Eotaxin Promoter Research Articles

Trans-activation of eotaxin-1 by Brg1 contributes to liver regeneration

Infiltration of eosinophils is associated with and contributes to liver regeneration. Chemotaxis of eosinophils is orchestrated by the eotaxin family of chemoattractants. We report here that expression of eotaxin-1 (referred to as eotaxin hereafter), but not that of either eotaxin-2 or eotaxin-3, were elevated, as measured by quantitative PCR and ELISA, in the proliferating murine livers compared to the quiescent livers. Similarly, exposure of primary murine hepatocytes to hepatocyte growth factor (HGF) stimulated eotaxin expression. Liver specific deletion of Brahma-related gene 1 (Brg1), a chromatin remodeling protein, attenuated eosinophil infiltration and down-regulated eotaxin expression in mice. Brg1 deficiency also blocked HGF-induced eotaxin expression in cultured hepatocytes. Further analysis revealed that Brg1 could directly bind to the proximal eotaxin promoter to activate its transcription. Mechanistically, Brg1 interacted with nuclear factor kappa B (NF-κB)/RelA to activate eotaxin transcription. NF-κB knockdown or pharmaceutical inhibition disrupted Brg1 recruitment to the eotaxin promoter and blocked eotaxin induction in hepatocytes. Adenoviral mediated over-expression of eotaxin overcame Brg1 deficiency caused delay in liver regeneration in mice. On the contrary, eotaxin depletion with RNAi or neutralizing antibodies retarded liver regeneration in mice. More important, Brg1 expression was detected to be correlated with eotaxin expression and eosinophil infiltration in human liver specimens. In conclusion, our data unveil a novel role of Brg1 as a regulator of eosinophil trafficking by activating eotaxin transcription.

Demethylation of the Human Eotaxin-3 Gene Promoter Leads to the Elevated Expression of Eotaxin-3

DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. In this article, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue. The demethylation of a specific CpG site (designated CpG 2), which is juxtaposed to a key cAMP-responsive element site, was significantly demethylated in patient-derived compared with normal control tissue-derived epithelial cells. Levels of methylation at CpG 2 inversely correlated with basal and IL-13-induced eotaxin-3 gene expression. Conversely, global inhibition of methylation with 5-azacytidine promoted eotaxin-3 production in association with decreasing CpG 2 methylation. In addition, the basal and IL-13-induced eotaxin-3 transcriptional activity was suppressed by promoter methylation using a methylation-free in vitro system. Furthermore, EMSAs demonstrated that the attachment of CREB binding protein and activating transcription factor 2 (ATF-2) to the cAMP-responsive element site was methylation dependent. Taken together, these data identify a contributory role for DNA methylation in regulating eotaxin-3 production in human allergic inflammation.

Omeprazole Blocks STAT6 Binding to the Eotaxin-3 Promoter in Eosinophilic Esophagitis Cells

BackgroundPatients who have esophageal eosinophilia without gastroesophageal reflux disease (GERD) nevertheless can respond to proton pump inhibitors (PPIs), which can have anti-inflammatory actions independent of effects on gastric acid secretion. In esophageal cell cultures, omeprazole has been reported to inhibit Th2 cytokine-stimulated expression of eotaxin-3, an eosinophil chemoattractant contributing to esophageal eosinophilia in eosinophilic esophagitis (EoE). The objective of this study was to elucidate molecular mechanisms underlying PPI inhibition of IL-4-stimulated eotaxin-3 production by esophageal cells.Methods/FindingsTelomerase-immortalized and primary cultures of esophageal squamous cells from EoE patients were treated with IL-4 in the presence or absence of acid-activated omeprazole or lansoprazole. We measured eotaxin-3 protein secretion by ELISA, mRNA expression by PCR, STAT6 phosphorylation and nuclear translocation by Western blotting, eotaxin-3 promoter activation by an exogenous reporter construct, and STAT6, RNA polymerase II, and trimethylated H3K4 binding to the endogenous eotaxin-3 promoter by ChIP assay. Omeprazole in concentrations ≥5 µM significantly decreased IL-4-stimulated eotaxin-3 protein secretion and mRNA expression. Lansoprazole also blocked eotaxin-3 protein secretion. Omeprazole had no effect on eotaxin-3 mRNA stability or on STAT6 phosphorylation and STAT6 nuclear translocation. Rather, omeprazole blocked binding of IL-4-stimulated STAT6, RNA polymerase II, and trimethylated H3K4 to the eotaxin-3 promoter.Conclusions/SignificancePPIs, in concentrations achieved in blood with conventional dosing, significantly inhibit IL-4-stimulated eotaxin-3 expression in EoE esophageal cells and block STAT6 binding to the promoter. These findings elucidate molecular mechanisms whereby patients with Th2 cytokine-driven esophageal eosinophilia can respond to PPIs, independent of effects on gastric acid secretion.

Association between three single nucleotide polymorphisms in eotaxin (CCL 11) gene, hexanucleotide repetition upstream, severity and course of coronary atherosclerosis

The impact of three single-nucleotide polymorphisms in eotaxin (SCYA11) gene promoter (-426C>T and -384A>G) and first exon (67G>A) and recently described hexanucleotide (GAAGGA)(n) 10.9 kb upstream on coronary atherosclerosis was investigated. Elective coronary angiography of 1050 consecutive subjects was performed. All patients were genotyped for the three SNPs. In a subset of the first 472 samples, the number of (GAAGGA)(n) repetitions was determined. For further evaluation, short and long variants were distinguished; the borderline corresponded with the median value of all alleles: ≤8 repetitions were considered as short sequence, ≥9 repetitions as long. Patients with bronchial asthma or insignificant atherosclerosis were excluded; the remaining group of 933 subjects was further investigated. Patients were grouped according to the form of CAD (ACS vs. stable angina) and the number of diseased vessels. The GG variant of 67 G>A polymorphism was associated with acute form of CAD compared to stable angina (p=0.0011, p(corr.)=0.013). The number of (GAAGGA)(n) repetitions in our set of patients ranged from 3 to 12. There were no subjects with 4 or 5 repetitions. The frequency of short repetition alleles increased with the number of affected vessels (1 vs. 3 diseased vessels: p=0.0043, p(corr)=0.034). In our study, the (GAAGGA)(n) hexanucleotide was associated with the severity of CAD. The 67 GG was associated with acute form of CAD. None of the two SNPs in eotaxin promoter had any relation to CAD. The number of (GAAGGA)(n) repetitions can thus be a novel genetic marker of the extent of CAD.

Omeprazole blocks eotaxin-3 expression by oesophageal squamous cells from patients with eosinophilic oesophagitis and GORD

ObjectiveEosinophilic oesophagitis (EoE) and gastro-oesophageal reflux disease (GORD) can have similar clinical and histological features. Proton pump inhibitors (PPIs) are used to distinguish the disorders, with the assumption that only...

599 in Esophageal Squamous Epithelial Cells From Patients With EoE, Omeprazole Blocks Cytokine-Stimulated Eotaxin-3 Secretion by Reducing Binding of STAT6 to the Eotaxin-3 Promoter.

599 in Esophageal Squamous Epithelial Cells From Patients With EoE, Omeprazole Blocks Cytokine-Stimulated Eotaxin-3 Secretion by Reducing Binding of STAT6 to the Eotaxin-3 Promoter.

Differences in Eotaxin-3 Expression After Stimulation With IL-13 and IL-4 in Esophageal Squamous Cell Lines From Patients With Eosinophilic Esophagitis and GERD.

Introduction: In eosinophilic esophagitis (EoE), food and other allergens appear to activate Th2 cells to release IL-13 and IL-4, cytokines that can induce esophageal squamous epithelial cells to produce the potent eosinophil chemoattractant eotaxin-3. GERD also can cause an eosinophilic infiltration of the esophagus, usually milder than that seen in EoE, through mechanisms that are not clear. In earlier studies, we showed that IL-13 and IL-4 caused a marked increase in eotaxin-3 protein secretion in an esophageal squamous cell line derived from a patient with EoE, but only a minimal increase in eotaxin-3 protein secretion in esophageal squamous cell lines derived from GERD patients. To explore this mechanism further, we studied the effects of IL-13 and IL-4 on transcriptional activation of eotaxin-3 in esophageal squamous cell lines from patients with EoE and GERD. Methods: Two telomerase-immortalized, non-neoplastic esophageal squamous cell lines derived from patients with GERD (NES-B10T & NES-G4T), and one derived from a patient with EoE (EoE1-T) were stimulated with IL-13 (100 ng/ml) or IL-4 (1 ng/ml) for times ranging from 24-48 hours. Eotaxin-3 mRNA expression levels were determined by RT-PCR, and an eotaxin-3 promoter (-800bp) construct attached to a luciferase reporter was used to measure transcriptional activity. Results: In the GERD cells, IL-13 and IL-4 induced only slight increases in eotaxin-3 mRNA expression levels at 24 and 48 hours. In contrast, both cytokines caused a marked increase in eotaxin-3 mRNA expression levels at 24 and 48 hours in the EoE cells. Both IL-13 and IL-4 significantly increased eotaxin-3 promoter activity in all 3 cell lines. There was no significant difference in the degree of eotaxin-3 promoter induction by IL-13 and IL-4 between the two GERD cell lines (NES-G4T cells IL-13: 431 ± 42.5 SEM RLUs % control, IL-4: 279± 38; NES-B10T cells IL-13: 554 ± 109, IL-4: 463 ± 54). In contrast, the cytokine-stimulated increase in eotaxin-3 promoter activity in the EoE1 cells (IL-13: 1522 ± 225, p<0.01; IL-4: 1714 ± 385, p<0.01) was significantly greater than that in the two GERD cell lines. Conclusions: In an EoE esophageal squamous cell line, stimulation with IL-13 and IL-4 caused a far greater increase in eotaxin-3 mRNA expression and promoter transcriptional activity than in esophageal squamous cell lines from GERD patients. These observations suggests that the esophagus of patients with EoE produces more eotaxin3 in response to Th2 cytokine stimulation than the esophagus of patients with GERD, and that differences among patients in the degree of cytokine-stimulated production of eotaxin3 by esophageal squamous cells might underlie the degree of esophageal infiltration by activated eosinophils.

Markers of Tyrosine Kinase Activity for Diagnosis and Treatment Response in Eosinophilic Esophagitis: A Pilot Study of pERK 1/2 and pSTAT5

Introduction: In eosinophilic esophagitis (EoE), food and other allergens appear to activate Th2 cells to release IL-13 and IL-4, cytokines that can induce esophageal squamous epithelial cells to produce the potent eosinophil chemoattractant eotaxin-3. GERD also can cause an eosinophilic infiltration of the esophagus, usually milder than that seen in EoE, through mechanisms that are not clear. In earlier studies, we showed that IL-13 and IL-4 caused a marked increase in eotaxin-3 protein secretion in an esophageal squamous cell line derived from a patient with EoE, but only a minimal increase in eotaxin-3 protein secretion in esophageal squamous cell lines derived from GERD patients. To explore this mechanism further, we studied the effects of IL-13 and IL-4 on transcriptional activation of eotaxin-3 in esophageal squamous cell lines from patients with EoE and GERD. Methods: Two telomerase-immortalized, non-neoplastic esophageal squamous cell lines derived from patients with GERD (NES-B10T & NES-G4T), and one derived from a patient with EoE (EoE1-T) were stimulated with IL-13 (100 ng/ml) or IL-4 (1 ng/ml) for times ranging from 24-48 hours. Eotaxin-3 mRNA expression levels were determined by RT-PCR, and an eotaxin-3 promoter (-800bp) construct attached to a luciferase reporter was used to measure transcriptional activity. Results: In the GERD cells, IL-13 and IL-4 induced only slight increases in eotaxin-3 mRNA expression levels at 24 and 48 hours. In contrast, both cytokines caused a marked increase in eotaxin-3 mRNA expression levels at 24 and 48 hours in the EoE cells. Both IL-13 and IL-4 significantly increased eotaxin-3 promoter activity in all 3 cell lines. There was no significant difference in the degree of eotaxin-3 promoter induction by IL-13 and IL-4 between the two GERD cell lines (NES-G4T cells IL-13: 431 ± 42.5 SEM RLUs % control, IL-4: 279± 38; NES-B10T cells IL-13: 554 ± 109, IL-4: 463 ± 54). In contrast, the cytokine-stimulated increase in eotaxin-3 promoter activity in the EoE1 cells (IL-13: 1522 ± 225, p<0.01; IL-4: 1714 ± 385, p<0.01) was significantly greater than that in the two GERD cell lines. Conclusions: In an EoE esophageal squamous cell line, stimulation with IL-13 and IL-4 caused a far greater increase in eotaxin-3 mRNA expression and promoter transcriptional activity than in esophageal squamous cell lines from GERD patients. These observations suggests that the esophagus of patients with EoE produces more eotaxin3 in response to Th2 cytokine stimulation than the esophagus of patients with GERD, and that differences among patients in the degree of cytokine-stimulated production of eotaxin3 by esophageal squamous cells might underlie the degree of esophageal infiltration by activated eosinophils.

Epigenetic Regulation of the IL-13-induced Human Eotaxin-3 Gene by CREB-binding Protein-mediated Histone 3 Acetylation

The etiology of a variety of chronic inflammatory disorders has been attributed to the interaction of genetic and environmental factors. Herein, we identified a link between epigenetic regulation and IL-13-driven eotaxin-3 in the pathogenesis of chronic allergic inflammation. We first demonstrated that the cAMP-responsive element (CRE) site in the eotaxin-3 promoter affects IL-13-induced eotaxin-3 promoter activity. Furthermore, the CRE-binding protein-binding protein (CBP), a histone acetyltransferase, induced base-line and IL-13-induced eotaxin-3 promoter activity. Additionally, IL-13 treatment promoted global histone 3 acetylation as well as the formation of a complex containing CBP and STAT6 and the subsequent acetylation of histone 3 at the eotaxin-3 promoter. CBP gene silencing decreased IL-13-induced transcription of eotaxin-3. Conversely, inhibition of histone deacetylation increased IL-13-induced eotaxin-3 production. Clinical studies demonstrated markedly increased global acetylation of histone 3 in the inflamed tissue of patients with allergic inflammation. Collectively, these results identify an epigenetic mechanism involving CBP and chromatin remodeling in regulating IL-13-induced chemokine transcription.

Glucocorticoid-regulated genes in eosinophilic esophagitis: A role for FKBP51

Eosinophilic esophagitis (EE) involves marked accumulation of eosinophils in the esophageal mucosa that responds to swallowed fluticasone propionate (FP) in a subset of patients. We aimed to uncover the mechanism of action of swallowed FP in patients with EE by providing evidence for a topical effect in the esophagus by identifying a molecular signature for FP exposure in vivo. Global microarray expression profiles, immunofluorescence microscopy, and cell signaling in esophageal tissue and cell lines were analyzed. Thirty-two transcripts exhibited altered expression in patients who responded to swallowed FP treatment. Esophageal FK506-binding protein 5 (FKBP51) mRNA levels were increased (P < .05) in FP responders compared with those seen in control subjects and patients with untreated active EE. After FP treatment of esophageal epithelial cells, FKBP51 mRNA and protein levels were increased in a dose- and time-dependent manner by FP treatment in vitro. FP-induced FKBP51 was steroid receptor dependent because RU486 completely inhibited gene and protein induction. The half-life of FKBP51 mRNA was 16 to 18 hours independent of FP treatment. FKBP51 overexpression reduced FP action as assessed by FP inhibition of IL-13-induced eotaxin-3 promoter activity. Our results suggest that swallowed glucocorticoid treatment directly affects esophageal gene expression in patients with EE. In particular, increased FKBP51 transcript levels identify glucocorticoid exposure in vivo and distinguish FP responders from untreated patients with active EE and patients without EE. In addition, FKBP51 reduces glucocorticoid-mediated inhibition of IL-13 signaling in epithelial cells in vitro, suggesting that FKBP51 might influence FP responsiveness. We propose that esophageal FKBP51 levels have diagnostic and prognostic significance in patients with EE.

Differential Regulation of Eotaxin Expression by Dexamethasone in Normal Human Lung Fibroblasts

Lung fibroblasts are a major source of several cytokines including CC chemokine eotaxin. We aimed to study the regulation of eotaxin-1/CCL11 production by dexamethasone and analyze its molecular mechanisms in human lung fibroblasts. Normal human lung fibroblast cells were exposed to IL-4 (40 ng/ml) and/or dexamethasone (10(-6)-10(-9) M), and eotaxin mRNA expression and production was evaluated. Mechanisms of transcriptional regulation were assessed by Western blotting and dual luciferase assay for eotaxin promoter. The effects of dexamethasone on suppressor of cytokine signaling (SOCS)-1 and eotaxin mRNA expression in the cells transfected with expression vector (pAcGFP1-C1) or short interfering RNA (siRNA) for SOCS-1 were also investigated. Within 24 hours, dexamethasone inhibited IL-4-induced eotaxin mRNA expression and protein production, while eotaxin production was markedly increased at 48 and 72 hours after coincubation with IL-4 and dexamethasone. IL-4-induced eotaxin promoter activity was inhibited by dexamethasone at 8 hours, but enhanced at 48 hours after coincubation. Dexamethasone suppressed SOCS-1 mRNA expression but enhanced IL-4-induced STAT6 phosphorylation at 36 to 48 hours after coincubation. Enhanced expression of eotaxin mRNA by dexamethasone 48 hours after coincubation was completely diminished in the cells transfected with either expression vector or siRNA for SOCS-1. These results indicated that dexamethasone, depending on the exposure duration, can either inhibit or enhance IL-4-induced expression and production of eotaxin in the lung fibroblasts. The mechanisms of later enhanced production may depend on the prolonged transcriptional activity of the eotaxin gene, in part due to inhibition of SOCS-1 expression.

IL-13 involvement in eosinophilic esophagitis: Transcriptome analysis and reversibility with glucocorticoids

Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. Early studies have established that esophageal eosinophilia occurs in association with T(H)2 allergic responses, and we recently identified an EE-specific esophageal transcriptome that included eotaxin-3. We sought to determine the mechanism by which this T(H)2 response leads to EE. Real-time PCR and microarray analysis were performed on RNA extracted from esophageal biopsy specimens and primary esophageal epithelial cell cultures stimulated with IL-13 (0-100 ng/mL). Transient transfections in esophageal cell lines were performed with plasmids containing the luciferase gene driven by eotaxin-3 promoter fragments and modified forms of signal transducer and activator of transcription 6. The IL-13 mRNA level was markedly increased (16-fold) in esophageal biopsy specimens from patients with EE compared with those from healthy individuals. Furthermore, IL-13 treatment of primary esophageal epithelial cells was sufficient to induce a global-expression transcript profile that remarkably overlapped with the EE-specific esophageal transcriptome. In addition, esophageal epithelial cells markedly produce eotaxin-3 after IL-13 stimulation through a transcriptional mechanism dependent on signal transducer and activator of transcription 6. Lastly, increased IL-13 mRNA levels and the EE transcriptome were largely reversible with glucocorticoid treatment in vivo. Taken together, we propose that the pathogenesis of EE is mediated by an IL-13-stimulated keratinocyte-derived transcriptome that is largely reversible with corticosteroid treatment. Furthermore, we identify an in vivo IL-13-induced transcriptome that has potential utility for target assessment after anti-IL-13 therapeutics. IL-13-induced pathways and genes are fundamental processes in the cause and manifestations of EE; as such, therapeutic agents that interfere with IL-13 might be particularly useful for disease treatment.

Differential Regulation of Chemokine Expression by Th1 and Th2 Cytokines and Mechanisms of Eotaxin/CCL-11 Expression in Human Airway Smooth Muscle Cells

Background: Airway smooth muscle (ASM) cells may contribute to the pathogenesis of asthma including airway inflammation and remodeling. We focused our study on the regulation of chemokine expression by cytokines and analyzed the mechanisms of eotaxin/CCL-11 expression in ASM cells. Methods: Human ASM cells were cultured in vitroand treated with IL-4, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα). Secretion of chemokines into the culture medium was analyzed by ELISA. Expression of eotaxin mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Binding of transcription factor signal transducer activator of transcription (STAT) 6 to the eotaxin promoter-derived DNA was analyzed by pull-down Western blot. To assess transcriptional regulation of eotaxin, cells were transfected with eotaxin promoter-luciferase reporter plasmids, and activity was determined by dual luciferase assay. Results: The Th2 cytokine IL-4 preferentially stimulated the expression of the CC chemokine receptor (CCR) 3-ligand chemokines eotaxin, eotaxin-3, and MCP-4. The Th1 cytokine IFNγ stimulated the expression of chemokines IP-10 and RANTES. IL-4 stimulated nuclear translocation of signal transducer activator of transcription 6 (STAT6) and its binding to the eotaxin promoter region. IL-4 activated the eotaxin promoter and its activity was inhibited by mutation of the binding site for STAT6 in the promoter. Conclusions: The Th2 cytokine IL-4 preferentially stimulated the expression of CCR3 ligand chemokines including eotaxin in ASM cells. The transcription factor STAT6 may play a pivotal role in the activation of eotaxin transcription in response to IL-4.

Expression of Eotaxin in Gastric Epithelial Cells Stimulated withHelicobacter pyloriVacuolating Cytotoxin

Helicobacter pylori-infected gastric mucosa is characterized by infiltration of various inflammatory cells such as neutrophils and eosinophils. Although several mechanisms for neutrophil infiltration are well known, there has been little known the role of eotaxin, which is a potent chemoattractant for eosinophils, on the inflammatory process of H. pylori infection. The present study was to investigate the mechanisms of eotaxin expression in gastric epithelial cells stimulated with H. pylori vacuolating cytotoxin (VacA). Stimulation with VacA purified from VacA + H. pylori slightly increased eotaxin expression in MKN-45 gastric epithelial cells. In contrast, the combined stimulation with VacA and IL-4 synergistically increased the eotaxin expression as determined by quantitative RT-PCR and ELISA. In MKN-45 cells transfected with an eotaxin promoter-luciferase reporter plasmid, costimulation with VacA and IL-4 induced more luciferase activity than either VacA or IL-4 alone did. However, such up-regulation was significantly decreased in the cells transfected with luciferase reporter plasmid bearing an eotaxin promoter which has a mutation at STAT6 binding site. These results suggest that the up-regulation of eotaxin in VacA-stimulated gastric epithelial cells may be synergistically facilitated by IL-4 via a STAT6-dependent mechanism.

Eotaxin-3/CCL26 gene expression in intestinal epithelial cells is up-regulated by interleukin-4 and interleukin-13 via the signal transducer and activator of transcription 6

Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at sites of inflammation. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. Eotaxin-3/CCL-26 belongs to a family of CC chemokines, which are potent chemoattractants for eosinophils. In this study, we hypothesized that intestinal epithelial cells could release eotaxin-3. We demonstrate that the T helper 2 type cytokines interleukin-4 or interleukin-13 increase eotaxin-3 mRNA levels and eotaxin-3 protein expression in the human intestinal epithelial cell lines HT-29 CL.19A and T84 in a dose-dependent manner. Addition of actinomycin-D prior to interleukin-4/-13 stimulation led to decreases in eotaxin-3 mRNA levels similar to those observed in controls without interleukin-4/-13. Interleukin-4 and interleukin-13 activated signal transducer and activator of transcription 6 which was found to bind the two canonical signal transducer and activator of transcription 6 binding sites located in the eotaxin-3 promoter. Experiments with the eotaxin-3 promoter luciferase constructs revealed that the most proximal signal transducer and activator of transcription 6 binding site located between positions −62 and −71 relative to the transcriptional start was necessary for full eotaxin-3 promoter activity. Importantly, we present evidence that the signal transducer and activator of transcription 6 is necessary and sufficient for interleukin-4 or interleukin-13 mediated eotaxin-3 gene up-regulation using HT-29 CL.19A cells expressing a dominant-negative signal transducer and activator of transcription 6. Overall, these results demonstrate that epithelial eotaxin-3 is up-regulated in the context of a T helper 2 mediated inflammatory bowel disease via the signal transducer and activator of transcription 6, thus suggesting that the intestinal epithelium actively participates in the recruitment of eosinophils at the site of inflammation.

β2-Adrenoceptor Agonists, Like Glucocorticoids, Repress Eotaxin Gene Transcription by Selective Inhibition of Histone H4 Acetylation

Eotaxin is a potent eosinophil chemoattractant implicated in various allergic inflammatory conditions including asthma, but relatively little is known about its regulation. Human airway smooth muscle cells are an important source of eotaxin in the airway. We have previously demonstrated that beta(2)-adrenoceptor agonists (beta(2)-agonists) and glucocorticoids additively inhibit eotaxin production in human airway smooth muscle cells, but the underlying mechanisms are unclear. Here, we studied the molecular mechanisms of their actions and interactions on eotaxin gene transcription. TNF-alpha-induced eotaxin gene transcription was mediated mainly by the transcription factor NF-kappaB (p65/p50) as analyzed by luciferase reporter gene assay, Western blotting, EMSA, and electrophoretic mobility supershift assay. Chromatin immunoprecipitation assay demonstrated that TNF-alpha also induced selective histone H4 acetylation on lysines 5 and 12 at the eotaxin promoter site and p65 binding to the eotaxin promoter, resulting in eotaxin gene transcription. The inhibition of eotaxin production by beta(2)-agonists and glucocorticoids was transcriptional and not due to altered NF-kappaB nuclear translocation or in vitro promoter binding capability, but due to their inhibition of TNF-alpha-induced histone H4 acetylation and p65 in vivo binding to the promoter. Additive inhibition was achieved when the two groups of drugs were combined. Our findings reveal a novel mechanism by which beta(2)-agonists, like glucocorticoids, regulate NF-kappaB-mediated inflammatory gene expression through inhibition of histone acetylation. This provides one explanation for the benefits that result when these agents are combined to treat asthma, and may have important implications in a wide range of inflammatory diseases.

Oncostatin M causes eotaxin-1 release from airway smooth muscle: Synergy with IL-4 and IL-13

Eotaxin is implicated in asthmatic eosinophilia. Oncostatin M (OSM) causes eotaxin release from fibroblasts. We sought to examine the effects and mechanism of action of OSM and other IL-6 family cytokines on eotaxin release from human airway smooth muscle cells. Eotaxin 1 release was measured by means of ELISA. Western blotting was used to examine mitogen-activated protein kinase and signal transducer and activator of transcription 3 (STAT-3) phosphorylation. Eotaxin promoter activity was analyzed in cells transfected with wild-type STAT-3, a mutant form of STAT-3 that cannot be phosphorylated, and a constitutively active form of STAT-3. The mRNA and protein expression of IL-4R alpha, the signaling receptor for IL-4 and IL-13, was evaluated by means of real-time PCR and flow cytometry, respectively. OSM increased eotaxin 1 release and augmented IL-4- or IL-13-induced eotaxin release, whereas other IL-6 family cytokines did not. OSM caused a greater increase in STAT-3 phosphorylation and STAT-3-mediated gene transcription than other IL-6 family cytokines. OSM increased eotaxin promoter activity and augmented IL-13- and IL-4-induced increases in promoter activity. The constitutively active form of STAT-3 increased eotaxin promoter activity, whereas the mutant form of STAT-3 that cannot be phosphorylated significantly reduced eotaxin promoter activity induced by OSM or IL-4 plus OSM. OSM increased IL-4R alpha mRNA and protein levels. OSM induces eotaxin 1 expression in human airway smooth muscle cells by a mechanism involving STAT-3. OSM synergizes with IL-13 and IL-4 to increase eotaxin 1 expression, possibly as a result of effects on IL-4R alpha expression.

Differential Regulation of Chemokine Expression by Peroxisome Proliferator-activated Receptor γ Agonists: INTERACTIONS WITH GLUCOCORTICOIDS AND β2-AGONISTS

Chemokine-mediated inflammatory cell infiltration is a hallmark of asthma. We recently demonstrated that glucocorticoids and beta(2)-agonists additively or synergistically suppress tumor necrosis factor-alpha (TNFalpha)-induced production of chemokines eotaxin and interleukin-8 (IL-8), respectively, in human airway smooth muscle (HASM) cells, which may partly explain their combined benefits in asthma. Peroxisome proliferator-activated receptors (PPARs) also modulate inflammatory gene expression. We reported here that the PPARgamma agonists 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and troglitazone, but not PPARalpha agonist WY-14643, inhibited TNFalpha-induced production of eotaxin and monocyte chemotactic protein-1 (MCP-1) but not IL-8. Eotaxin inhibition was transcriptional and additively enhanced by the glucocorticoid fluticasone and the beta(2)-agonist salmeterol, whereas MCP-1 inhibition was post-transcriptional and additively and synergistically enhanced by fluticasone and salmeterol, respectively. Coimmunoprecipitation revealed that 15d-PGJ(2) induced a protein-protein interaction between PPARgamma and the glucocorticoid receptor (GR) in TNFalpha-treated HASM cells, which was enhanced by fluticasone and salmeterol. 15d-PGJ(2), fluticasone, and salmeterol all inhibited TNFalpha-induced histone H4 acetylation at the eotaxin promoter and NF-kappaB p65 binding to the eotaxin promoter and induced PPARgamma and GR association with the eotaxin promoter, as analyzed by chromatin immunoprecipitation assay. Our data suggest that chemokine expression in HASM cells is differentially regulated by PPARgamma agonists and that the interaction between PPARgamma and GR may be responsible for the additive and synergistic inhibition of chemokine expression by PPARgamma agonists, glucocorticoids, and beta(2)-agonists, particularly the chromatin-dependent suppression of eotaxin gene transcription. The interaction may have wide applications and may provide a potential target for pharmacological and molecular intervention.

Molecular Mechanisms of Repression of Eotaxin Expression with Fluticasone Propionate in Airway Epithelial Cells

Background: Glucocorticoids are known to repress the expression of CC chemokine eotaxin in airway epithelial cells. We focused our study on the molecular mechanisms of the glucocorticoid, fluticasone, in the inhibition of the expression of the eotaxin gene in the cells. Methods: The airway epithelial cell line, BEAS-2B, was stably transfected with signal transducers and activators of transcription 6 (STAT6)-expressing vector and used in the following experiments to clarify the function of STAT6. Levels of eotaxin mRNA and protein expression were determined with RT-PCR and ELISA. Mechanisms of transcriptional regulation were assessed by the electrophoretic mobility shift assay and dual luciferase assay using eotaxin promoter-luciferase reporter plasmids. Results: Fluticasone significantly inhibited the induction of eotaxin protein stimulated with TNF-α and IL-4 in the cells. Fluticasone also repressed the induction of eotaxin mRNA with these stimuli. It partially inhibited the activity of eotaxin promoter; however, it did not inhibit the nuclear translocation and binding of transcription factors, nuclear factor-kappa B (NF-ĸB) or STAT6, to the DNA derived from the proximal promoter region of the eotaxin gene. Moreover, the inhibitory effect was also conserved in the experiments using the reporter plasmid of which the putative glucocorticoid-responsive element was mutated. Conclusions: Fluticasone inhibits the expression of eotaxin gene in airway epithelial cells in part through repression of the transcription. However, the mechanisms depend neither on the inhibition of transcription factors’ translocation into nuclei nor the function of the putative glucocorticoid-responsive element in the promoter, indicating that other mechanisms would be related to the transcriptional repression of the eotaxin gene in airway epithelial cells.

Retinoic acid inhibits interleukin-4-induced eotaxin production in a human bronchial epithelial cell line.

Retinoic acid (RA) is known to accelerate wound healing and induce cell differentiation. All-trans RA (ATRA) exerts its effect by binding retinoic acid receptors, which are members of the nuclear receptor family. We investigated whether RA can alter expression of eotaxin, a potent eosinophil chemoattractant that is regulated by the transcription factors signal transducer and activator of transcription 6 (STAT6) and NF-kappaB. We examined the effects of RA on eotaxin expression in a human bronchial epithelial cell line BEAS-2B. ATRA and its stereodimer 9-cis retinoic acid (9-cis RA) inhibited IL-4-induced release of eotaxin at 10(-6) M by 78.0 and 52.0%, respectively (P < 0.05). ATRA and 9-cis RA also significantly inhibited IL-4-induced eotaxin mRNA expression at 10(-6) M by 52.3 and 53.5%, respectively (P < 0.05). In contrast, neither ATRA nor 9-cis RA had any effects on TNF-alpha-induced eotaxin production. In transfection studies using eotaxin promoter luciferase plasmids, the inhibitory effect of ATRA on IL-4-induced eotaxin production was confirmed at the transcriptional level. Interestingly, ATRA had no effects on IL-4-induced tyrosine phosphorylation, nuclear translocation, or DNA binding activity of STAT6. Activating protein-1 was not involved in ATRA-mediated transrepression of eotaxin with IL-4 stimulation. The mechanism of the inhibitory effect of ATRA on IL-4-induced eotaxin production in human bronchial epithelial cells has not been elucidated but does not appear to be due to an effect on STAT6 activation. These findings raise the possibility that RA may reduce eosinophilic airway inflammation, one of the prominent pathological features of allergic diseases such as bronchial asthma.