Hepatocyte NF-kappaB activation is hepatoprotective during ischemia-reperfusion injury and is augmented by ischemic hypothermia

The present study examined the role of hepatocyte NF-kappaB activation during ischemia-reperfusion injury. Second, we evaluated the effects of ischemic hypothermia on NF-kappaB activation and liver injury. C57BL/6 mice underwent 90 min of partial hepatic ischemia and up to 8 h of reperfusion. Body temperature was regulated during the ischemic period between 35 and 37 degrees C, 33 and 35 degrees C, 29 and 33 degrees C or unregulated, where temperature fell to <29 degrees C. Liver injury, as measured by serum alanine aminotransferase as well as liver histopathology, was inversely proportional to regulated body temperature, with the unregulated group (<29 degrees C) being highly protected and the normothermic group (35-37 degrees C) displaying the greatest injury. Inflammation, as measured by production of TNF-alpha and liver recruitment of neutrophils, was greatest in the normothermic groups and lowest in the ischemic hypothermia groups. Interestingly, hepatocyte NF-kappaB activation was highest in the hypothermic group and least in the normothermic group. Paradoxically, degradation of IkappaB proteins, IkappaB-alpha and IkappaB-beta, was greatest in the normothermic group, suggesting an alternate NF-kappaB regulatory mechanism during ischemia-reperfusion injury. Subsequently, we found that NF-kappaB p65 protein was increasingly degraded in normothermic versus hypothermic groups, and this degradation was specific for hepatocytes and was associated with decreased expression of the peptidyl-prolyl isomerase Pin1. The data suggest that NF-kappaB activation in hepatocytes is a protective response during ischemia-reperfusion and can be augmented by ischemic hypothermia. Furthermore, it appears that Pin1 promotes NF-kappaB p65 protein stability such that decreased expression of Pin1 during ischemia-reperfusion results in p65 degradation, reduced nuclear translocation of NF-kappaB, and enhanced hepatocellular injury.     

Surfactant protein A activation of atypical protein kinase C zeta in IkappaB-alpha-dependent anti-inflammatory immune regulation

The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-kappaB activation in vivo and in vitro are only partially understood. We previously demonstrated that SP-A stabilizes IkappaB-alpha, the primary regulator of NF-kappaB, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IkappaB-alpha knockout/IkappaB-beta knockin mice, SP-A fails to inhibit LPS-induced TNF-alpha production and p65 nuclear translocation, confirming a critical role for IkappaB-alpha in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) zeta as a pivotal upstream regulator of SP-A-mediated IkappaB-alpha/NF-kappaB pathway modulation deduced from blocking experiments and confirmed by using AM from PKCzeta-/- mice. SP-A transiently triggers aPKCThr(410/403) phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IkappaB-alpha by SP-A critically depends on PKCzeta activity, and thus attribute a novel, stimulus-specific signaling function to PKCzeta in SP-A-modulated pulmonary immune response.     

Kainic acid-induced apoptosis in rat striatum is associated with nuclear factor-kappaB activation

The present study evaluated whether nuclear factor-kappaB (NF-kappaB) activation contributes to the apoptotic-like death of striatal neurons induced by kainic acid (KA) receptor stimulation. Intrastriatally infused KA (1.25-5.0 nmol) produced substantial neuronal loss as indicated by an 8-73% decrease in 67-kDa glutamic acid decarboxylase (p<0.05). KA (1.25-5.0 nmol) elicited internucleosomal DNA fragmentation that was inhibited by the AMPA/KA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dibenzo[f]quinoxaline-7-sulfonamide) but not by the NMDA receptor antagonist MK-801. A decrease in IkappaB-alpha protein levels, which was accompanied by an increase in NF-kappaB binding activity, was found from 6 to 72 h after KA (2.5 nmol) infusion. NF-kappaB was composed mainly of p65 and c-Rel as revealed by supershift assay. In addition, c-Myc and p53 increased from five- to sevenfold from 24 to 72 h after KA (2.5 nmol) administration. Immunohistochemistry revealed high levels of c-Myc and p53 immunoreactivity, mainly in medium-sized striatal neurons. Pretreatment with the cell-permeable recombinant peptide NF-kappaB SN50 (5-20 microg) blocked NF-kappaB nuclear translocation, but had no effect on AP-1 binding. NF-kappaB SN50 also inhibited the KA-induced up-regulation of c-Myc and p53, as well as internucleosomal DNA fragmentation. The apoptotic-like destruction of rat striatal neurons induced by KA receptor stimulation thus appears to involve biochemical mechanisms similar to those mediating the excitotoxic response to NMDA receptor stimulation. The present results provide additional support for the view that NF-kappaB activation contributes to c-Myc and p53 induction and subsequent apoptosis in an excitotoxic model of Huntington's disease.     

Mechanisms of persistent NF-kappa B activity in the bronchi of an animal model of asthma

In most cells trans-activating NF-kappaB induces many inflammatory proteins as well as its own inhibitor, IkappaB-alpha, thus assuring a transient response upon stimulation. However, NF-kappaB-dependent inflammatory gene expression is persistent in asthmatic bronchi, even after allergen eviction. In the present report we used bronchial brushing samples (BBSs) from heaves-affected horses (a spontaneous model of asthma) to elucidate the mechanisms by which NF-kappaB activity is maintained in asthmatic airways. NF-kappaB activity was high in granulocytic and nongranulocytic BBS cells. However, NF-kappaB activity highly correlated to granulocyte percentage and was only abrogated after granulocytic death in cultured BBSs. Before granulocytic death, NF-kappaB activity was suppressed by simultaneous addition of neutralizing anti-IL-1beta and anti-TNF-alpha Abs to the medium of cultured BBSs. Surprisingly, IkappaB-beta, whose expression is not regulated by NF-kappaB, unlike IkappaB-alpha, was the most prominent NF-kappaB inhibitor found in BBSs. The amounts of IkappaB-beta were low in BBSs obtained from diseased horses, but drastically increased after addition of the neutralizing anti-IL-1beta and anti-TNF-alpha Abs. These results indicate that sustained NF-kappaB activation in asthmatic bronchi is driven by granulocytes and is mediated by IL-1beta and TNF-alpha. Moreover, an imbalance between high levels of IL-1beta- and TNF-alpha-mediated IkappaB-beta degradation and low levels of IkappaB-beta synthesis is likely to be the mechanism preventing NF-kappaB deactivation in asthmatic airways before granulocytic death.     

Nuclear factor kappaB/p49 is a negative regulatory factor in nerve growth factor-induced choline acetyltransferase promoter activity in PC12 cells

Anovel nuclear factor kappaB (NF-kappaB) binding site has been identified within the promoter region of the mouse gene encoding choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine and has been implicated in the cognitive deficits associated with aging and Alzheimer's disease. This binding site, which is located within the nerve growth factor (NGF)-responsive enhancer element, was recognized by the NF-kappaB protein p49 but not p65 or p50. p49 from both basal forebrain and PC12 nuclear extracts interacted with this specific sequence in electrophoretic mobility shift assays. Mutation of the NF-kappaB site caused an increase in NGF-induced promoter activation, whereas overexpression of p49 in NGF-differentiated PC12 cells caused a decrease in endogenous ChAT enzyme activity and a decrease in promoter activity that was specifically mediated through this NF-kappaB binding site. Treatment of PC12 cells with NGF resulted in a drastic reduction in nuclear p49 binding to the ChAT NF-kappaB site after 24 h, but nuclear p49 levels were not altered, suggesting that late NGF-mediated events prevent binding of p49 to the ChAT promoter by an unknown mechanism other than nuclear translocation. Decreased ChAT expression and increased NF-kappaB activity in the brain are associated with aging and Alzheimer's disease. These data indicate that p49 is a negative regulator of ChAT expression and suggest a possible mechanism for aging-associated declines in cholinergic function.     

Induction of nitric oxide synthase in raw 264.7 macrophages by lipoteichoic acid from<Emphasis Type="Italic">Staphylococcus aureus:</Emphasis> Involvement of protein kinase C- and nuclear factor-kB-dependent mechanisms

This study investigates the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release caused by Staphylococcus aureus lipoteichoic acid (LTA) in RAW 264.7 macrophages. A phosphatidylcholine-phospholipase C (PC-PLC) inhibitor (D-609) and a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor (U-73122) attenuated LTA-induced iNOS expression and NO release. Two PKC inhibitors (Go 6976 and Ro 31-8220), an NF-kappaB inhibitor (pyrrolidine dithiocarbamate; PDTC), and long-term (24 h) 12-phorbol-13-myristate acetate (PMA) treatment each also inhibited LTA-induced iNOS expression and NO release. Treatment of cells with LTA caused an increase in PKC activity; this stimulatory effect was inhibited by D-609, U-73122, or Ro 31-8220. Stimulation of cells with LTA caused IkappaB-alpha phosphorylation and IkappaB-alpha degradation in the cytosol, and translocation of p65 and p50 NF-kappaB from the cytosol to the nucleus. Treatment of cells with LTA caused NF-kappaB activation by detecting the formation of NF-kappaB-specific DNA-protein complexes in the nucleus; this effect was inhibited by Go 6976, Ro 31-8220, long-term PMA treatment, PDTC, L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), and calpain inhibitor I. These results suggest that LTA might activate PC-PLC and PI-PLC to induce PKC activation, which in turn initiates NF-kappaB activation, and finally induces iNOS expression and NO release in RAW 264.7 macrophages.     

Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation

Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity.     

NF-kappaB p65 regulates nuclear translocation of Ku70 via degradation of heat shock cognate protein 70 in pancreatic acinar AR42J cells

Ku proteins such as Ku70 and Ku80 play key roles in multiple nuclear processes. Nuclear translocation of Ku70 is independent of Ku80 translocation and mediated by nuclear localization signal (NLS) receptors including importin-alpha. In the present study using pancreatic acinar AR42J cells, heat shock cognate protein 70 (Hsc70) was identified as the protein associated with NLS of Ku70. Interaction of Ku70 with importin-alpha and nuclear translocation of Ku70 was suppressed by overexpression of Hsc70, but enhanced by downregulation of Hsc70. The results suggest that the formation of Ku70 complex with Hsc70 prevents NLS of Ku70 from access of importin-alpha and inhibits nuclear translocation of Ku70. Since NF-kappaB p65 activation induced the decrease of Hsc70 level, the interaction of Ku70 with importin-alpha and nuclear translocation of Ku70 increased upon the activation of NF-kappaB p65. NF-kappaB p65 induced cell proliferation through decrease of Hsc70 levels and increase of nuclear translocation of Ku70. In the cells treated with cerulein as a physiological stimulus to activate NF-kappaB p65, nuclear translocation of Ku70 increased through NF-kappaB p65-mediated decrease of Hsc70 level. The results suggest that the involvement of NF-kappaB p65 in nuclear translocation of Ku70 may be mediated by Hsc70 degradation, which may play a key role in cell proliferation of pancreatic acinar AR42J cells.