNFR family that can inhibit the suppressive function of regulatory T cells and promote the survival and activation of T cells. However, little is known about the molecular mechanisms regulating T cell survival and activation downstream of GITR. To gain further insight into the cellular events and signaling pathways triggered by GITR, survival, proliferation, and cytokine production as well as activation of MAPKs and NF-kappaB were monitored after cross-linking of the receptor on naive and activated T cells. GITR cross-linking provided costimulation of naive and activated T cells and resulted in activation of MAPKs and NF-kappaB. Although GITR-induced signaling pathways augmented the survival of naive T cells, they were not sufficient to inhibit activation-induced cell death triggered by CD3 cross-linking of activated T cells. Differences in the contributions of GITR to cell survival between naive and activated T cells suggest that the receptor triggers specific pathways depending on the activation state of the T cell.     

Calcineurin negatively regulates TLR-mediated activation pathways

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proinflammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-kappaB, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-kappaB, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. We further found that IkappaB-alpha degradation, MAPK activation, and TNF-alpha production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs.     

Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities

In the present study, we have examined the insulin-signaling pathways involved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular system that expresses high number of high affinity insulin receptors. Insulin (50 nM) rapidly (5 min) stimulated beta-chain insulin receptor, activated the phosphatidylinositol (PI) 3-kinase/Akt/p70S6-kinase signaling pathway, as well as phosphorylated both p44/p42- and p38-mitogen-activated protein kinases (MAPKs). Preconfluent cells were differentiated in a serum-free medium in response to 50 nM insulin for 72 h, as revealed by the formation of multinucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA content and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor-kappaB (NF-kappaB) DNA binding activity and down-regulated activating protein-1 (AP-1) DNA binding activity throughout the differentiation process. The use of specific inhibitors of the insulin-signaling pathways indicated that myogenesis was precluded by treatment for 72 h with LY294002 (an inhibitor of PI 3-kinase), rapamycin (a p70S6-kinase blocker), and SB203580 or PD169316 (p38-MAPK inhibitors). These inhibitors abolished insulin induction of NF-kappaB DNA binding activity and kappaB-chloramphenicol acetyltransferase (CAT) promoter activity, maintaining expressed cytosolic IkappaB-alpha protein, and increased AP-1 DNA binding activity and TRE-CAT promoter activity. These data suggest that insulin induces myogenesis in C2C12 through PI 3-kinase/ p70S6-kinase and p38-MAPK pathways, the signaling through p44/p42-MAPK being inhibited.     

Carbon monoxide mediates heme oxygenase 1 induction via Nrf2 activation in hepatoma cells

Carbon monoxide (CO) and nitric oxide (NO) are two gas molecules which have cytoprotective functions against oxidative stress and inflammatory responses in many cell types. Currently, it is known that NO produced by nitric oxide synthase (NOS) induces heme oxygenase 1 (HO1) expression and CO produced by the HO1 inhibits inducible NOS expression. Here, we first show CO-mediated HO1 induction and its possible mechanism in human hepatocytes. Exposure of HepG2 cells or primary hepatocytes to CO resulted in dramatic induction of HO1 in dose- and time-dependent manner. The CO-mediated HO1 induction was abolished by MAP kinase inhibitors (MAPKs) but not affected by inhibitors of PI3 kinase or NF-kappaB. In addition, CO induced the nuclear translocation and accumulation of Nrf2, which suppressed by MAPKs inhibitors. Taken together, we suggest that CO induces Nrf2 activation via MAPKs signaling pathways, thereby resulting in HO1 expression in HepG2 cells.     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

Epidermal growth factor and interleukin-1beta synergistically stimulate the production of nitric oxide in rat intestinal epithelial cells

Epidermal growth factor (EGF) is one of the trophic factors for intestinal adaptation after small bowel transplantation (SBT). A recent report indicates that nitric oxide (NO) has cytoprotective effects on bacterial translocation (BT) after SBT. We hypothesized that EGF stimulates the expression of the inducible NO synthase (iNOS) gene in the graft after SBT, followed by increased production of NO, resulting in the decrease of BT. Intestinal epithelial cells (IEC)-6 were treated with EGF and/or IL-1beta in the presence and absence of phosphatidylinositol 3-kinase (PI3-kinase) and EGF receptor kinase inhibitors (LY-294002 and tyrphostin A25). The induction of NO production and iNOS and its signal molecules, including the inhibitory protein of NF-kappaB (IkappaB), NF-kappaB, and Akt, were analyzed. IL-1beta stimulated the degradation of IkappaB and the activation of NF-kappaB but had no effect on iNOS induction. EGF, which had no effect on the NF-kappaB activation and iNOS induction, stimulated the upregulation of type 1 IL-1 receptor (IL-1R1) through PI3-kinase/Akt. Simultaneous addition of EGF and IL-1beta stimulated synergistically the induction of iNOS, leading to the increased production of NO. Our results indicate that EGF and IL-1beta stimulate two essential signals for iNOS induction in IEC-6 cells: the upregulation of IL-1R1 through PI3-kinase/Akt and the activation of NF-kappaB through IkappaB kinase, respectively. Simultaneous addition of EGF and IL-1beta can enhance the production of NO, which may contribute to the cytoprotective effect of EGF against intestinal injury.     

Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells

Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.     

Role of mitogen-activated protein kinases in the iNOS production and cytokine secretion by Salmonella enterica serovar Typhimurium porins

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both physiological and pathological functions. The present study examined the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and proinflammatory cytokine production in RAW 264.7 cells in response to Salmonella enterica serovar Typhimurium porins. By use of Western blotting for iNOS detection and enzyme-linked immunosorbent assay (ELISA) for quantization of cytokine secretion, selective pharmacological inhibitors of MAPK pathways were tested for dissecting the molecular mechanisms underlying the mediation of these signaling in porins-stimulated murine macrophages. S. enterica serovar Typhimurium porins activated iNOS expression, NO production and interleukin (IL)-6, IL-8 and tumor necrosis factor-α (TNF-α) release. Treatment of cells with SB203580 and SP600125 (inhibitors of p38 and JNK, respectively) significantly affected porin-stimulated iNOS and NO production. Concomitant decrease in the proinflammatory cytokine secretion was detected. These data confirm the importance of the MAPKs cascade in macrophage activation by bacterial product opening up new strategies for therapy of septic shock.