Hypoxia modulates lipopolysaccharide induced TNF-alpha expression in murine macrophages

The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-alpha) together with tissue hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-alpha biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known. Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-induced TNF-alpha expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38 MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-alpha. Furthermore, hypoxia inducible factor-1alpha (HIF-1alpha) was detected in vivo after LPS administration but its expression was not affected by SB203580. Our data indicate that LPS induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-alpha secretion. Furthermore, the induction of HIF-1alpha in mice with endotoxemia suggested a synergistic effect on p38 mediated TNF-alpha expression. These findings provide new insights on the pathophysiological effects of hypoxia in sepsis and septic shock.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

Mitogen-activated protein kinases in cell-cycle control

The mitogen-activated protein kinase (MAPK) family of kinases connects extracellular stimuli with diverse cellular responses ranging from activation or suppression of gene expression to the regulation of cell mortality, growth, and differentiation. The MAPK family has been studied extensively; however, the role of these kinases in cell growth and cell-cycle control has become increasingly complex. Patterns have begun to emerge from these studies that show the functions of MAPK subfamilies at different stages of the cell cycle. Their patterns of subcellular localization and movement during the cell cycle are subfamily-specific and have raised many questions about possible cell-cycle functions that have yet to be demonstrated. This article will compare and contrast our current understanding of the functions and localization patterns of the MAPK subfamilies (ERK, BMK, p38, and JNK) in cell-cycle control.     

IFN-gamma sensitizes MIN6N8 insulinoma cells to TNF-alpha-induced apoptosis by inhibiting NF-kappaB-mediated XIAP upregulation

Although X-linked inhibitor of apoptosis protein (XIAP) is an important intracellular suppressor of apoptosis in a variety of cell types, its role in cytokine-induced pancreatic beta-cell apoptosis remains unclear. Here, we found that: (i) XIAP level was inversely correlated with tumor necrosis factor (TNF)-alpha-induced apoptosis in MIN6N8 insulinoma cells; (ii) adenoviral XIAP overexpression abrogated the TNF-alpha-induced apoptosis through inhibition of caspase activity; (iii) downregulation of XIAP by antisense oligonucleotide or Smac peptide sensitized MIN6N8 cells to TNF-alpha-induced apoptosis; (iv) XIAP expression was induced by TNF-alpha through a nuclear factor-kappaB (NF-kappaB)-dependent pathway, and interferon (IFN)-gamma prevented such an induction in a manner independent of NF-kappaB, which presents a potential mechanism underlying cytotoxic IFN-gamma/TNF-alpha synergism. Taken together, our results suggest that XIAP is an important modulator of TNF-alpha-induced apoptosis of MIN6N8 cells, and XIAP regulation in pancreatic beta-cells might play an important role in pancreatic beta-cell apoptosis and in the pathogenesis of type 1 diabetes.     

Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is induced by glucocorticoids (GCs), but it was not previously known if MIF regulates cellular sensitivity to GC. Here we show in GC and LPS-treated peritoneal macrophages derived from MIF-/- and wt mice that the absence of endogenous MIF is associated with increased sensitivity to GC of TNF release. This is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), concomitant decreased phosphorylation of p38 MAPK, but no effect of MIF on nuclear factor kappaB (NF-kappaB). These results demonstrate that MIF regulates GC sensitivity by phosphorylation of p38, and provides a cellular mechanism for this observation, indicating that MKP-1 is a central target of this regulation.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

Differential regulation of IGF-II-induced IL-8 by extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases in human keratinocytes

In order to study the relationship between insulin like growth factor-II (IGF-II) and interleukin-8 (IL-8) that are upregulated in psoriasis, we monitored IL-8 expression in IGF-II-treated human keratinocytes and explored the signaling pathways of IL-8 expression by IGF-II. IGF-II increased the IL-8 mRNA and protein levels in human keratinocytes. The upregulation of IL-8 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src, PI3-kinase, and ERK, but not by p38. Furthermore, IGF-II remarkably increased the DNA binding activities of NF-kappaB and AP-1, and the IL-8 promoter activity. However, cotransfection with IkappaB mutant blocked the IGF-II-induced IL-8 promoter activity. In addition, cotransfection with dominant negative MEK1 mutant, but not with dominant negative p38 mutant, blocked the IGF-II-induced IL-8 promoter activity. These results suggest that IGF-II is involved in the pathogenesis of psoriasis by inducing IL-8 gene expression through the tyrosine kinase-Src-ERK1/2-AP-1 pathway, and the PI3-kinase and NF-kappaB pathway.     

Following in vitro activation of mitogen-activated protein kinases by mass spectrometry and tryptic peptide analysis: purifying fully activated p38 mitogen-activated protein kinase alpha

p38 mitogen-activated protein kinase alpha (MAPKalpha) belongs to the MAPK subfamily, which plays a pivotal role in cell signal transduction, where it mediates responses to cell stresses and, to a lesser extent, growth factors. Although its cellular function has been under intense scrutiny since its initial discovery, little progress has been made in understanding its kinetic mechanism. A contributory factor has been the lack of a fast and rigorous method for the purification of activated p38 MAPKalpha in sufficient quantity and purity for biophysical studies. Here we present a method for the preparation of milligram quantities of activated p38 MAPKalpha, specifically phosphorylated on Thr180 and Tyr182. Purification of the inactive (unphosphorylated) p38 MAPKalpha is facilitated by an N-terminal hexahistidine tag. Removal of this tag from His6-p38 MAPKalpha, prior to its activation, is essential to ensure preparation of high yields of homogeneous, dually phosphorylated enzyme. Activation is achieved on incubation with a glutathione S-transferase (GST) fusion of the constitutively active mutant of the upstream activator, MKK6b (GST-MKK6b S207E T211E), in the presence of MgATP2-. Notably, we show that specific formation of activated p38 MAPKalpha can be quantified by following the formation of the bis-phosphorylated tryptic peptide, 173-HTDDEMT*GY*VATR-186, using [gamma-32P]adenosine triphosphate (ATP) as the phosphate source and reverse-phase high-performance liquid chromatography (HPLC) to separate the phosphopeptides. This approach offers the only means to specifically determine both stoichiometry and specificity of p38 MAPKalpha phosphorylation.     

The α2AR/Caveolin-1/p38MAPK/NF-κB axis explains dexmedetomidine protection against lung injury following intestinal ischaemia-reperfusion

Intestinal ischaemia-reperfusion (I/R) injury can result in acute lung injury due to ischaemia and hypoxia. Dexmedetomidine (Dex), a highly selective alpha2-noradrenergic receptor (α2AR) agonist used in anaesthesia, is reported to regulate inflammation in organs. This study aimed to investigate the role and mechanism of Dex in lung injury caused by intestinal I/R. After establishing a rat model of intestinal I/R, we measured the wet-to-dry specific gravity of rat lungs upon treatments with Dex, SB239063 and the α2AR antagonist Atipamezole. Moreover, injury scoring and histopathological studies of lung tissues were performed, followed by ELISA detection on tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 expression. Correlation of Caveolin-1 (Cav-1) protein expression with p38, p-p38, p-p65 and p65 in rat lung tissues was analysed, and the degree of cell apoptosis in lung tissues after intestinal I/R injury was detected by TUNEL assay. The lung injury induced by intestinal I/R was a dynamic process. Moreover, Dex had protective effects against lung injury by mediating the expression of Cal-1 and α_2A-AR. Specifically, Dex promoted Cav-1 expression via α_2A-AR activation and mitigated intestinal I/R-induced lung injury, even in the presence of Atipamezole. The protective effect of Dex on intestinal I/R-induced lung injury was also closely related to α_2A-AR/p38 mitogen-activated protein kinases/nuclear factor-kappaB (MAPK/NF-κB) pathway. Dex can alleviate pulmonary inflammation after in intestinal I/R by promoting Cav-1 to inhibit the activation of p38 and NF-κB. In conclusion, Dex can reduce pulmonary inflammatory response even after receiving threats from both intestinal I/R injury and Atipamezole.     

Mitogen-activated protein kinases and cerebral ischemia

Mitogen-activated protein kinases (MAPKs) have crucial roles in signal transduction from the cell surface to the nucleus and regulate cell death and survival. Recent papers support the hypothesis that neuronal apoptosis and cerebral ischemia induce the robust activation of MAPK cascades. Although extracellular signal-regulated kinases pathways promote cell survival and proliferation, and c-Jun N-terminal protein kinases/p38 pathways induce apoptosis in general, the roles of MAPK cascades in neuronal death and survival seem to be complicated and altered by the type of cells and the magnitude and timing of insults. Some specific inhibitors of MAPK cascades provide important information in clarifying the roles of each molecule in neuronal death and survival, but the results are still controversial. Further studies are necessary to elucidate the activated signal transduction upstream and downstream of the cascades in cerebral ischemia, and to define the crosstalk between the cascades and other signaling pathways, before MAPK cascades can be candidate molecules in the treatment of cerebral ischemia.