Interleukin-6 Gene Transcription Research Articles

Shiga Toxin 2 and Flagellin from Shiga-ToxigenicEscherichia coliSuperinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation

Shiga toxins expressed in the intestinal lumen during infection with Shiga-toxigenic Escherichia coli must translocate across the epithelium and enter the systemic circulation to cause systemic (pathological) effects, including hemolytic uremic syndrome. The transepithelial migration of polymorphonuclear leukocytes in response to chemokine expression by intestinal epithelial cells is thought to promote uptake of Stx from the intestinal lumen by compromising the epithelial barrier. In the present study, we investigated the hypothesis that flagellin acts in conjunction with Shiga toxin to augment this chemokine expression. We investigated the relative contributions of nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) signaling to transcription and translation of interleukin-8. Using reporter gene constructs, we showed that flagellin-mediated interleukin-8 gene transcription is heavily dependent on both NF-kappaB and extracellular signal-regulated kinase 1 and 2 (ERK-1/2) activation. In contrast, inhibition of p38 has no detectable effect on interleukin-8 gene transcription, even though flagellin-mediated activation of host p38 is critical for maximal interleukin-8 protein expression. Inhibition of MAPK-interacting kinase 1 suggests that p38 signaling affects the posttranscriptional regulation of interleukin-8 protein expression induced by flagellin. Cotreatment with Stx2 and flagellin results in a synergistic upregulation of c-Jun N-terminal protein kinases (JNKs), p38 activation, and a superinduction of interleukin-8 mRNA. This synergism was also evident at the protein level, with increased interleukin-8 protein detectable following cotreatment with flagellin and Stx2. We propose that flagellin, in conjunction with Shiga toxin, synergistically upregulates stress-activated protein kinases, resulting in superinduction of interleukin-8 and, ultimately, absorption of Stx into the systemic circulation.

Targeted Knockdown of EGR-1 Inhibits IL-8 Production and IL-8-mediated Invasion of Prostate Cancer Cells through Suppressing EGR-1/NF-κB Synergy

IL-8 produced by prostate cancer cells may be responsible for the androgen-independent growth of advanced prostate cancers. Accumulating evidence from microarray analyses and animal genetic models highlights the central involvement of the transcription factor early growth response-1 (EGR-1) in prostate carcinoma progression. It is unknown, however, whether knockdown of EGR-1 inhibits IL-8 production and IL-8-mediated tumor metastasis. Here we show that EGR-1 knockdown by a specific shRNA-Egr1 inhibited gene transcription and production of IL-8 by the human prostate cancer cell line DU145. Conversely, enforced expression of EGR-1 in EGR-1-lacking PC3 prostate cancer cells markedly enhanced IL-8 transcription and secretion. By using wild type and a series of mutant IL-8 promoter luciferase constructs, we found that the NF-kappaB binding site is important for EGR-1 regulation of IL-8. Furthermore, silencing EGR-1 suppressed a synergistically functional interaction between EGR-1 and NF-kappaB. Consequently, knockdown of EGR-1 inhibited IL-8-mediated tumor colony formation and invasion. Thus, targeted knockdown of EGR-1 could be an effective therapeutic approach against prostate cancer.

The pilot study on the correlation between human cytomegalovirus infection and the secretion of chemokines in the cultured human fibroblasts

To investigate the effect of HCMV infection on the expression of interleukin-8 (IL-8) and regulated on activation, normal T expressed and secreted (RANTES) so as to explore the possible mechanism of the immune-pathological changes caused by HCMV infection. Expression of both IL-8 and RANTES mRNA was detected by RT-PCR. Secretion of IL-8 and RANTES protein was quantitated by enzyme linked immune-sororbant assay (ELISA). After HCMV infection, the expression of IL-8 in HEL cells was found to be increased gradually concomitant with the prolonged infection time at both the transcriptional level and the protein level. By the time of 72 h.p.i, as compared with mock-infected cells, IL-8 mRNA expression was increased up to 12-fold and IL-8 protein up to 9-fold in HCMV-infected cells. The expression of RANTES mRNA was occurred at 8 h.p.i, with a maximum at 24 h.p.i, since then it remained relatively high level. The expression of RANTES protein peaked at 24 h.p.i, then dropped sharply and was almost undetectable by the time at 72 h.p.i. HCMV infection of HEL cells may induce the transcription of IL-8 gene as well as the production of IL-8 orotein. HCMV may down-regulate extra-cellular production of RANTES protein.

Expression of interleukin-6 is downregulated by 17-(allylamino)-17-demethoxygeldanamycin in human prostatic carcinoma cells

Interleukin-6 (IL-6) is a pleiotropic cytokine that is associated with tumor metastasis and prostate cancer. We evaluated the mechanism and effect of 17-(allylamino)-17-demethoxygeldanamycin (17AAG), a novel inhibitor of heat shock protein 90 (Hsp90), on the IL-6 gene expression in human prostatic carcinoma (PC-3) cells. Quantitative IL-6 and IL-6 receptor (IL-6R) expressions were assessed using RT-PCR. The deregulation of 17AAG and phorbol 12-myristate 13-acetate (PMA) on the IL-6 gene was determined by ELISA and transient gene expression assays using an IL-6 reporter vector. Although the IL-6R is ubiquitously expressed by prostatic epithelium cells, the IL-6 expression is only found in advanced prostatic carcinoma cells, such as PC-3 and DU145. Further studies using RT-PCR indicated that 17AAG downregulated the gene expression of IL-6. ELISA and the transient gene expression assay revealed that 17AAG blocked the stimulation of PMA of IL-6 gene expression in PC-3 cells. The PMA-induced IL-6 gene expression is dependent on the NF-kappaB response element. However, the effect of 17AAG appears to be mediated via a region located at -149 to +8 bp upstream of the transcriptional starting site of the IL-6 gene, and might not be through the NF-kappaB signaling pathway. The present study reveals that IL-6 is transcriptionally downregulated in human prostatic carcinoma cells in response to 17AAG. This result suggests the presence of a novel Hsp90 mediation pathway that is involved in the deregulation on the transcription of the human IL-6 gene in human prostate cancer.

Interleukin (IL) 1β Induction of IL-6 Is Mediated by a Novel Phosphatidylinositol 3-Kinase-dependent AKT/IκB Kinase α Pathway Targeting Activator Protein-1

Here we describe a novel role for the phosphatidylinositol 3-kinase/AKT pathway in mediating induction of interleukin-6 (IL-6) in response to IL-1. Pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited IL-6 mRNA and protein production. Overexpression of either dominant-negative AKT or IkappaB kinase alpha mutant, IKKalphaT23A, containing a mutation in a functional AKT phosphorylation site, shown previously to be important for NFkappaB activation, completely abrogated IL-6 promoter activation in response to IL-1. However, mutation of the consensus NFkappaB site on the IL-6 promoter did not abrogate promoter activation by IL-1 in contrast to the AP-1 site mutation. IL-1 induces phosphorylation of IKKalpha on the NFkappaB inducing kinase (NIK) phosphorylation sites Ser(176)/Ser(180) and on the Thr(23) site, and although phosphorylation of IKKalphaT23 is inhibited both by LY294002 and wortmannin, phosphorylation of Ser(176)/Ser(180) is not. Neither inhibition of PI 3-kinase/AKT nor IKKalphaT23A overexpression affected IkappaBalpha degradation in response to IL-1. Only partial inhibition by dominant-negative AKT and no inhibitory effect of IKKalphaT23A was observed on an IL-6 promoter-specific NFkappaB site in contrast to significant inhibitory effects on the AP-1 site. Taken together, we have discovered a novel PI 3-kinase/AKT-dependent pathway in response to IL-1, encompassing PI 3-kinase/AKT/IKKalphaT23 upstream of AP-1. This novel pathway is a parallel pathway to the PI 3-kinase/AKT upstream of NFkappaB and both are involved in IL-6 gene transcription in response to IL-1.

Cigarette Smoke Condensate Enhances Respiratory Syncytial Virus–Induced Chemokine Release by Modulating NF-kappa B and Interferon Regulatory Factor Activation

Exposure to cigarette smoke is a risk factor contributing to the severity of respiratory tract infections associated with respiratory syncytial virus (RSV). Stimulation of airway epithelial cells by either RSV or cigarette smoke condensate (CSC) has been shown to induce secretion of the proinflammatory chemokines. However, the effect of coexposure of airway epithelial cells to CSC and RSV on inducible chemokine production has not been previously investigated. The results of this study indicate that CSC costimulation significantly increased RSV-induced interleukin-8 (IL-8) and monocyte chemoattactant protein-1 gene and protein expression when compared with each stimulus alone. Promoter deletion studies identified the interferon stimulatory response element (ISRE) of the IL-8 promoter as a critical region responsible for the synergistic increase of IL-8 gene transcription during mixed exposure. CSC costimulation enhanced RSV-induced activation of interferon regulatory factor (IRF)-1 and IRF-7, which bind to the ISRE site. CSC also furthered RSV-induced activation of the transcription factor nuclear factor kappa B (NF-kappaB), as shown by increased NF-kappaB DNA binding to its specific site of the IL-8 promoter and increased NF-kappaB-driven gene transcription. Therefore, our data demonstrate that a combined exposure to CSC and RSV synergistically increases chemokine expression in airway epithelial cells, suggesting that CSC contributes to an exuberant immune response to RSV by stimulating overlapping signal transduction pathways.

Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells

BackgroundLegionella pneumophila is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of L. pneumophila on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines.ResultsInfection of L. pneumophila strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with L. pneumophila lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient Legionella. Activation of the IL-8 promoter by L. pneumophila infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited L. pneumophila-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed L. pneumophila-induced IL-8 mRNA due to deactivation of NF-κB.ConclusionCollectively, these results suggest that L. pneumophila induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in L. pneumophila-induced IL-8 expression, presumably contributing to immune response in L. pneumophila. The presence of flagellin and a type IV secretion system are critical for Legionella to induce IL-8 expression in lung epithelial cells.

Pituitary adenylate cyclase-activating polypeptide stimulates corticotropin-releasing factor, vasopressin and interleukin-6 gene transcription in hypothalamic 4B cells

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic-pituitary-adrenal axis. CRF, produced in the hypothalamic paraventricular nucleus (PVN) in response to stress, is secreted into the pituitary portal circulation, resulting in the release of adrenocorticotropic hormone from the anterior pituitary. AVP is synthesized in the PVN and supraoptic nucleus by various stressors. Hypothalamic 4B cells coexpress CRF and AVP. In 4B cells transfected with either a CRF or an AVP promoter-luciferase construct, forskolin increased the transcriptional activity of CRF or AVP. In the present study, we tried to determine whether pituitary adenylate cyclase-activating polypeptide (PACAP) regulates both CRF and AVP genes in the hypothalamic cells, because receptors for PACAP were expressed in the hypothalamic cells. PACAP stimulated activity of both CRF and AVP promoter via protein kinase A pathway. PACAP stimulated interleukin (IL)-6 promoter activity and the levels of IL-6 mRNA and protein. IL-6 stimulated activity of both CRF and AVP promoter in a dose-dependent manner. Finally, we found that the stimulatory effects of PACAP on both activities were significantly inhibited by treatment with anti-IL-6 monoclonal antibody. These data suggest that PACAP is involved in regulating the synthesis of IL-6 mRNA and IL-6 protein, and that the increase in endogenous IL-6 also contributes to stimulate the expression of both CRF and AVP genes. Taken together, these findings indicate that PACAP stimulates the transcription of CRF, AVP, and IL-6 genes in hypothalamic 4B cells.

Contraction‐induced interleukin‐6 transcription in rat slow‐type muscle is partly dependent on calcineurin activation

The present work aimed at determining whether interleukin-6 (IL-6) produced by skeletal muscle during exercise is related, at least partly, to calcineurin activity. Rats were treated with two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, or vehicle (Vhl); they were then subjected to exhaustive treadmill running. Modulatory Calcineurin-Interacting Protein-1 (MCIP-1) mRNA levels, a reliable indicator of calcineurin activity, and IL-6 mRNA levels were measured by real-time RT-PCR in soleus muscles, and IL-6 protein concentration was measured in the plasma. Because low carbohydrates availability enhances IL-6 transcription through p38 Mitogen Activated Protein Kinase (MAPK) pathway, muscle glycogen content and glycaemia were measured and p38 MAPK phosphorylation was determined in skeletal muscle by western blotting. As expected, exercise induced an increase in IL-6 (P < 0.01) and MCIP-1 mRNA (P < 0.01) in soleus muscle of Vhl rats, and enhanced p38 phosphorylation and plasmatic IL-6 protein (P < 0.05). Calcineurin inhibition did not affect running time, glycemia or soleus glycogen content. CsA administration totally inhibited the exercise-induced increase in MCIP-1 mRNA (P < 0.01), blunted the IL-6 gene transcription related to muscle activity, and suppressed the changes in IL-6 protein in plasma. In addition to its inhibition of calcineurin activity, FK506 administration totally suppressed the exercise-induced IL-6 gene transcription, likely by an inhibition of p38 activation. Taken together, these results demonstrate that in addition to p38 MAPK, increased calcineurin activity is one of the signalling events involved in IL-6 gene transcription.

Fluid Resuscitation Increases Inflammatory Gene Transcription After Traumatic Injury

The debate continues over type and quantity of fluid to administer for resuscitation after traumatic injury. This study aimed to examine effects of resuscitation with lactated Ringer's (LR) and Hextend (HEX) on the inflammatory response after uncontrolled hemorrhagic shock (UHS). There were 38 swine randomized. Control swine were anesthetized and killed. Sham swine underwent laparotomy, splenectomy, and 2 hours of anesthesia. UHS swine received a grade V liver injury after laparotomy and splenectomy and were randomized to no fluid (NF) resuscitation or to blinded resuscitation 30 minutes after injury with LR or HEX. Fluids were infused as needed to maintain baseline blood pressure for 90 minutes. Lung tissue mRNA levels of interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), and tumor necrosis factor alpha (TNF-alpha) were determined. Lung sections were examined for neutrophils (PMNs) sequestered within alveolar walls. All UHS animals survived and initial blood loss was similar between groups. Mean arterial pressures (MAPs) were similar for all UHS animals until resuscitation was initiated. MAPs of resuscitated animals remained similar and were significantly higher than MAPs of the NF animals. Sequestered PMNs were equally elevated in all UHS animals. Cytokine analysis showed increased IL-6, G-CSF, and TNF-alpha gene transcription in resuscitated swine compared with NF swine. LR and HEX resuscitated swine tissue mRNA levels showed no differences. Fluid resuscitation after solid organ injury and uncontrolled hemorrhage results in greater proinflammatory gene transcription than no resuscitation. LR and HEX resuscitation have equivalent effects on indices of inflammation in the lungs.

Interleukin‐6 g.−174G&gt;C Promoter Polymorphism Is Associated with Obesity in the EPIC‐Potsdam Study

Homozygosity for the interleukin-6 (IL-6) g.-174G>C promoter polymorphism has recently been associated with indices of overweight. Homozygous subjects were observed to have reduced energy expenditure, suggesting that lower IL-6 gene transcription, caused by the IL-6 g.-174G>C promoter polymorphism, may be associated with obesity. The aim of this study was to investigate the association of this polymorphism with long-term weight gain. For 334 normal weight (20 < BMI < or = 25 kg/m2) and 334 obese (BMI > 30 kg/m2) subjects matched by age and sex originating from the population-based EPIC-Potsdam Study, recalled weight change from age 25 to study enrollment was determined, the IL-6 g.-174G>C promoter polymorphism was defined, and plasma concentrations of IL-6 and C-reactive protein were measured. The IL-6 g.-174G>C promoter polymorphism was significantly associated with obesity (chi2 = 7,34, p = 0.026). Odds ratios for subjects with GC and CC genotypes for obesity were 1.19 (95% CI: 0.84 to 1.68; p = 0.323) and 1.91 (95% CI: 1.19 to 3.08; p = 0.007), respectively. Recalled weight change from age 25 years to study enrollment differed significantly according to genotype (p = 0.044) and was most pronounced in subjects with the CC genotype, suggesting that the IL-6 g.-174G>C promoter polymorphism is a susceptibility or modifying locus for common obesity and weight gain.

Depolarization-induced slow Ca2+ transients stimulate transcription of IL-6 gene in skeletal muscle cells

Contracting skeletal muscle produces and releases interleukin-6 (IL-6) in high amounts. Nevertheless, the mechanisms underlying IL-6 expression are not understood. Because inositol-1,4,5-trisphosphate (IP(3))-mediated slow Ca(2+) signals evoked by depolarization of skeletal myotubes appears to play a role in the regulation of gene expression, we examined its involvement on IL-6 transcription. With the use of semiquantitative RT-PCR, we have shown that K(+) depolarization of myotubes induces a transient increase in IL-6 mRNA level, which peaks at 3-4 h and is independent of extracellular Ca(2+). Inhibitors of IP(3)-dependent Ca(2+) signals, like 2-aminoethoxydiphenyl borate (2-APB) and U-73122, decreased activation of IL-6 gene expression as did Ca(2+) signals inhibitor BAPTA-AM, whereas ryanodine, a fast Ca(2+) transient inhibitor, had no effect on IL-6 induction. Depolarization of myotubes transiently transfected with a reporter gene construct, containing 651 bp of IL-6 promoter, induced a twofold increase in promoter activity, which was abolished by either 2-APB or U-73122 and remained unaffected after ryanodine treatment. Site-directed mutagenesis of parental construct allowed us to identify activator protein-1 and NF-kappaB sequences as regulatory elements involved in IL-6 upregulation. Our results provide evidence for involvement of IP(3)-mediated Ca(2+) signals on IL-6 transcription in skeletal muscle cells.

Differential regulation of interleukin-8 gene transcription by death receptor 3 (DR3) and type I TNF receptor (TNFRI)

TL1A induces interleukin-8 (IL-8) secretion in human peripheral blood monocyte-derived macrophage in a dose- and time-dependent manner. Overexpression of its cognate receptor DR3 can induce a higher amount of IL-8 protein secretion than that induced by TNFRI even though both receptors activate IL-8 gene transcription in a similar fashion. The underlying mechanism for the regulation of the IL-8 gene transcription by DR3 has not been investigated yet. Here, we used HEK293 cells as a model system to dissect the possible signaling components that are involved in the regulation of DR3-mediated IL-8 gene expression. Although both DR3 and TNFRI activated TRAF2 and NF-kappaB to induce IL-8 gene transcription, the kinase cascades that transduce signals for DR3- and TNFRI-induced IL-8 gene transcription are different. The axis TAK1/ASK1-MKK4/MKK7-JNK2 is responsible for DR3-mediated IL-8 gene expression whereas the axis ASK1-MKK4-JNK1/JNK2/p38MAPK is the choice for TNFRI-mediated activation of IL-8 gene expression. This indicates that the downstream signaling pathways of DR3 and TNFRI for IL-8 secretion are divergent even though both receptors contain death-domain and induce IL-8 secretion via TRAF2.

Fibre‐type specificity of interleukin‐6 gene transcription during muscle contraction in rat: association with calcineurin activity

In this study, we quantified the transcription of the interleukin-6 (IL-6) gene in individual fibres and the associated changes in calcineurin activity assessed at the cellular level during prolonged muscle contraction. Individual myofibres were isolated from plantaris and soleus muscles of rats at the end of an exhaustive running exercise test (n = 10), categorized according to their myosin heavy chain isoform content, and compared to those of resting rats (n = 10). Using real-time PCR analysis in individual fibres, a marked rise in IL-6 transcript levels occurred in type I and IIa fibres at the end of exercise (P < 0.05). Transcription of the gene encoding for the modulatory calcineurin-interacting protein-1 (MCIP-1), a sensitive indicator of calcineurin activity, also mainly increased in type I and IIa fibres (P < 0.05). Moreover, a slight increase in MCIP-1 mRNA levels was observed in type IIx (P < 0.05). Fibre types determined by immunohistochemistry were qualitatively examined for glycogen content using periodic acid-Shiff staining, and no direct relationship was found, at the cellular level, between glycogen content, fibre-type and IL-6 transcription. Our data clearly suggest that IL-6 gene transcription was mainly observed in early recruited myofibres and that contraction-induced IL-6 transcription could be associated with enhanced calcineurin activity.

Prostaglandin E2 Induces Interleukin-8 Gene Transcription by Activating C/EBP Homologous Protein in Human T Lymphocytes

The effect of prostaglandin E(2) (PGE(2)) in regulating the synthesis of the pro-inflammatory chemokine interleukin-8 (IL-8) in T lymphocytes is not yet defined, even though it may reduce or enhance IL-8 synthesis in other cell types. Here, we demonstrate that, in human T cells, PGE(2) induced IL-8 mRNA transcription through prostaglandin E(2) receptors 1- and 4-dependent signal transduction pathways leading to the activation of the transcription factor C/EBP homologous protein (CHOP), never before implicated in IL-8 transcription. Several kinases, including protein kinase C, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and p38 MAPK, were involved in PGE(2)-induced CHOP activation and IL-8 production. The transactivation of the IL-8 promoter by CHOP was NF-kappaB-independent. Our data suggest that PGE(2) acts as a potent pro-inflammatory mediator by inducing IL-8 gene transcription in activated T cells through different signal transduction pathways leading to CHOP activation. These findings show the complexity with which PGE(2) regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions.

Impact of the Mitogen-activated Protein Kinase Pathway on Parathyroid Hormone-related Protein Actions in Osteoblasts

Parathyroid hormone-related protein (PTHrP) regulates proliferation and differentiation of osteoblastic cells via binding to the parathyroid hormone receptor (PTH-1R). The cAMP-dependent protein kinase A pathway governs the majority of these effects, but recent evidence also implicates the MAPK pathway. MC3T3-E1 subclone 4 cells (MC4) were treated with the MAPK inhibitor U0126 and PTHrP. In differentiated MC4 cells, osteocalcin and bone sialoprotein gene expression were both down-regulated by PTHrP and also by inhibition of the MAPK pathway. PTHrP-mediated down-regulation of PTH-1R mRNA and up-regulation of c-fos mRNA were MAPK-independent, whereas PTHrP stimulation of fra-2 and interleukin-6 (IL-6) mRNA was MAPK-dependent. Luciferase promoter assays revealed that regulation of IL-6 involved the cAMP-dependent protein kinase A and MAPK pathways with a potential minor role of the protein kinase C pathway, and a promoter region containing an activator protein-1 site was necessary for PTHrP-induced IL-6 gene transcription. An alternative pathway, through cAMP/Epac/Rap1/MAPK, mediated ERK phosphorylation but was not sufficient for IL-6 promoter activation. Phosphorylation of the transcription factor CREB was also necessary but not sufficient for PTHrP-mediated IL-6 promoter activity. Most interesting, a bidirectional effect was found with PTHrP increasing phosphorylated ERK in undifferentiated MC4 cells but decreasing phosphorylated ERK in differentiated cells. These data indicate that inactivation of the MAPK pathway shows differential regulation of PTHrP-stimulated activator protein-1 members, blocks PTHrP-stimulated IL-6, and synergistically down-regulates certain osteoblastic markers associated with differentiation. These novel findings indicate that the MAPK pathway plays a selective but important role in the actions of PTHrP.

Hypoxia-induced activation of p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase signaling pathways contributes to expression of interleukin 8 in human ovarian carcinoma cells.

Overexpression of interleukin 8 (IL-8) is associated with disease progression in human ovarian cancer. Hypoxia, a common feature in solid tumors, induces IL-8 expression in human ovarian carcinoma cells through activation of nuclear factor-kappa B and activating protein-1. Here we show the upstream components of these signal transduction pathways that lead to IL-8 expression under hypoxia. We incubated Hey-A8 human ovarian carcinoma cells under hypoxic condition (1% O(2)) and determined hypoxia regulation of phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, mitogen-activated protein kinases (MAPKs), and effects of ras and vascular endothelial growth factor by Western and Northern blots, the use of specific inhibitors, in vitro kinase assays, luciferase reporter genes, and ELISA. While investigating the upstream signaling pathways, we found that Akt kinase and p38 MAPK are activated by hypoxia. Both hypoxia-induced Akt and p38 MAPK functional activity, and IL-8 mRNA and protein expression were reduced with the inhibition of PI3K and p38 MAPK. Oncogenic ras overexpression resulted in an increase in the hypoxia-induced IL-8 expression, whereas the inhibition of ras by transfection of dominant-negative ras inhibited the hypoxia-induced IL-8 expression. These results show that hypoxia activates ras, PI3K/Akt pathway, and p38 MAPK pathway to enhance IL-8 gene transcription under hypoxia, and suggest these signaling pathways as potential targets for controlling IL-8 expression and angiogenesis by human ovarian carcinoma cells.

Inhibition of Helicobacter pylori-induced nuclear factor-kappa B activation and interleukin-8 gene expression by ecabet sodium in gastric epithelial cells.

Helicobacter pylori stimulates nuclear factor-kappa B (NF-kappa B) activation and chemokine interleukin-8 (IL-8) expression in gastric epithelial cells. Ecabet sodium (ecabet), a locally acting antiulcer drug, is known to have anti-H. pylori activity. However, there is little understanding of how ecabet induces anti-inflammatory activity in gastric epithelial cells infected with H. pylori. The aim of this study was to investigate the effects of ecabet on IL-8 gene expression and NF-kappa B activation in human gastric epithelial cells infected with H. pylori. After Hs746T, MKN-45, or SNU-5 gastric epithelial cell lines had been infected with cagA+cytotoxin+H. pylori in the presence of ecabet, IL-8 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction, and IL-8 secretion was measured by enzyme-linked immunosorbent assay. NF-kappa B and inhibitory kappa B-alpha (I kappa B alpha) signals were assayed by electrophoretic mobility shift assay and Western blot, respectively. The activation of NF-kappa B and IL-8 reporter genes was determined by luciferase assay. Ecabet showed no antimicrobial activiy against Gram-positive or -negative bacteria. However, ecabet inhibited transcription of the IL-8 gene and secretion of IL-8 by gastric epithelial cells infected with H. pylori at a concentration of 5 micro g/ml. Moreover, ecabet inhibited the activation of NF- kappa B and the degradation of I kappa B alpha in gastric epithelial cells in response to H. pylori infection. In addition, the NF-kappa B signal inhibited by ecabet was comprised predominantly of heterodimers of p65/p50. Ecabet inhibited H. pylori-induced IL-8 gene transcription and secretion by suppressing the NF-kappa B signal. This inhibition might be one pathway by which ecabet exerts its anti-inflammatory effect on H. pylori-induced gastric inflammation.

Suppression of IL-8 gene transcription by resveratrol in phorbol ester treated human monocytic cells

Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and other food products, has promising anti-inflammatory and anticancer effects. To observe the modulation of interleukin-8 (IL-8) production in human monocytic cells by resveratrol and explore its mechanism at the gene transcription level, U937 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 h. IL-8 protein in supernatants was measured by radioimmunoassay. The cytotoxicity of PMA, dexamethasone and resveratrol was accessed by MTT cell proliferation assay. The RNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and IL-8 were detected by RT-PCR using specific primers. DNA binding activities of NF-κB and AP-1 were examined by electrophoretic mobility shift assay (EMSA). 0.01–100 nM PMA could significantly induce IL-8 production in U937 cells; 10 μM Dexamethasone and 10, 1, 0.1 μM resveratrol could inhibit PMA-induced IL-8 protein production and mRNA accumulation. The cytotoxicity did not contribute to their inhibitory effect. The DNA binding activity of AP-1 was inhibited by dexamethasone and resveratrol, but resveratrol has little effect on PMA-induced NF-κB activation. Resveratrol could inhibit PMA-induced IL-8 production in U937 cells at protein and mRNA levels. The suppression of IL-8 gene transcription by resveratrol was, at least partly, due to inhibition of AP-1 activation.

Interleukin-8 secretion from Mycobacterium tuberculosis-infected monocytes is regulated by protein tyrosine kinases but not by ERK1/2 or p38 mitogen-activated protein kinases.

Mycobacterium tuberculosis upregulates NF-kappaB binding and interleukin-8 (IL-8) gene expression and secretion in primary human monocytes. Inhibition of tyrosine protein kinases but not of ERK1/2 or p38 mitogen-activated protein kinases downregulates tuberculosis-induced IL-8 secretion. The inhibitor genistein decreased NF-kappaB nuclear translocation and IL-8 gene transcription in addition to acting on posttranscriptional processing.