Egr-1 DNA Binding Activity Research Articles

N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway.

Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthine oxidase-hypoxanthine (XO/HX) activated all three MAPKs (ERK1/2, JNK, p38), and induced Egr-1 expression and Egr-1 DNA-binding activity in CMECs, whereas ROS scavengers (EDA and NAC) had the opposite effect following H/R. Inhibitors of all three MAPKs individually inhibited induction of Egr-1 expression by H/R in CMECs. Moreover, N-n-butyl haloperidol (F2), previously shown to protect cardiomyocytes subjected to I/R, dose-dependently downregulated H/R-induced ROS generation, MAPK activation, and Egr-1 expression and activity in CMECs, whereas XO/HX and MAPK activators (EGF, anisomycin) antagonized the effects of F2. Inhibition of the ROS/MAPK/Egr-1 signaling pathway, by either F2, NAC, or inhibition of MAPK, increased CMEC viability and the GSH/GSSG ratio, and decreased Egr-1 nuclear translocation. These results show that the ROS/MAPK/Egr-1 signaling pathway mediates H/R injury in CMECs, and F2 blocks this pathway to protect against H/R injury and further alleviate myocardial I/R injury.

Hypochlorite-modified high-density lipoprotein promotes induction of HO-1 in endothelial cells via activation of p42/44 MAPK and zinc finger transcription factor Egr-1

Modification/chlorination of high-density lipoprotein (HDL) by hypochlorous acid (HOCl), formed by the myeloperoxidase–H2O2–chloride system of activated phagocytes, converts an anti-atherogenic lipoprotein into a pro-inflammatory lipoprotein particle. Chlorinated HDL is present in human lesion material, binds to and is internalized by endothelial cells and impairs expression and activity of endothelial nitric oxide synthase. The present study aimed at clarifying whether exposure of endothelial cells to pro-inflammatory HOCl–HDL impacts on expression of heme oxygenase-1, a potential rescue pathway against endothelial dysfunction. Our findings revealed that HDL modified by HOCl, added as reagent or generated enzymatically, induced phosphorylation of p42/44 mitogen-activated protein kinase, expression of transcription factor early growth response-1 (Egr-1) and enhanced expression of heme oxygenase-1 in human endothelial cells. Upregulation of heme oxygenase-1 could be blocked by an inhibitor upstream of p42/44 mitogen-activated protein kinase and/or knockdown of Egr-1 by RNA-interference. Electrophoretic mobility shift assays demonstrated HOCl–HDL-mediated induction of the Egr-1 DNA binding activity. Immunocytochemical and immunoblotting experiments demonstrated HOCl–HDL-induced translocation of Egr-1 to the nucleus. The present study demonstrates a novel compensatory pathway against adverse effects of HOCl–HDL, providing cytoprotection in a number of pathological conditions including cardiovascular disease.

A novel anticancer effect of Astragalus saponins: Transcriptional activation of NSAID‐activated gene

Astragalus membranaceus has been used to ameliorate the side effects of antineoplastic drugs because of its immunomodulating nature. We had recently demonstrated that total Astragalus saponins (AST) possess anticarcinogenic and proapoptotic properties in human colon cancer cells and tumor xenograft. In this study, we identified NSAID-activated gene (NAG-1) as a potential molecular target of AST. The growth-inhibitory and proapoptotic effects of AST were assessed in a panel of human cancer cell lines. Hoechst 33342 nuclear staining, Annexin V-FITC/propidium iodide staining, Western immunoblotting, real-time PCR, luciferase reporter assay and electrophoretic mobility shift assay were conducted to determine the association of NAG-1 and related transcription factors with AST during its regulation of apoptotic activities. Moreover, the combined proapoptotic and NAG-1 promoting activities of AST and/or inhibitors of the PI3K-Akt pathway were also examined. AST caused overexpression of NAG-1, leading to PARP cleavage and apoptosis. The induction of NAG-1 promoter activity by the drug was associated with increased gene expression, in addition to prior increase in Egr-1 expression and DNA binding activity. AST-induced NAG-1 activation was intensified when PI3K inhibitor LY294002 or Akt inhibitor was co-treated and reversed by NAG-1 siRNA transfection. Nevertheless, the extent of NAG-1 induction could not be altered by the ERK inhibitor PD98059. Our results indicate that NAG-1 is a potential molecular target of AST in its antitumorigenic and proapoptotic actions, which would have additive effects when used along with PI3K-Akt inhibitors. The information obtained could facilitate future development of a novel target-specific chemotherapeutic agent with known molecular pathway.

Early Growth Response-1 Regulates Angiopoietin-1-Induced Endothelial Cell Proliferation, Migration, and Differentiation

Angiopoietin-1 (Ang-1) is an important regulator of angiogenesis in endothelial cells. It promotes migration, proliferation, and differentiation of cells, although the regulating factors involved in these processes remain unclear. In this study, we evaluated the contribution of the transcription factor early growth response-1 (Egr-1) to Ang-1-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Expression of Egr-1 was evaluated with real-time PCR and immunoblotting, whereas Egr-1 DNA binding activity was monitored with electrophoretic mobility shift assays. Cell migration was measured with wound healing and Boyden chamber assays, whereas cell proliferation and differentiation of cells into capillary-like tube structures were monitored with cell counting, BrdU incorporation and Matrigels. To selectively inhibit Egr-1 expression, we used both siRNA oligonucleotides and specific DNAzymes. Egr-1 mRNA expression rose approximately 9-fold within 2 hours of Ang-1 exposure and declined thereafter. Upregulation of Egr-1 expression was accompanied by an increase in nuclear mobilization and augmented DNA binding. These processes were mediated through the Erk1/2, PI-3 kinase/AKT, and mTOR pathways. Knockdown of Egr-1 expression completely abrogated Ang-1-induced endothelial migration and significantly reduced proliferation and capillary-like tube formation of HUVECs that overexpress Ang-1. Ang-1 triggers significant and transient induction of Egr-1, and Egr-1 contributes to Ang-1-induced endothelial cell migration and proliferation.

Suppression of Lipopolysaccharide-stimulated Tumor Necrosis Factor-α Production by Adiponectin Is Mediated by Transcriptional and Post-transcriptional Mechanisms

Adiponectin is an adipokine with potent anti-inflammatory properties. Treatment of macrophages with adiponectin results in a suppression of lipopolysaccharide (LPS)-stimulated cytokine production. Here we investigated the transcriptional and post-transcriptional mechanisms by which adiponectin suppresses LPS-stimulated tumor necrosis factor (TNF)-alpha production. Treatment of RAW 264.7 macrophages with LPS increased TNF-alpha promoter-driven luciferase activity (TNF-alpha promoter/Luc activity) by 20-fold over basal. After culture with 1 mug/ml globular adiponectin (gAcrp) for 18 h, TNF-alpha promoter/Luc activity was increased even in the absence of LPS; further challenge with LPS only increased TNF-alpha promoter/Luc activity by 1.4-fold. Treatment with gAcrp decreased LPS-stimulated ERK1/2 phosphorylation and IkappaB degradation and suppressed the ability of LPS to increase the DNA binding activity of Egr-1 and p65. gAcrp also suppressed LPS-mediated stabilization of TNF-alpha mRNA. In controls cells, the half-life of TNF-alpha mRNA was increased from approximately 30 min at base line to approximately 80 min in response to LPS. After treatment with gAcrp for 18 h, LPS failed to increase TNF-alpha mRNA stability. This gAcrp-mediated loss of stimulus-induced stabilization of TNF-alpha mRNA required the presence of the TNF-alpha 3'-untranslated region and was associated with an increase in expression and RNA binding activity of tristetraprolin, an mRNA-binding protein that destabilizes TNF-alpha mRNA. In summary, these data characterize the complex transcriptional and post-transcriptional effects of gAcrp on LPS-stimulated TNF-alpha expression in macrophages. gAcrp treatment profoundly suppressed the ability of LPS to increase TNF-alpha transcription and reduced the stimulus-induced stabilization of TNF-alpha mRNA in response to LPS.

Targeting of p38 mitogen-activated protein kinases to early growth response gene 1 (EGR-1) in the human paclitaxel-resistance ovarian carcinoma cells

To investigate the relationship between the expression of early growth response gene 1 (EGR-1) and p38MAPK pathway in the paclitaxel resistance of ovarian carcinoma cells, the effect of p38MAPK inhibitor SB203580 on cell apoptosis was examined by using Hoechst 33258 staining. The intracellular Rh123 (Rhodamine 123) accumulation was detected by the flow cytometry (FCM). The 50% inhibition concentration (IC50) of paclitaxel for A2780/Taxol cells was determined by MTT method. Electrophoretic motility shift assay (EMSA) was employed to examine the EGR-1DNA binding activity. MDR1 and EGR-1 mRNA were assessed by RT-PCR. The expressed of p-gp, phosphorylated p53 and p38 were detected by Western blotting. SB203580 could remarkably promote the apoptosis of A2780/Taxol cells, and the cell apoptosis was in a time-dependent manner. Cellular Rh123 accumulation was increased, and the IC50 of paclitaxel for A2780/Taxol cells was decreased significantly. A2780/Taxol cell line after SB203580 treatment was shown to have a significantly higher level of EGR-1 DNA binding activity. SB203580 down-regulated the activity of p38MAPK pathway, but up-regulated EGR-1 expression. SB203580 significantly increased the level of cellular phosphorylated p53 protein, but decreased the p-gp protein level and MDR1 mRNA level in A2780/Taxol cells. There existed a close relationship between p38MAPK pathway and the paclitaxel resistance of ovarian carcinoma cells. The expression of EGR-1 mediated by p38MAPK pathway plays a critical role in paclitaxel resistance of ovarian carcinoma cells.

Membrane type-1 matrix metalloproteinase is induced following cyclic compression of in vitro grown bovine chondrocytes

To determine if membrane type-1 matrix metalloproteinase (MT1-MMP) will respond to cyclic compression of chondrocytes grown in vitro and the regulatory mechanisms underlying this response. Cyclic compression (30min, 1kPa, 1Hz) was applied to bovine chondrocytes (6-9-month-old animals) grown on top of a biodegradable substrate within 3 days of initiating culture. Luciferase assays using bovine articular chondrocytes were undertaken to demonstrate the mechanosensitivity of MT1-MMP. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to establish the time course of gene and protein upregulation in response to cyclic compression. The regulation of MT1-MMP was assessed by electrophoretic mobility shift assays, RT-PCR and western blot analysis. As well, an MT1-MMP decoy oligonucleotide and an extracellular signal-regulated kinase 1/2 (ERK1/2) pharmacological inhibitor were utilized to further characterize MT1-MMP regulation. After cyclic compression, MT1-MMP showed a rapid and transient increase in gene expression. Elevated protein levels were detected within 2h of stimulation which returned to baseline by 6h. During cyclic compression, phosphorylation of the mitogen activated protein kinase ERK1/2 increased significantly. This was followed by increased gene and protein expression of the transcription factor; early growth factor-1 (Egr-1) and Egr-1 binding to the MT1-MMP promoter. Blocking Egr-1 DNA binding with a decoy MT1-MMP oligonucleotide, downregulated MT1-MMP gene expression. The ERK1/2 inhibitor U0126 also reduced Egr-1 DNA binding activity to MT1-MMP promoter sequences and subsequent transcription of MT1-MMP. These data suggest that cyclic compression of chondrocytes in vitro upregulates MT1-MMP via ERK1/2 dependent activation of Egr-1 binding. Delineation of the regulatory pathways activated by mechanical stimulation will further our understating of the mechanisms influencing tissue remodeling.

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

EARLY GROWTH RESPONSE 1 MEDIATES THE SYSTEMIC AND HEPATIC INFLAMMATORY RESPONSE INITIATED BY HEMORRHAGIC SHOCK

Hemorrhagic shock (HS) is a major cause of morbidity and mortality in trauma patients. The early growth response 1 (Egr-1) transcription factor is induced by a variety of cellular stresses, including hypoxia, and may function as a master switch to trigger the expression of numerous key inflammatory mediators. We hypothesized that HS would induce hepatic expression of Egr-1 and that Egr-1 upregulates the inflammatory response after HS. The Egr-1 mice and wild-type (WT) controls (n>or=5 for all groups) were subjected to HS alone or HS followed by resuscitation (HS/R). Other mice were subjected to a sham procedure which included general anesthesia and vessel cannulation but no shock (sham). After the HS, HS/R, or sham procedures, mice were euthanized for determination of serum concentrations of interleukin (IL) 6, IL-10, and alanine aminotransferase. Northern blot analysis was performed to evaluate Egr-1 messenger RNA (mRNA) expression. Liver whole cell lysates were evaluated for Egr-1 protein expression by Western blot analysis. Hepatic expression of IL-6, granulocyte colony-stimulating factor, and intracellular adhesion molecule 1 mRNA was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The Egr-1 DNA binding was assessed using the electrophoretic mobility shift assay. Hemorrhagic shock results in a rapid and transient hepatic expression of Egr-1 mRNA in WT mice by 1 h, whereas protein and DNA binding activity was evident by 2.5 h. The Egr-1 mRNA expression diminished after 4 h of resuscitation, whereas Egr-1 protein expression and DNA binding activity persisted through resuscitation. The Egr-1 mice exhibited decreased levels of hepatic inflammatory mediators compared with WT controls with a decrease in hepatic mRNA levels of IL-6 by 42%, granulocyte colony-stimulating factor by 39%, and intracellular adhesion molecule 1 by 43%. Similarly, Egr-1 mice demonstrated a decreased systemic inflammatory response and hepatic injury after HS/R compared with their WT counterparts. Early growth response 1 is rapidly upregulated in the liver during and after resuscitation from HS. Our results showing a blunted inflammatory response in Egr-1 mice provides evidence that Egr-1 functions as a proximal signal transduction mechanism responding to shock by amplifying the systemic inflammatory response.

Hepatocyte Growth Factor/Scatter Factor Differentially Regulates Expression of Proangiogenic Factors through Egr-1 in Head and Neck Squamous Cell Carcinoma

Hepatocyte growth factor/scatter factor (HGF) and the angiogenesis factors platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) are found in elevated concentrations in serum or tumor tissue of patients with head and neck squamous cell carcinomas (HNSCC), suggesting these factors may be coregulated. A cDNA microarray analysis for HGF-inducible genes revealed that HGF also modulates PDGFA expression, a gene recently shown to be inducible by the transcription factor, early growth response-1 (Egr-1). In the present study, we investigated the potential role of HGF-induced Egr-1 in expression of PDGF, VEGF, and IL-8. HGF induced expression of all three factors and Egr-1 expression and DNA-binding activity. The analysis of promoter sequences showed putative Egr-1 binding sites in the PDGFA or VEGF but not in the IL-8 promoter, and HGF-induced Egr-1-binding activity was confirmed by chromatin immunoprecipitation (ChIP) assay. The maximal basal and HGF-induced promoter activity for the PDGFA gene existed within -630 bp of the promoter region, and overexpression of Egr-1 significantly increased such activity. Consistent with this, expression of PDGFA and VEGF but not IL-8 showed corresponding differences with Egr-1 expression in HNSCC tumor specimens and were strongly suppressed by transfection of Egr-1-antisense or small interference RNA (siRNA) oligonucleotides. HGF-induced expression of Egr-1, PDGFA, and VEGF was suppressed by pharmacologic and siRNA inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and protein kinase C (PKC) pathways. We conclude that the HGF-induced activation of transcription factor Egr-1 by MEK1/2- and PKC-dependent mechanisms differentially contributes to expression of PDGF and VEGF, which are important angiogenesis factors and targets for HNSCC therapy.

Early Growth Response-1 Transcription Factor Is Essential for Ethanol-Induced Fatty Liver Injury in Mice

Early growth response-1 (Egr-1), an immediate early gene/zinc-finger transcription factor, is required for maximal stimulation of tumor necrosis factor alpha (TNF-alpha) transcription in response to lipopolysaccharide (LPS). Because chronic ethanol exposure sensitizes macrophages to LPS-stimulated TNF-alpha expression, we have investigated the role of Egr-1 in mediating increased LPS-stimulated TNF-alpha expression after chronic ethanol feeding. Furthermore, because TNF-alpha contributes to alcoholic liver injury, we tested the hypothesis that Egr-1 is required for the development of ethanol-induced fatty liver injury in wild type and egr-1 -/- mice. Wild-type and egr-1 -/- mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. Wild-type mice fed the ethanol diet developed hepatic steatosis characterized by micro- and macrovesicular lipid accumulation. However, egr-1 -/- mice did not develop steatosis after ethanol feeding. Alanine transferase and TNF-alpha concentrations in serum were increased after ethanol feeding in wild-type but not egr-1 -/- mice. In wild-type mice, challenge with LPS increased Egr-1 messenger RNA (mRNA) and DNA binding activity in liver; this response to LPS was enhanced after chronic ethanol feeding. LPS challenge also increased hepatic TNF-alpha mRNA and serum TNF-alpha to a greater extent after ethanol feeding compared with pair-fed wild-type mice. However, chronic ethanol feeding did not enhance LPS-stimulated TNF-alpha mRNA or serum TNF-alpha in egr-1 -/- mice. These data show that Egr-1 contributes to increased LPS-mediated TNF-alpha expression after chronic ethanol and that the absence of Egr-1 prevents chronic ethanol-induced fatty liver, as well as increased sensitivity to LPS.

Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: Contribution of vesicular ATP release

Extracellular nucleotides such as ATP and UTP are released in response to mechanical stimulation in different cell systems. It is becoming increasingly evident that ATP release plays a role in autocrine and paracrine stimulation of osteoblasts. Mechanical stimulation, as shear stress, membrane stretch or hypo-osmotic swelling, as well as oscillatory fluid flow, stimulates ATP release from different osteoblastic cell lines. Human osteoblast-like initial transfectant (HOBIT) cells release ATP in response to mechanical stimulation. In the present study, we show that HOBIT cells are activated by nanomolar levels of extracellular ATP, concentrations that can be detected under resting conditions and increase following hypotonic shock. Cell activation by hypotonic medium induced intracellular Ca 2+ oscillations, and Egr-1 synthesis and DNA-binding activity. Quinacrine staining of living, resting cells revealed a granular fluorescence, typical of ATP-storing vesicles. Monensin prevented quinacrine staining and considerably inhibited hypotonic-induced ATP release. Finally, elevated levels of cytosolic Ca 2+ activated massive ATP release and a dose-dependent loss of quinacrine granules. The contribution of a vesicular mechanism for ATP release is proposed to sustain paracrine osteoblast activation.

Mechanism of amyloid peptide induced CCR5 expression in monocytes and its inhibition by siRNA for Egr-1

In Alzheimer's disease (AD), one finds increased presence of monocytes/macrophages and activated microglial cells in the brain. Immunohistochemical studies show increased expression of chemokine receptor 5 (CCR5) on reactive microglia associated with amyloid deposits in AD, suggesting that CCR5 may play a role in the regulation of the immune response in AD. In this study, we used peripheral blood monocytes and human monocytic THP-1 cell line as a model of microglia to delineate the cellular signaling mechanism of Abeta-induced CCR5 expression and the latter's role in the chemotaxis of monocytes. We observed that Abeta peptides at pathophysiological concentrations (125 nM) increased CCR5 mRNA and cell surface protein expression. The cellular signaling involved activation of c-Raf, ERK-1/ERK-2, and c-Jun NH(2)-terminal kinase. Analysis of some transcription factors associated with CCR5 promoter revealed that Abeta increased DNA binding activity of Egr-1 and AP-1. In addition, we show that CCR5 promoter contains an Egr-1 like consensus sequence GCGGGGGTG as demonstrated by 1) electrophoretic mobility shift assay, 2) transfection studies with truncated CCR5 gene promoter construct, and 3) chromatin immunoprecipitation analysis. Moreover, transfection of Egr-1 siRNA, but not of scrambled Egr-1 siRNA, in THP-1 cells resulted in >75% reduction in both Abeta-mediated CCR5 expression and concomitant chemotaxis to its ligands. We suggest that inhibition of Egr-1 by either Egr-1 siRNA or pharmacological agents may reduce activation of monocytes/microglia and possibly ameliorate the inflammation and progression of AD.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: Evidence for an autoregulatory loop

The Early Growth Response protein (Egr-1) is a C2H2–zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation–reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H2O2 stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H2O2 stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

Stretch-inducible Expression of the Angiogenic Factor CCN1 in Vascular Smooth Muscle Cells Is Mediated by Egr-1

CCN1 is an angiogenic factor that promotes cell adhesion, proliferation, and differentiation. CCN1-deficient mice suffer embryonic death because of vascular defects, demonstrating that CCN1 is required for vessel development. Because mechanical stretch may act as a trigger for vessel development, we investigated the impact of mechanical stretch on the regulatory mechanism of CCN1 expression. Mechanical stretch rapidly enhances CCN1 expression and release in vascular smooth muscle cells (VSMC) in vitro and CCN1 expression in murine aortic segments in vivo. Transfection experiments of VSMC with deletion constructs of the CCN1 promoter revealed the regulatory region responsible for the stretch-induced CCN1 expression in the approximately 200-bp promoter region upstream of the TATA-box containing potential binding sites for early growth response-1 (Egr-1), nuclear factor of activated T-cells and cAMP response element binding protein. Decoy oligonucleotides to Egr-1, but not to nuclear factor of activated T-cells or cAMP response element binding protein, abolished the stretch-induced transcription of CCN1. In addition, mutagenesis of the Egr-1 binding site within the CCN1 promoter completely blunted the stretch-induced activation of the promoter. Furthermore, mechanical stretch induced the expression and DNA-binding activity of Egr-1 in VSMC as demonstrated by Western blot and electromobility shift assay. Moreover, a pressure overload-dependent de novo synthesis of Egr-1 was observed after aortic banding. These findings indicate that mechanical stretch leads to enhanced expression of CCN1 via the mechanosensitive transcription factor Egr-1, suggesting a central role for mechanical stretch in the regulation of CCN1-dependent pro-angiogenic potency.

Curcumin, the active constituent of turmeric, inhibits amyloid peptide‐induced cytochemokine gene expression and CCR5‐mediated chemotaxis of THP‐1 monocytes by modulating early growth response‐1 transcription factor

Epidemiological studies show reduced risk of Alzheimer's disease (AD) among patients using non-steroidal inflammatory drugs (NSAID) indicating the role of inflammation in AD. Studies have shown a chronic CNS inflammatory response associated with increased accumulation of amyloid peptide and activated microglia in AD. Our previous studies showed that interaction of Abeta1-40 or fibrilar Abeta1-42 caused activation of nuclear transcription factor, early growth response-1 (Egr-1), which resulted in increased expression of cytokines (TNF-alpha and IL-1beta) and chemokines (MIP-1beta, MCP-1 and IL-8) in monocytes. We determined whether curcumin, a natural product known to have anti-inflammatory properties, suppressed Egr-1 activation and concomitant expression of cytochemokines. We show that curcumin (12.5-25 microm) suppresses the activation of Egr-1 DNA-binding activity in THP-1 monocytic cells. Curcumin abrogated Abeta1-40-induced expression of cytokines (TNF-alpha and IL-1beta) and chemokines (MIP-1beta, MCP-1 and IL-8) in both peripheral blood monocytes and THP-1 cells. We found that curcumin inhibited Abeta1-40-induced MAP kinase activation and the phosphorylation of ERK-1/2 and its downstream target Elk-1. We observed that curcumin inhibited Abeta1-40-induced expression of CCR5 but not of CCR2b in THP-1 cells. This involved abrogation of Egr-1 DNA binding in the promoter of CCR5 by curcumin as determined by: (i) electrophoretic mobility shift assay, (ii) transfection studies with truncated CCR5 gene promoter constructs, and (iii) chromatin immunoprecipitation analysis. Finally, curcumin inhibited chemotaxis of THP-1 monocytes in response to chemoattractant. The inhibition of Egr-1 by curcumin may represent a potential therapeutic approach to ameliorate the inflammation and progression of AD.

Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease.

To better understand the molecular basis of chronic obstructive pulmonary disease (COPD), we used serial analysis of gene expression (SAGE) and microarray analysis to compare the gene expression patterns of lung tissues from COPD and control smokers. A total of 59,343 tags corresponding to 26,502 transcripts were sequenced in SAGE analyses. A total of 327 genes were differentially expressed (1.5-fold up- or down-regulated). Microarray analysis using the same RNA source detected 261 transcripts that were differentially expressed to a significant degree between GOLD-2 and GOLD-0 smokers. We confirmed the altered expression of a select number of genes by using real-time quantitative RT-PCR. These genes encode for transcription factors (EGR1 and FOS), growth factors or related proteins (CTGF, CYR61, CX3CL1, TGFB1, and PDGFRA), and extracellular matrix protein (COL1A1). Immunofluorescence studies on the same lung specimens localized the expression of Egr-1, CTGF, and Cyr61 to alveolar epithelial cells, airway epithelial cells, and stromal and inflammatory cells of GOLD-2 smokers. Cigarette smoke extract induced Egr-1 protein expression and increased Egr-1 DNA-binding activity in human lung fibroblast cells. Cytomix (tumor necrosis factor alpha, IL-1beta, and IFN-gamma) treatment showed that the activity of matrix metalloproteinase-2 (MMP-2) was increased in lung fibroblasts from EGR1 control (+/+) mice but not detected in that of EGR1 null (-/-) mice, whereas MMP-9 was regulated by EGR1 in a reverse manner. Our study represents the first comprehensive analysis of gene expression on GOLD-2 versus GOLD-0 smokers and reveals previously unreported candidate genes that may serve as potential molecular targets in COPD.

Activation of Peroxisome Proliferator-activated Receptor γ Inhibits Interleukin-1β-induced Membrane-associated Prostaglandin E2 Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1

Membrane-associated prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the conversion of PGH(2) to PGE(2), which contributes to many biological processes. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. Here, we examined the effect of PPARgamma ligands on interleukin-1beta (IL-1beta)-induced mPGES-1 expression in human synovial fibroblasts. PPARgamma ligands 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinedione troglitazone (TRO), but not PPARalpha ligand Wy14643, dose-dependently suppressed IL-1beta-induced PGE(2) production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ(2) and TRO suppressed IL-1beta-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARgamma further enhanced, whereas overexpression of a dominant negative PPARgamma alleviated, the suppressive effect of both PPARgamma ligands. Furthermore, pretreatment with an antagonist of PPARgamma, GW9662, relieves the suppressive effect of PPARgamma ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARgamma. We demonstrated that PPARgamma ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPARgamma ligands was enhanced in the presence of a PPARgamma expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ(2) and TRO suppressed IL-1beta-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPARgamma and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPARgamma regulates inflammatory responses.

Activation of peroxisome proliferator-activated receptor γ inhibits IL-1β-induced mPGES-1 expression in human synovial fibroblasts by interfering with Egr-1

Membrane-associated prostaglandin (PG) E synthase-1 (mPGES-1) catalyzes the conversion of PGH2 to PGE2, which plays a critical role in the pathogenesis of arthritis. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor and was shown to regulate a number of inflammatory genes in several cell types. In this study, we examined the effect of PPARγ ligands on IL-1β-induced mPGES-1 expression in human synovial fibroblasts. The PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and the thiazolidinedione troglitazone (TRO), but not the PPARα ligand Wy14643, dose-dependently suppressed IL-1β-induced PGE2 production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ2 and TRO suppressed IL-1β-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARγ further enhanced, whereas overexpression of a dominant negative PPARγ alleviated, the suppressive effect of both PPARγ ligands. Furthermore, pretreatment with an antagonist of PPARγ, GW9662, relieved the suppressive effect of PPARγ ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARγ. Previous studies have shown that Egr-1 plays a pivotal role in transactivation of mPGES-1 gene. We demonstrated that PPARγ ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. Electrophoretic mobility shift and super-shift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ2 and TRO suppressed IL-1β-induced DNA binding activity of Egr-1. This occurs without interfering with Egr-1 expression. These data define mPGES-1 and Egr-1 as novel targets of PPARγ and provide further support for the promising application of PPARγ ligands in the treatment of arthritis.

Amyloid peptide-induced cytokine and chemokine expression in THP-1 monocytes is blocked by small inhibitory RNA duplexes for early growth response-1 messenger RNA.

In Alzheimer's disease (AD) one finds increased deposition of A beta and also an increased presence of monocytes/macrophages in the vessel wall and activated microglial cells in the brain. AD patients show increased levels of proinflammatory cytokines by activated microglia. Here we used a human monocytic THP-1 cell line as a model for microglia to delineate the cellular signaling mechanism involved in amyloid peptides (A beta(1-40) and A beta(1-42))-induced expression of inflammatory cytokines and chemokines. We observed that A beta peptides at physiological concentrations (125 nM) increased mRNA expression of cytokines (TNF-alpha, and IL-1 beta) and chemokines (monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1 beta (MIP-1 beta)). The cellular signaling involved activation of c-Raf, extracellular signal-regulated kinase-1 (ERK-1)/ERK-2, and c-Jun N-terminal kinase, but not p38 mitogen-activated protein kinase. This is further supported by the data showing that A beta causes phosphorylation of ERK-1/ERK-2, which, in turn, activates Elk-1. Furthermore, A beta mediated a time-dependent increase in DNA binding activity of early growth response-1 (Egr-1) and AP-1, but not of NF-kappa B and CREB. Moreover, A beta-induced Egr-1 DNA binding activity was reduced >60% in THP-1 cells transfected with small interfering RNA duplexes for Egr-1 mRNA. We show that A beta-induced expression of TNF-alpha, IL-1 beta, MCP-1, IL-8, and MIP-1 beta was abrogated in Egr-1 small inhibitory RNA-transfected cells. Our results indicate that A beta-induced expression of cytokines (TNF-alpha and IL-1 beta) and chemokines (MCP-1, IL-8, and MIP-1 beta) in THP-1 monocytes involves activation of ERK-1/ERK-2 and downstream activation of Egr-1. The inhibition of Egr-1 by Egr-1 small inhibitory RNA may represent a potential therapeutic target to ameliorate the inflammation and progression of AD.