IkappaBalpha Phosphorylation Research Articles

Hepatitis C virus NS4B induces unfolded protein response and endoplasmic reticulum overload response-dependent NF-κB activation

Hepatitis C virus nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane associated protein and a potent causative factor of ER stress. Here we reported that unfolded protein response (UPR) can be activated by HCV NS4B through inducing both XBP1 mRNA splicing and ATF6 cleavage in human hepatic cells. Flow cytometric analysis revealed that HCV NS4B stimulates the production of reactive oxygen species (ROS) by perturbing intracellular Ca(2+) homeostasis. Luciferase assay showed that HCV NS4B also activates the multifunctional transcription factor, NF-kappaB, in a dose-dependent manner through Ca(2+) signaling and ROS. Further immunoblot analysis showed that HCV NS4B promotes NF-kappaB translocation into the nucleus via protein-tyrosine kinase (PTK) mediated phosphorylation and subsequent degradation of IkappaBalpha. These studies provide an important insight into the implication of NS4B in HCV life cycle and HCV-associated liver disease by affecting host intracellular signal transduction pathways.

NF-kappaB inhibitors impair platelet activation responses.

Although platelets are anucleated cells, they express several transcription factors that exert non-genomic functions, including the positive and negative regulation of platelet activation. NF-kappaB is a major transcriptional regulator of genes involved in survival, proliferation and inflammation. Because platelets play a critical role not only in hemostasis, but also in inflammation and tumor progression, we evaluated the role of NF-kappaB in platelet physiology. Immunofluorescence, Western blotting and ELISA studies revealed that platelets express IkappaBalpha and NF-kappaB, and that stimulation with thrombin triggers IkappaBalpha phosphorylation and degradation and the binding of platelet NF-kappaB p65 subunit to synthetic oligonucleotides containing the consensus sequence for NF-kappaB. Two specific unrelated inhibitors of NF-kappaB activation, BAY 11-7082 and Ro 106-9920, reduced PAC-1 and fibrinogen binding to integrin alpha(IIb)beta3 and restricted platelet spreading on immobilized fibrinogen. Both inhibitors impaired aggregation mediated by ADP, epinephrine, collagen or thrombin, but not arachidonic acid. ATP release, TXB2 formation, P-selectin expression, ERK phosphorylation and cPLA2 activity stimulated by thrombin were reduced in BAY 11-7082- or Ro 106-9920-treated platelets. Although bleeding time was not affected, ADP-induced platelet aggregation was impaired in mice treated with BAY 11-7082. Our results suggest that NF-kappaB may be a novel mediator of platelet responses. The blockade of platelet function by NF-kappaB inhibitors might be relevant in those clinical situations where these drugs are being considered for anti-tumor and/or anti-inflammatory therapy.

Anandamide enhances IL‐10 production in activated microglia by targeting CB2 receptors: Roles of ERK1/2, JNK, and NF‐κB

The endocannabinoid system exhibits anti-inflammatory properties by regulating cytokine production. Anandamide (AEA) down-regulates proinflammatory cytokines in a viral model of multiple sclerosis (MS). However, little is known about the mechanisms by which AEA exerts these effects. Microglial cells are the main source of cytokines within the brain and the first barrier of defense against pathogens by acting as antigen presenting cells. IL-10 is a key physiological negative regulator of microglial activation. In this study we show that AEA enhances LPS/IFNgamma-induced IL-10 production in microglia by targeting CB(2) receptors through the activation of ERK1/2 and JNK MAPKs. AEA also inhibits NF-kappaB activation by interfering with the phosphorylation of IkappaBalpha, which may result in an increase of IL-10 production. Moreover, endogenously produced IL-10 negatively regulates IL-12 and IL-23 cytokines, which in its turn modify the pattern of expression of transcription factors involved in Th commitment of splenocytes. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the central nervous system (CNS). Accordingly, pharmacological modulation of AEA uptake and degradation might be a useful tool for treating neuroinflammatory diseases.

Comprehensive evaluation of a novel nuclear factor‐κB inhibitor, quinoclamine, by transcriptomic analysis

The transcription factor nuclear factor-kappaB (NF-kappaB) has been linked to the cell growth, apoptosis and cell cycle progression. NF-kappaB blockade induces apoptosis of cancer cells. Therefore, NF-kappaB is suggested as a potential therapeutic target for cancer. Here, we have evaluated the anti-cancer potential of a novel NF-kappaB inhibitor, quinoclamine (2-amino-3-chloro-1,4-naphthoquinone). In a large-scale screening test, we found that quinoclamine was a novel NF-kappaB inhibitor. The global transcriptional profiling of quinoclamine in HepG2 cells was therefore analysed by transcriptomic tools in this study. Quinoclamine suppressed endogenous NF-kappaB activity in HepG2 cells through the inhibition of IkappaB-alpha phosphorylation and p65 translocation. Quinoclamine also inhibited induced NF-kappaB activities in lung and breast cancer cell lines. Quinoclamine-regulated genes interacted with NF-kappaB or its downstream genes by network analysis. Quinoclamine affected the expression levels of genes involved in cell cycle or apoptosis, suggesting that quinoclamine exhibited anti-cancer potential. Furthermore, quinoclamine down-regulated the expressions of UDP glucuronosyltransferase genes involved in phase II drug metabolism, suggesting that quinoclamine might interfere with drug metabolism by slowing down the excretion of drugs. This study provides a comprehensive evaluation of quinoclamine by transcriptomic analysis. Our findings suggest that quinoclamine is a novel NF-kappaB inhibitor with anti-cancer potential.

Anti-allergic effects of scoparone on mast cell-mediated allergy model

Scoparone is known to have a wide range of pharmacological properties in vitro. However, the roles of scoparone in immediate-type allergic reactions have not yet been investigated. In this study, we demonstrated that scoparone attenuated IgE-mediated allergic response in mast cells. Oral administration of scoparone inhibited passive cutaneous anaphylaxis in rats. Presence of scoparone dose-dependently decreased histamine release from rat peritoneal mast cells (RPMC) stimulated by anti-dinitrophenyl IgE. Moreover, scoparone reduced the expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-6 in RPMC. Pretreatment with scoparone inhibited the calcium uptake and p38 mitogen-activated protein kinase (MAPK) activity. Furthermore, scoparone blocked translocation of nuclear factor-kappa B (NF-kappaB) p65 subunit by suppressing IkappaBalpha phosphorylation in RPMC. Reduced calcium uptake as well as the suppressed activity of p38 MAPK and NF-kappaB might be involved in the inhibitory effect of scoparone on the secretory response. Our findings suggest that scoparone may serve as an effective therapeutic agent for allergic diseases.

Tea polyphenols inhibit cyclooxygenase-2 expression and block activation of nuclear factor-kappa B and Akt in diethylnitrosoamine induced lung tumors in Swiss mice

Due to lack of validated screening methods and hence poor prognosis, treatment of lung cancer has not still improved up to the expectations. Therefore, risk of lung cancer needs to be minimized by efficient preventive measures. Tea (Camellia sinensis) and its bioactive polyphenols have been associated with prevention of human cancer for several organs. Thus, intake of tea polyphenols seems to be a viable mean to control lung cancer burden. In the present study, we studied the chemopreventive effects of green tea polyphenols (GTP) and black tea polyphenols (BTP) against diethylnitrosoamine (DEN) induced lung tumors in Swiss albino mice. Chemopreventive potential of tea polyphenols, was recorded as evident by, low incidence of alveologenic tumors in lungs of animals at tested doses (0.1% and 0.2% of both GTP and BTP) when compared with DEN (20 mg/kg b wt) treated animals. As a mechanism of cancer chemoprevention cellular signaling pathways were also targeted. GTP and BTP treatment inhibited the expression of Akt, cyclooxygenase-2 and inactivated nuclear factor-kappa B via blocking phosphorylation and subsequent degradation of IkappaB alpha. Thus, the study suggests that polyphenolic constituents of both cultivars of tea, i.e. green and black, have chemopreventive effects in DEN induced lung tumorigenesis in Swiss albino mice.

Cellular Markers of Muscle Atrophy in Chronic Obstructive Pulmonary Disease

Skeletal muscle atrophy in individuals with advanced chronic obstructive pulmonary disease (COPD) is associated with diminished quality of life, increased health resource use, and worsened survival. Muscle wasting results from an imbalance between protein degradation and synthesis, and is enhanced by decreased regenerative repair. We investigated the activation of cellular signaling networks known to mediate muscle atrophy and regulate muscle regenerative capacity in rodent models, in individuals with COPD (FEV(1) < 50% predicted). Nine patients with COPD and nine control individuals were studied. Quadriceps femoris muscle isometric contractile force and cross-sectional area were confirmed to be significantly smaller in the patients with COPD compared with control subjects. The vastus lateralis muscle was biopsied and muscle transcript and/or protein levels of key components of ubiquitin-mediated proteolytic systems (MuRF1, atrogin-1, Nedd4), inflammatory mediators (IkappaBalpha, NF-kappaBp65/p50), AKT network (AKT, GSK3beta, p70S6 kinase), mediators of autophagy (beclin-1, LC3), and myogenesis (myogenin, MyoD, Myf5, myostatin) were determined. Atrogin-1 and Nedd4, two ligases regulating ubiquitin-mediated protein degradation and myostatin, a negative regulator of muscle growth, were significantly increased in the muscle of patients with COPD. MuRF1, Myf5, myogenin, and MyoD were not differentially expressed. There were no differences in the level of phosphorylation of AKT, GSK3beta, p70S6kinase, or IkappaBalpha, activation of NF-kappaBp65 or NF-kappaBp50, or level of expression of beclin-1 or LC3, suggesting that AKT signaling was not down-regulated and the NF-kappaB inflammatory pathway and autophagy were not activated in the COPD muscle. We conclude that muscle atrophy associated with COPD results from the recruitment of specific regulators of ubiquitin-mediated proteolytic pathways and inhibition of muscle growth.

Allylmethylsulfide Down-Regulates X-Ray Irradiation-Induced Nuclear Factor-kappaB Signaling in C57/BL6 Mouse Kidney.

Allylmethylsulfide (AMS), a volatile organosulfur derivative from garlic, has been shown to have radioprotective effects in radiation-challenged cell and animal models, but the mechanism of radioprotection is not well understood. To determine the mechanism of radioprotection in an in vivo model, we first verified the antioxidant capacity of AMS using 2,2'-azobis(2-amidinopropane) dihydrochloride-induced human embryonic kidney 293T cells by measuring reactive oxygen species generation, reduced glutathione, protein tyrosine kinase/protein tyrosine phosphatase balance, and nuclear factor-kappaB (NF-kappaB) protein levels. We then investigated the protective effects of AMS (55 and 275 micromol/kg, intraperitoneal treatment) on 15 Gy X-ray-irradiated mouse kidney. The results showed that AMS decreased the free radical-induced lipid peroxidation in mice exposed to X-rays. Moreover, the antioxidative AMS suppressed the activation of NF-kappaB and its dependent genes such as vascular cell adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2 through inhibition of IkappaBalpha phosphorylation and activation of IkappaB kinase alpha/beta and mitogen-activated protein kinases (MAPKs). Based on these results, AMS may be a useful radioprotective agent by down-regulating the MAPKs and NF-kappaB signaling pathway that can be induced via X-ray irradiation.

Inhibitory effects of flavonoids on TNF-α-induced IL-8 gene expression in HEK 293 cells

Due to their multiple biological activities, flavonoids have gained attention as potentially useful therapeutics for a variety of diseases including cancer, cardiovascular diseases, and autoimmune diseases. In this study, we demonstrated that several flavonoids, including kaempferol, quercetin, fisetin, and chrysin block TNF-alpha induced IL-8 promoter activation and gene expression in HEK 293 cells. In addition, phosphorylation and degradation of IkappaBalpha and translocation of NF-kappaB p65 were inhibited by these flavonoids in TNF-alpha-stimulated HEK 293 cells. Furthermore, generation of reactive oxygen species (ROS) in response to TNF-alpha was reduced by the flavonoids. Moreover, although pretreatment with fisetin, quercetin, or chrysin decreased cell viability, kaempferol did not. Taken together, these findings suggest that kaempferol would be useful for the treatment of TNF-alpha-induced inflammatory diseases.

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin, FAK, ERK, and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, alphavbeta3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)-9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, alphavbeta3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN-induced increase of the migration and MMP-9 up-regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, treatment of JJ012 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited OPN-induced cell migration and MMP-9 up-regulation. Stimulation of JJ012 cells with OPN also induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OPN-mediated increases in MMP-9 and kappaB-luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP-9 expression through the alphavbeta3 integrin, FAK, MEK, ERK and NF-kappaB signal transduction pathway.

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)-gamma-induced protein 10 (IP-10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN-gamma depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN-gamma and TNF-alpha have not been adequately elucidated in human monocytes. In this study, we showed that TNF-alpha had more potential than IFN-gamma to induce CXCL10 production in THP-1 monocytes. Furthermore, IFN-gamma synergistically enhanced the production of CXCL10 in parallel with the activation of NF-kappaB in TNF-alpha-stimulated THP-1 cells. Blockage of STAT1 or NF-kappaB suppressed CXCL10 production. JAKs inhibitors suppressed IFN-gamma plus TNF-alpha-induced production of CXCL10 in parallel with activation of STAT1 and NF-kappaB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF-kappaB, but not that of STAT1. IFN-gamma-induced phosphorylation of JAK1 and JAK2, whereas TNF-alpha induced phosphorylation of ERK1/2. Interestingly, IFN-gamma alone had no effect on phosphorylation and degradation of IkappaB-alpha, whereas it significantly promoted TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha. These results suggest that TNF-alpha induces CXCL10 production by activating NF-kappaB through ERK and that IFN-gamma induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF-alpha-induced NF-kappaB may be the primary pathway contributing to CXCL10 production in THP-1 cells. IFN-gamma potentiates TNF-alpha-induced CXCL10 production in THP-1 cells by increasing the activation of STAT1 and NF-kappaB through JAK1 and JAK2.

Tax1 enhances cancer cell proliferation via Ras–Raf–MEK–ERK signaling pathway

Erbin is an ErbB2 binding protein, which belongs to the LAP (leucine-rich repeat (LRR) and PDZ domain) protein family. We previously reported that Tax1, a protein of the human T-cell leukemia virus type I (HTLV-I), associated with Erbin by using Erbin PDZ domain as a bait to screen a human T lymphocyte cDNA library by a yeast two hybrid strategy. In the present study, we demonstrated that Tax1 enhances cancer cell proliferation via Ras-Raf-MEK-ERK signaling pathway by using molecular section strategy. The pull-down assay showed that the four amino acid domain, that is, Tax1 350-353, might specifically interact with Erbin, but not any other Tax1 deletion mutants. The coimmunoprecipitation assay confirmed that Tax1 350-353 domain bound with Erbin in vivo. Functional study demonstrated that overexpression of Tax1 in cancer cell lines of liver cancer SMMC-7721, colon cancer HCT-116, and breast cancer MCF-7 facilitated the cell proliferation. And the transfection of Tax1 353 in MCF-7 cells with endogenous Erbin expression markedly increased phosphorylation of Ras, Raf, MEK1/2, ERK1/2, PI3K, and IkappaBalpha, suggesting that Tax1-enhanced cell proliferation tracks Ras-Raf-MEK-ERK signaling pathway.

Systemic Inhibition of NF-κB Activation Protects from Silicosis

BackgroundSilicosis is a complex lung disease for which no successful treatment is available and therefore lung transplantation is a potential alternative. Tumor necrosis factor alpha (TNFα) plays a central role in the pathogenesis of silicosis. TNFα signaling is mediated by the transcription factor, Nuclear Factor (NF)-κB, which regulates genes controlling several physiological processes including the innate immune responses, cell death, and inflammation. Therefore, inhibition of NF-κB activation represents a potential therapeutic strategy for silicosis.Methods/FindingsIn the present work we evaluated the lung transplant database (May 1986–July 2007) at the University of Pittsburgh to study the efficacy of lung transplantation in patients with silicosis (n = 11). We contrasted the overall survival and rate of graft rejection in these patients to that of patients with idiopathic pulmonary fibrosis (IPF, n = 79) that was selected as a control group because survival benefit of lung transplantation has been identified for these patients. At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFα expressing macrophage and NF-κB activation in epithelial cells. Patients with silicosis had poor survival (median survival 2.4 yr; confidence interval (CI): 0.16–7.88 yr) compared to IPF patients (5.3 yr; CI: 2.8–15 yr; p = 0.07), and experienced early rejection of their lung grafts (0.9 yr; CI: 0.22–0.9 yr) following lung transplantation (2.4 yr; CI:1.5–3.6 yr; p<0.05). Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-κB activation with a pharmacologic inhibitor (BAY 11-7085) of IκBα phosphorylation decreased silica-induced inflammation and collagen deposition. In contrast, transgenic mice expressing a dominant negative IκBα mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica.ConclusionsAlthough limited by its size, our data support that patients with silicosis appear to have poor outcome following lung transplantation. Experimental data indicate that while the systemic inhibition of NF-κB protects from silica-induced lung injury, epithelial cell specific NF-κB inhibition appears to aggravate the outcome of experimental silicosis.

Activation of p38 MAPK in the substantia nigra leads to nuclear translocation of NF‐κB in MPTP‐treated mice: implication in Parkinson’s disease

Activation and translocation of the transcription factor nuclear factor kappa B (NF-kappaB) from cytoplasm to the nucleus has been reported in models of Parkinson's disease (PD). Our focus was to discern the upstream events which ultimately lead to NF-kappaB nuclear translocation using animal model of PD. We demonstrate that p38 activation results in downstream phosphorylation of NF-kappaB and accumulation of p65 subunit of NF-kappaB selectively in ventral midbrain but not in striatum. Treatment with p38 inhibitor, SB239063, prevented downstream phosphorylation of IkappaB alpha and p65 translocation to the nucleus in the ventral midbrain. Phosphorylation of anti-apoptotic Bcl2, an NF-kappaB target gene by p38 to inactive pBcl2ser87 was also attenuated by SB239063. Increased staining of p65 in the nuclei of cells in the substantia nigra but not in the ventral tegmental area of MPTP-treated mice further suggests a role for NF-kappaB in PD. In agreement with the above, sustained caspase activation is seen in the ventral midbrain but not in striatum. We demonstrate the region specific p38-mediated activation of NF-kappaB following MPTP treatment demonstrating the role of p38/NF-kappaB signaling in the pathogenesis and progression of the disease. Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression.

Jagged1 regulates the activation of astrocytes via modulation of NFκB and JAK/STAT/SOCS pathways

The Notch pathway is implicated in many aspects of the central nervous system (CNS) development and functions. Recently, we and others identified the Notch pathway to be involved in inflammatory events of the CNS. To understand the implication of this pathway on astrocytes, we have studied the Jagged-Notch-Hes pathway under inflammatory conditions. LPS exposure induced an upregulation of Jagged1 expression on cultured astrocytes. To address the role of Jagged1 in the modulation of inflammation, we used a siRNA mediated silencing of Jagged1 (siRNA J1). Jagged1 inhibition induced important variations on the Notch pathway components like Hes1, Hes5, Notch3, and RBP-Jkappa. siRNA J1 repressed the mRNA expression of genes known as hallmarks of the gliosis like GFAP and endothelin(B) receptor. On activated astrocytes, the inhibition of Jagged1 had antiinflammatory effects and resulted in a decrease of LPS-induced proinflammatory cytokines (IL1beta, IL1alpha, and TNFalpha) as well as the iNOS expression. The inhibition of Jagged1 induced a modulation of the JAK/STAT/SOCS signaling pathway. Most interestingly, the siRNA J1 decreased the LPS-induced translocation of NFkappaB p65 and this could be correlated to the phosphorylation of IkappaBalpha. These results suggest that during inflammatory and gliotic events of the CNS, Jagged1/Notch signaling sustains the inflammation mainly through NFkappaB and in part through JAK/STAT/SOCS signaling pathways.

Interleukin‐1β enhances nucleotide‐induced and α‐secretase‐dependent amyloid precursor protein processing in rat primary cortical neurons via up‐regulation of the P2Y2 receptor

The heterologous expression and activation of the human P2Y(2) nucleotide receptor (P2Y(2)R) in human 1321N1 astrocytoma cells stimulates alpha-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPPalpha). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y(2)R-mediated production of sAPPalpha occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y(2)R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) up-regulated both P2Y(2)R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y(2)R was abrogated by pre-incubation with Bay 11-7085, an IkappaB-alpha phosphorylation inhibitor, which suggests that P2Y(2)R mRNA transcript levels are regulated through nuclear factor-kappa-B (NFkappaB) signaling. Furthermore, the P2Y(2)R agonist Uridine-5'-triphosphate (UTP) enhanced the release of sAPPalpha in rPCNs treated with IL-1beta or transfected with P2Y(2)R cDNA. UTP-induced release of sAPPalpha from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y(2)R-mediated release of sAPPalpha from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1beta, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y(2)R in neurons.

Ischaemia/reperfusion induced cardiac stem cell homing to the injured myocardium by stimulating stem cell factor expression via NF‐κB pathway

Ischaemia/reperfusion (I/R) is a major cause of heart failure. Recently cardiac stem cells (CSCs) were proposed as the most appropriate cell type for heart disease therapy. However, it is still unclear whether I/R can stimulate the CSCs homing to the injured myocardium. Male Sprague-Dawley rats were subjected to a 30-min ischaemia followed by reperfusion of different intervals. RT-PCR, western blotting and immunohistochemistry were performed to detect stem cell factor (SCF) expression at mRNA and protein levels respectively. Activation of nuclear factor-kappaB (NF-kappaB) was determined by electrophoretic mobility shift assay. To assess the homing of CSCs in vivo, BrdU-labelled CSCs were injected into AV-groove before induction of ischaemia and examined by immunofluorescent staining in the injured myocardium after I/R. From day 3 to day 6 after reperfusion, the accumulation of CSCs was significantly elevated in the injured area, which was matched with the increased SCF expression during I/R. Pretreatment of rats with NF-kappaB inhibitor, N-acetyl-L-cysteine (NAC) not only suppressed NF-kappaB activation induced by I/R but also attenuated SCF expression. Further analysis revealed that I/R induced phosphorylation of IkappaBalpha after 15 min of reperfusion, and the raised phosphor-IkappaBalpha returned to the basal level at 2 h of reperfusion. In simulated I/R(SI/R) in vitro, it enhanced NF-kappaB activation and SCF expression in cultured neonatal rat cardiomyocytes, which was markedly inhibited by NF-kappaB decoy oligodeoxynucleotide or NAC. Taken together, our results demonstrated that I/R induced CSCs homing to the injured myocardium by stimulating myocardial SCF expression via activation of NF-kappaB.

Adenosine Signaling Mediates SUMO-1 Modification of IκBα during Hypoxia and Reoxygenation

Small ubiquitin-like modifier 1 (SUMO-1) modification of IkappaBalpha has been described to actively participate in NFkappaB regulation. Following proteosomal degradation of IkappaBalpha, an auto-regulatory loop consisting of transcriptional activation of IkappaBalpha gene and SUMO-1 modification of newly synthesized IkappaBalpha proceeds. The SUMOylated IkappaBalpha form is resistant to signal-induced degradation, consequently halting NFkappaB activation. We describe a mechanistic model by which adenosine (Ado) signaling results in significant accumulation of SUMO-1 modified IkappaBalpha with subsequent attenuation of NFkappaB activation. Using models of hypoxia followed by reoxygenation (H/R), we have documented an H/R cycle-dependent increase in extracellular Ado correlating with increases in the cytoplasmic pool of IkappaBalpha/SUMO-1. We demonstrate a dose-dependent increase in IkappaBalpha/SUMO in cells treated with the general Ado receptor agonist NECA and abolished by Ado receptor antagonists. Experiments in cells exposed to cycles of H/R followed by hypoxia demonstrated differential patterns of SUMOylation and phosphorylation of IkappaBalpha, greatly impacting its proteosomal degradation by the 26 S proteasome. Assays targeting knockdown and overexpression of SUMO-1 demonstrated significant regulation of NFkappaB activation and NFkappaB-mediated gene transcription (interleukin-6). These results were confirmed in vivo using wild type and cd73 null mouse lung tissue. In summary, we present an endogenous mechanism by which cells and tissues acquire anti-inflammatory properties by recruiting a nondegradable form of IkappaBalpha, a major control point for NFkappaB activation via Ado signaling.

The antibiotics doxycycline and minocycline inhibit the inflammatory responses to the Lyme disease spirochete Borrelia burgdorferi.

Tetracyclines moderate inflammatory responses of various etiologies. We hypothesized that tetracyclines, in addition to their antimicrobial function, could exert control over the inflammation elicited by Borrelia burgdorferi. To model systemic effects, we used the human monocytic cell line THP-1; to model effects in the central nervous system, we used rhesus monkey brain astrocytes and microglia. Cells were stimulated with live or sonicated B. burgdorferi or with the lipoprotein outer surface protein A in the presence of increasing concentrations of doxycycline or minocycline. Both antibiotics significantly reduced the production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8 in a dose-dependent manner in all cell types. Microarray analyses of the effect of doxycycline on gene transcription in spirochete-stimulated monocytes revealed that the NFKB and CHUK (alias, IKKA) genes were down-regulated. Functionally, phosphorylation of IkappaBalpha and binding of NF-kappaB to target DNA were both reduced in these cells. Our results suggest that tetracyclines may have a dual therapeutic effect in Lyme disease.

Genomic analysis of sulfasalazine effect in experimental colitis is consistent primarily with the modulation of NF-κB but not PPAR-γ signaling

Sulfasalazine (SAZ) is a widely used drug in inflammatory bowel disease patients but its mechanism of action is incompletely understood. The objective of our study was to further characterize SAZ mechanism by studying its effect on the colonic transcriptome in a suitable preclinical model of inflammatory bowel disease. The trinitrobenzenesulfonic acid model of colitis in rats was used. The effect of SAZ on mRNA expression was assessed with Affymetrix Rat Expression 230 2.0 arrays used in triplicate (sextuplicate for controls), validated in separate samples with quantitative reverse-time PCR analysis. Some nongenomic experiments were also carried out. SAZ (500 mg/kg) had a marked anti-inflammatory effect as expected, which was correlated with a dramatic impact on colonic gene expression. In addition to immune/inflammatory genes, SAZ responsive genes were involved in distinct metabolic and signaling pathways. The effect of sulfasalazine was generally of normalization of colitis-modulated genes to control levels, with very few exceptions. Postgenomic validation showed an excellent correlation with microarray data and seemed to be slightly more sensitive. SAZ generally modulated the expression of nuclear factor-kappaB-driven genes. SAZ was also shown to inhibit IkappaB-alpha phosphorylation in rat primary splenocytes and in HT29 and IEC18 cells. In contrast, SAZ had only a modest effect on peroxisome proliferator-activated receptor (PPAR)-gamma-regulated genes and it was confirmed to induce PPAR-gamma in enterocytes but not splenocytes. Mechanistically, our data are consistent primarily with nuclear factor-kappaB inhibition, and there is little evidence of a prominent role of activation of PPAR-gamma receptors or antioxidative actions.