Intercellular Adhesion Molecule-1 Promoter Research Articles

Alantolactone derivatives inhibit the tumor necrosis factor α-induced nuclear factor κB pathway by a different mechanism from alantolactone

Alantolactone is a eudesmane-type sesquiterpene lactone that exerts various biological effects, including anti-inflammatory activity. In the present study, screening using the RIKEN Natural Products Depository chemical library identified alantolactone derivatives that inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells stimulated with proinflammatory cytokines and Toll-like receptor ligands. In human lung adenocarcinoma A549 cells stimulated with tumor necrosis factor-α (TNF-α), six alantolactone derivatives inhibited ICAM-1 expression in a dose-dependent manner and at IC50 values of 13–21 μM, whereas that of alantolactone was 5 μM. Alantolactone possesses an α-methylene-γ-lactone moiety, whereas alantolactone derivatives do not. In the nuclear factor κB (NF-κB) signaling pathway, alantolactone prevented the TNF-α-induced phosphorylation and degradation of the inhibitor of NF-κB α (IκBα) protein, and its downstream signaling pathway. In contrast, alantolactone derivatives neither reduced TNF-α-induced IκBα degradation nor the nuclear translocation of the NF-κB subunit RelA, but inhibited the binding of RelA to the ICAM-1 promoter. The inhibitory activities of alantolactone and alantolactone derivatives were attenuated by glutathione. These results indicate that alantolactone derivatives inhibit the TNF-α-induced NF-κB pathway by a different mechanism from alantolactone.

CX3CL1 induces cell migration and invasion through ICAM-1 expression in oral squamous cell carcinoma cells.

Human oral squamous cell carcinoma (OSCC) has been associated with a relatively low survival rate over the years and is characterized by a poor prognosis. C-X3-C motif chemokine ligand 1 (CX3CL1) has been involved in advanced migratory cells. Overexpressed CX3CL1 promotes several cellular responses related to cancer metastasis, including cell movement, migration and invasion in tumour cells. However, CX3CL1 controls the migration ability, and its molecular mechanism in OSCC remains unknown. The present study confirmed that CX3CL1 increased cell movement, migration and invasion. The CX3CL1-induced cell motility is upregulated through intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. These effects were significantly suppressed when OSCC cells were pre-treated with CX3CR1 monoclonal antibody (mAb) and small-interfering RNA (siRNA). The CX3CL1-CX3CR1 axis activates promoted PLCβ/PKCα/c-Src phosphorylation. Furthermore, CX3CL1 enhanced activator protein-1 (AP-1) activity. The CX3CR1 mAb and PLCβ, PKCα, c-Src inhibitors reduced CX3CL1-induced c-Jun phosphorylation, c-Jun translocation into the nucleus and c-Jun binding to the ICAM-1 promoter. The present results reveal that CX3CL1 induces the migration of OSCC cells by promoting ICAM-1 expression through the CX3CR1 and the PLCβ/PKCα/c-Src signal pathway, suggesting that CX3CL1-CX3CR1-mediated signalling is correlated with tumour motility and appealed to be a precursor for prognosis in human OSCC.

EPAS1 (Endothelial PAS Domain Protein 1) Orchestrates Transactivation of Endothelial ICAM1 (Intercellular Adhesion Molecule 1) by Small Nucleolar RNA Host Gene 5 (SNHG5) to Promote Hypoxic Pulmonary Hypertension.

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Biological Evaluation of Alkyl Triphenylphosphonium Ostruthin Derivatives as Potential Anti-Inflammatory Agents Targeting the Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

Ostruthin (6-geranyl-7-hydroxycoumarin) is one of the constituents isolated from Paramignya trimera and has been classified as a simple coumarin. We recently reported the synthesis of alkyl triphenylphosphonium (TPP) derivatives from ostruthin and evaluated their anticancer activities. In the present study, we demonstrated that alkyl TPP ostruthin derivatives inhibited the up-regulation of cell-surface intercellular adhesion molecule-1 (ICAM-1) in human lung adenocarcinoma A549 cells stimulated with tumor necrosis factor-α (TNF-α) without affecting cell viability, while ostruthin itself exerted cytotoxicity against A549 cells. The heptyl TPP ostruthin derivative (termed OS8) attenuated the up-regulation of ICAM-1 mRNA expression at concentrations higher than 40 µM in TNF-α-stimulated A549 cells. OS8 inhibited TNF-α-induced nuclear factor κB (NF-κB)-responsive luciferase reporter activity at concentrations higher than 40 µM, but did not affect the translocation of the NF-κB subunit RelA in response to the TNF-α stimulation at concentrations up to 100 µM. A chromatin immunoprecipitation assay showed that OS8 at 100 µM prevented the binding of RelA to the ICAM-1 promoter. We also showed that OS8 at 100 µM inhibited the TNF-α-induced phosphorylation of RelA at Ser 536. Moreover, the TNF-α-induced phosphorylation of an inhibitor of NF-κB α and extracellular signal-regulated kinase was reduced by OS8. These results indicate that OS8 has potential as an anti-inflammatory agent that targets the NF-κB signaling pathway.

α-Conidendrin inhibits the expression of intercellular adhesion molecule-1 induced by tumor necrosis factor-α in human lung adenocarcinoma A549 cells

α-Conidendrin is a lignan isolated from Taxus wallichiana and other species. In the present study, we demonstrated that α-conidendrin inhibited the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) at an IC50 value of 40–60 μM in human lung adenocarcinoma A549 cells. α-Conidendrin decreased ICAM-1 protein and mRNA expression levels at concentrations of 40–100 μM in TNF-α-stimulated A549 cells. The TNF-α-induced mRNA expression of vascular cell adhesion molecule-1, E-selectin, and cyclooxygenase-2 was also reduced by α-conidendrin. In the TNF-α-induced nuclear factor κB (NF-κB) signaling pathway, α-conidendrin did not influence the translocation of the NF-κB subunit RelA from the cytoplasm to the nucleus at concentrations up to 100 μM. A chromatin immunoprecipitation assay revealed that α-conidendrin at 100 μM reduced the binding of RelA to the ICAM-1 promoter in response to a stimulation with TNF-α. Collectively, these results indicated that α-conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter, thereby reducing ICAM-1 transcription.

AF1q inhibited T cell attachment to breast cancer cell by attenuating Intracellular Adhesion Molecule-1 expression.

Aim:To investigate whether AF1q, overexpressed in metastatic cells compared with the primary tumor cells, plays a pivotal role in breast cancer metastasis.Methods:To investigate whether AF1q has a responsibility in the acquisition of a metastatic phenotype, we performed RNA-sequencing (RNA-Seq) to identify the gene signature and applied the Metacore direct interactions network building algorithm with the top 40 amplicons of RNA-Seq.Results:Most genes were directly linked with intercellular adhesion molecule-1 (ICAM-1). Likewise, we identified that ICAM-1 expression is attenuated in metastatic cells compared to primary tumor cells. Moreover, overexpression of AF1q attenuated ICAM-1 expression, whereas suppression of AF1q elicited the opposite effect. AF1q had an effect on ICAM-1 promoter region and regulated its transcription. Decreased ICAM-1 expression affected the attachment of T cells to a breast cancer cell monolayer. We confirmed the finding by performing the analysis on Burkitt’s lymphoma.Conclusion:Attenuation of ICAM-1 by AF1q on tumor cells disadvantages host anti-tumor defenses through the trafficking of lymphocytes, which affects tumor progression and metastasis.

AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro.

PurposePathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)–mediated CRISPR (clustered regularly interspaced short palindromic repeats)–associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events.MethodsThe dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined.ResultsAAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs.ConclusionsAAV-CRISRP/Cas9–mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.

Quinacrine Inhibits ICAM-1 Transcription by Blocking DNA Binding of the NF-κB Subunit p65 and Sensitizes Human Lung Adenocarcinoma A549 Cells to TNF-α and the Fas Ligand.

Quinacrine has been used for therapeutic drugs in some clinical settings. In the present study, we demonstrated that quinacrine decreased the expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor (TNF)-α and interleukin-1 (IL-1) α in human lung adenocarcinoma A549 cells. Quinacrine inhibited ICAM-1 mRNA expression and nuclear factor κB (NF-κB)-responsive luciferase reporter activity following a treatment with TNF-α and IL-1α. In the NF-κB signaling pathway, quinacrine did not markedly affect the TNF-α-induced degradation of the inhibitor of NF-κB or the TNF-α-induced phosphorylation of the NF-κB subunit, p65, at Ser-536 and its subsequent translocation to the nucleus. In contrast, a chromatin immunoprecipitation assay showed that quinacrine prevented the binding of p65 to the ICAM-1 promoter following TNF-α stimulation. Moreover, TNF-α and the Fas ligand effectively reduced the viability of A549 cells in the presence of quinacrine only. Quinacrine down-regulated the constitutive and TNF-α-induced expression of c-FLIP and Mcl-1 in A549 cells. These results revealed that quinacrine inhibits ICAM-1 transcription by blocking the DNA binding of p65 and sensitizes A549 cells to TNF-α and the Fas ligand.

Changes in the DNA Methylation and Hydroxymethylation Status of the Intercellular Adhesion Molecule 1 Gene Promoter in Thyrocytes from Autoimmune Thyroiditis Patients

The intercellular adhesion molecule 1 (ICAM1) gene is not expressed in normal thyroid tissue but was shown to be expressed in the thyroid tissue of autoimmune thyroiditis (AIT) patients. This study aimed to explore whether the DNA methylation and hydroxymethylation status of the ICAM1 promoter are aberrantly altered in the thyroid cells of AIT patients and whether this change is associated with dysfunctional expression of ICAM1. A total of 35 AIT patients and 35 sex- and age-matched controls were studied. After the isolation of thyrocytes via density-gradient centrifugation, ICAM1 mRNA expression was measured using real-time PCR. The DNA methylation and hydroxymethylation status were assessed using quantitative PCR following T4 β-glucosyltransferase treatment and MspI/HpaII cleavage at -937 bp, -701 bp, -226 bp, and -65 bp upstream of the transcription start site (TSS). The DNA methylation level was verified via pyrosequencing. The AIT group showed increased DNA hydroxymethylation at -937 bp and -226 bp and decreased methylation at -937 bp, -701 bp, and -226 bp upstream of the TSS. Pyrosequencing also revealed DNA hypomethylation at -708 bp, -692 bp, -690 bp, and -688 bp upstream of the TSS. The DNA methylation status at -708 bp, -692 bp, and -226 bp upstream of the TSS was negatively associated with ICAM1 mRNA expression. In summary, we identified aberrant DNA methylation and hydroxymethylation of the ICAM1 gene promoter in the thyrocytes of AIT patients. This aberrant epigenetic modification is associated with increased expression of the ICAM1 gene.

Ablation of Interferon Regulatory Factor 3 Protects Against Atherosclerosis in Apolipoprotein E-Deficient Mice.

The secretion of adhesion molecules by endothelial cells, as well as the subsequent infiltration of macrophages, determines the initiation and progression of atherosclerosis. Accumulating evidence suggests that IRF3 (interferon regulatory factor 3) is required for the induction of proinflammatory cytokines and for endothelial cell proliferation. However, the effect and underlying mechanism of IRF3 on atherogenesis remain unknown. Our results demonstrated a moderate-to-strong immunoreactivity effect associated with IRF3 in the endothelium and macrophages of the atherosclerotic plaques in patients with coronary heart disease and in hyperlipidemic mice. IRF3-/-ApoE-/- mice showed significantly decreased atherosclerotic lesions in the whole aorta, aortic sinus, and brachiocephalic arteries. The bone marrow transplantation further suggested that the amelioration of atherosclerosis might be attributed to the effects of IRF3 deficiency mainly in endothelial cells, as well as in macrophages. The enhanced stability of atherosclerotic plaques in IRF3-/-ApoE-/- mice was characterized by the reduction of necrotic core size, macrophage infiltration, and lipids, which was accompanied by increased collagen and smooth muscle cell content. Furthermore, multiple proinflammatory cytokines showed a marked decrease in IRF3-/-ApoE-/- mice. Mechanistically, IRF3 deficiency suppresses the secretion of VCAM-1 (vascular cell adhesion molecule 1) and the expression of ICAM-1 (intercellular adhesion molecule 1) by directly binding to the ICAM-1 promoter, which subsequently attenuates macrophage infiltration. Thus, our study suggests that IRF3 might be a potential target for the treatment of atherosclerosis development.

Essential role of ICAM-1 in aldosterone-induced atherosclerosis

ObjectiveElevated aldosterone is associated with increased risk of atherosclerosis complications, whereas treatment with mineralocorticoid receptor (MR) antagonists decreases the rate of cardiovascular events. Here we test the hypothesis that aldosterone promotes early atherosclerosis by modulating intercellular adhesion molecule-1 (ICAM-1) expression and investigate the molecular mechanisms by which aldosterone regulates ICAM-1 expression. Methods and resultsApolipoprotein-E (ApoE)−/− mice fed an atherogenic diet and treated with aldosterone for 4weeks showed increased vascular expression of ICAM-1, paralleled by enhanced atherosclerotic plaque size in the aortic root. Moreover, aldosterone treatment resulted in increased plaque lipid and inflammatory cell content, consistent with an unstable plaque phenotype. ApoE/ICAM-1 double knockout (ApoE−/−/ICAM-1−/−) littermates were protected from the aldosterone-induced increase in plaque size, lipid content and macrophage infiltration. Since aldosterone is known to regulate ICAM-1 transcription via MR in human endothelial cells, we explored MR regulation of the ICAM-1 promoter. Luciferase reporter assays performed in HUVECs using deletion constructs of the human ICAM-1 gene promoter showed that a region containing a predicted MR-responsive element (MRE) is required for MR-dependent transcriptional regulation of ICAM-1. ConclusionsPro-atherogenic effects of aldosterone are mediated by increased ICAM-1 expression, through transcriptional regulation by endothelial MR. These data enhance our understanding of the molecular mechanism by which MR activation promotes atherosclerosis complications.

Peroxisome proliferator-activated receptor (PPAR) α and δ activators induce ICAM-1 expression in quiescent non stimulated endothelial cells.

BackgroundPeroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are implicated in the regulation of lipid and glucose homeostasis. PPAR agonists have been shown to control inflammatory processes, in part by inhibiting the expression of distinct proinflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1), IL-8, and intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is an important endothelial membrane receptor that facilitates the transmigration of leukocytes across the endothelium. To date, the influence of PPARα and δ activators on the expression of ICAM-1 in non-induced, quiescent endothelial cells has been unclear. Therefore, we examined the effects of various PPARα and δ agonists on the expression of ICAM-1 in non-stimulated primary human endothelial cells.ResultsWe found that PPARα and PPARδ agonists significantly induced ICAM-1 surface, intracellular protein, and mRNA expression in a time and concentration-dependent manner. The PPARδ induced ICAM-1 expression could be paralleled with a significantly increased T-cell adherence to the endothelial cells whereas PPARα failed to do so. Transcriptional activity studies using an ICAM-1 reporter gene constructs revealed that PPARδ, but not PPARα agonists induced gene expression by stimulating ICAM-1 promoter activity via an Sp1 transcription factor binding site and inhibit the binding of the transcription factors Sp1 and Sp3. Furthermore, we performed mRNA stability assays and found that PPARα and PPARδ agonists increased ICAM-1 mRNA stability.ConclusionTherefore, our data provide the first evidence that PPARα and PPARδ agonists induce ICAM-1 expression in non-stimulated endothelial cells via transcriptional and posttranscriptional mechanisms.

Amphiregulin enhances intercellular adhesion molecule-1 expression and promotes tumor metastasis in human osteosarcoma.

Osteosarcoma is a common, high malignant, and metastatic bone cancer. Amphiregulin (AREG) has been associated with cancer cellular activities. However, the effect of AREG on metastasis activity in human osteosarcoma cells has yet to be determined. We determined that AREG increases the expression of intercellular adhesion molecule-1 (ICAM-1) through PI3K/Akt signaling pathway via its interaction with the epidermal growth factor receptor, thus resulting in the enhanced cell migration of osteosarcoma. Furthermore, AREG stimulation increased the association of NF-κB to ICAM-1 promoter which then up-regulated ICAM-1 expression. Finally, we observed that shRNA silencing of AREG decreased osteosarcoma metastasis in vivo. Our findings revealed a relationship between osteosarcoma metastatic potential and AREG expression and the modulating effect of AREG on ICAM-1 expression.

TNF-α mediates PKCδ/JNK1/2/c-Jun-dependent monocyte adhesion via ICAM-1 induction in human retinal pigment epithelial cells.

Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases.

The saponin DT-13 attenuates tumor necrosis factor-α-induced vascular inflammation associated with Src/NF-кB/MAPK pathway modulation.

This study aimed to explore the effect of DT-13 (25(R,S)-ruscogenin- 1-O- [β-d-glucopyranosyl- (1→2)][β-d-xylopyranosyl-(1→3)]-β -d- fucopyranoside) on tumor necrosis factor (TNF)-α-induced vascular inflammation and the potential molecular mechanisms. In vitro, DT-13 suppressed TNF-α-induced adhesion and migration of human umbilical vein endothelial cells (HUVECs) by inhibiting the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). DT-13 markedly suppressed NF-кB p65 phosphorylation, and when NF-кB p65 was over-expressed, the inhibitory effect of DT-13 on adhesion molecular decreased. DT-13 also suppressed TNF-α induced luciferase activities of ICAM-1 and VCAM-1 promoter containing NF-κB binding sites. Furthermore, DT-13 markedly suppressed p38 phosphorylation and Src degradation induced by TNF-α, whereas had no significant effect on ERK and JNK activation. In vivo, DT-13 at 4 mg/kg prevented vascular inflammation and the expression of adhesion molecules induced by TNF-α in mice. These findings suggest that DT-13 abrogates vascular inflammation by down-regulating adhesion molecules associated with modulating the NF-кB, p38MAPK, Src signaling pathways, and NF-κB binding site is at least one of the targets of DT-13. This study provides novel information regarding the mechanism by which DT-13 exerts its effects on vascular inflammation, which is important for the onset and progression of various diseases.

Thrombin induces ICAM-1 expression in human lung epithelial cells via c-Src/PDGFR/PI3K/Akt-dependent NF-κB/p300 activation

Up-regulation of ICAM-1 (intercellular adhesion molecule-1) is frequently implicated in lung inflammation and lung diseases, such as IPF (idiopathic pulmonary fibrosis). Thrombin has been shown to play a key role in inflammation via the induction of adhesion molecules, which then causes lung injury. However, the mechanisms underlying thrombin-induced ICAM-1 expression in HPAEpiCs (human pulmonary alveolar epithelial cells) remain unclear. In the present study, we have shown that thrombin induced ICAM-1 expression in HPAEpiCs. Pre-treatment with the inhibitor of thrombin [PPACK (D-Phe-Pro-Arg-chloromethyl ketone)], c-Src (PP1), PDGFR (platelet-derived growth factor receptor) (AG1296), PI3K (phosohinositide 3-kinase) (LY294002), NF-κB (nuclear factor κB) (Bay11-7082) or p300 (GR343) and transfection with siRNAs of c-Src, PDGFR, Akt, p65 and p300 markedly reduced thrombin-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with thrombin. In addition, we established that thrombin stimulated the phosphorylation of c-Src, PDGFR, Akt and p65, which were inhibited by pre-treatment with their respective inhibitors PP1, AG1296, LY294002 or Bay11-7082. In addition, thrombin also enhanced Akt and NF-κB translocation from the cytosol to the nucleus, which was reduced by PP1, AG1296 or LY294002. Thrombin induced NF-κB promoter activity and the formation of the p65-Akt-p300 complex, which were inhibited by AG1296, LY294002 or PP1. Finally, we have shown that thrombin stimulated in vivo binding of p300, Akt and p65 to the ICAM-1 promoter, which was reduced by AG1296, LY294002, SH-5 or PP1. These results show that thrombin induced ICAM-1 expression and monocyte adherence via a c-Src/PDGFR/PI3K/Akt/NF-κB-dependent pathway in HPAEpiCs. Increased understanding of the signalling mechanisms underlying ICAM-1 gene regulation will create opportunities for the development of anti-inflammatory therapeutic strategies.

Attenuation of Inflammatory Response and Reduction in Infarct Size by Postconditioning Are Associated With Downregulation of Early Growth Response 1 During Reperfusion in Rat Heart

Early growth response 1 (EGR-1) works as a master regulator that plays a key role in triggering inflammation-induced tissue injury after ischemia and reperfusion. This study tested the hypothesis that postconditioning (Postcon) or anti-inflammatory compound, curcumin, ameliorates inflammatory responses and further reduces infarct size by normalizing EGR-1 expression during reperfusion. In the control group, male Sprague-Dawley rats were subjected to 30-min ischemia and 180-min reperfusion. Postcon with four cycles of 10-s/10-s reperfusion/ischemia was applied at the onset of reperfusion. Curcumin (150 mg/kg per day) was fed 5 days before ischemia. Relative to the control, Postcon reduced expression of EGR-1 mRNA and protein, as further identified by less EGR-1 immunoreactivity in myocardial nuclei and microvessels during reperfusion. Along with EGR-1 downregulation, levels of plasma and myocardial tumor necrosis factor α and interleukin 6 (IL-6) were significantly decreased. Upregulated P-selectin and intercellular adhesion molecule 1 mRNA and protein as well as their immunoreactivity at area at risk myocardium were significantly attenuated. Neutrophil extravasation identified by myeloperoxidase immunohistochemical staining was inhibited. Infarct size, determined with triphenyltetrazolium chloride staining, was smaller in the Postcon group than that in the control. The protection achieved with pretreatment with curcumin was comparable to the benefits gained by Postcon in all end points measured. In H9C2 rat cardiomyoblast cell line, EGR-1 siRNA downregulated hydrogen peroxide-induced EGR-1 mRNA expression and subsequently reduced tumor necrosis factor α mRNA level. These results suggest that EGR-1 seems to play a critical role in myocardial reperfusion injury because downregulation of EGR-1 either by Postcon or the use of pharmacological intervention reduces infarct size, most likely through an inhibition of inflammation-mediated processes.

Syk/JNK/AP-1 Signaling Pathway Mediates Interleukin-6-Promoted Cell Migration in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) typically migrates and metastasizes. Interleukin-6 (IL-6) is a multifunctional cytokine associated with disease status and cancer outcomes. The effect of IL-6 on human OSCC cells, however, is unknown. Here, we showed that IL-6 increased cell migration and Intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. Pretreatment of OSCC cells with IL-6R monoclonal antibody (mAb) significantly abolished IL-6-induced cell migration and ICAM-1 expression. By contrast, IL-6-mediated cell motility and ICAM-1 upregulation were attenuated by the Syk and c-Jun N-terminal kinase (JNK) inhibitors. Stimulation of OSCC cells with IL-6 promoted Syk and JNK phosphorylation. Furthermore, IL-6 enhanced AP-1 activity, and the IL-6R mAb, Syk inhibitor, or JNK inhibitor all reduced IL-6-mediated c-Jun phosphorylation, c-Jun binding to the ICAM-1 promoter, and c-Jun translocation into the nucleus. Our results indicate that IL-6 enhances the migration of OSCC cells by increasing ICAM-1 expression through the IL-6R receptor and the Syk, JNK, and AP-1 signal transduction pathways.

Cigarette smoke increases BLT2 receptor functions in bronchial epithelial cells: in vitro and ex vivo evidence

Leukotriene B(4) (LTB(4)) is a neutrophil chemotactic molecule with important involvement in the inflammatory responses of chronic obstructive pulmonary disease (COPD). Airway epithelium is emerging as a regulator of innate immune responses to a variety of insults including cigarette smoke, the major risk factor for COPD. In this study we have explored whether cigarette smoke extracts (CSE) or soluble mediators present in distal lung fluid samples (mini-bronchoalveolar lavages) from smokers alter the expression of the LTB(4) receptor 2 (BLT2) and peroxisome proliferator-activated receptor-α (PPAR-α) in bronchial epithelial cells. We also evaluated the effects of CSE on the expression of intercellular adhesion molecule 1 (ICAM-1) and on the binding of signal transducer and activator of transcription 1 (STAT-1) to ICAM-1 promoter as well as the adhesiveness of neutrophils to bronchial epithelial cells. CSE and mini-bronchoalveolar lavages from smokers increased BLT2 and ICAM-1 expression as well as the adhesiveness of neutrophils to bronchial epithelial cells and decreased PPAR-α expression. CSE induced the activation of STAT-1 and its binding to ICAM-1 promoter. These findings suggest that, in bronchial epithelial cells, CSE promote a prevalent induction of pro-inflammatory BLT2 receptors and activate mechanisms leading to increased neutrophil adhesion, a mechanism that contributes to airway neutrophilia and to tissue damage.

SIRT1 suppresses PMA and ionomycin-induced ICAM-1 expression in endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the recruitment of leukocytes to the endothelium, which causes inflammation and initiation of atherosclerosis. We have previously shown that endothelium-specific over-expression of class III deacetylase SIRT1 decreases atherosclerosis. We therefore addressed the hypothesis that SIRT1 suppresses ICAM-1 expression in the endothelial cells. Here, we found that expression of SIRT1 and ICAM-1 was significantly induced by PMA and ionomycin (PMA/Io) in human umbilical vein endothelial cells (HUVECs). Adenovirus-mediated over-expression of SIRT1 significantly inhibited PMA/Io-induced ICAM-1 expression in HUVECs. Knockdown of SIRT1 by RNA interference (RNAi) resulted in increased expression of ICAM-1 in HUVECs. Luciferase report assay showed that over-expression of SIRT1 suppressed ICAM-1 promoter activity both in basic and in PMA/Io-induced conditions. We further found that SIRT1 was involved in transcription complex binding on the ICAM-1 promoter by chromatin immunoprecipitation (ChIP) assays. Furthermore, SIRT1 RNAi increased NF-κB p65 binding ability to the ICAM-1 promoter by ChIP assays. Overall, these data suggests that SIRT1 inhibits ICAM-1 expression in endothelial cells, which may contribute to its anti-atherosclerosis effect.