Intercellular Adhesion Molecule-1 Promoter Activity Research Articles

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.

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.

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.

CXC chemokine ligand 12/Stromal cell-derived factor-1 regulates cell adhesion in human colon cancer cells by induction of intercellular adhesion molecule-1

BackgroundThe CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor-1 (SDF-1) and CXC receptor 4 (CXCR4) axis is involved in human colorectal cancer (CRC) carcinogenesis and can promote the progression of CRC. Interaction between CRC cells and endothelium is a key event in tumor progression. The aim of this study was to investigate the effect of SDF-1 on the adhesion of CRC cells.MethodsHuman CRC DLD-1 cells were used to study the effect of SDF-1 on intercellular adhesion molecule-1 (ICAM-1) expression and cell adhesion to endothelium.ResultsSDF-1 treatment induced adhesion of DLD-1 cells to the endothelium and increased the expression level of the ICAM-1. Inhibition of ICAM-1 by small interfering RNA (siRNA) and neutralizing antibody inhibited SDF-1-induced cell adhesion. By using specific inhibitors and short hairpin RNA (shRNA), we demonstrated that the activation of ERK, JNK and p38 pathways is critical for SDF-1-induced ICAM-1 expression and cell adhesion. Promoter activity and transcription factor ELISA assays showed that SDF-1 increased Sp1-, C/EBP-β- and NF-κB-DNA binding activities in DLD-1 cells. Inhibition of Sp1, C/EBP-β and NF-κB activations by specific siRNA blocked the SDF-1-induced ICAM-1 promoter activity and expression. The effect of SDF-1 on cell adhesion was mediated by the CXCR4.ConclusionOur findings support the hypothesis that ICAM-1 up-regulation stimulated by SDF-1 may play an active role in CRC cell adhesion.

Deoxypodophyllotoxin Inhibits the Expression of Intercellular Adhesion Molecule-1 Induced by Tumor Necrosis Factor-.ALPHA. in Murine Lung Epithelial Cells

Intercellular adhesion molecule-1 (ICAM-1) is associated with processes of inflammation. We investigated the effects of deoxypodophyllotoxin (DPT) on tumor necrosis factor-alpha (TNF-alpha) induced ICAM-1 expression in the mouse lung epithelial cell line, LA4. DPT (5 to 20 nM) inhibited TNF-alpha-induced ICAM-1 expression through nuclear factor-kappa B (NF-kappaB) in a dose-dependent manner and repressed ICAM-1 promoter activity. NF-kappaB reporter gene activity and DNA binding activity were also strongly inhibited. In addition, DPT inhibited degradation by the TNF-alpha induced inhibitory kappaB-alpha (IkappaB-alpha) in a concentration-dependent manner. Taken together with our previous results suggest DPT might provide a basis for novel anti-inflammatory drug development.

Glutathione regulation of redox-sensitive signals in tumor necrosis factor-α-induced vascular endothelial dysfunction

We investigated the regulatory role of glutathione in tumor necrosis factor-alpha (TNF-α)-induced vascular endothelial dysfunction as evaluated by using vascular endothelial adhesion molecule expression and monocyte–endothelial monolayer binding. Since TNF-α induces various biological effects on vascular cells, TNF-α dosage could be a determinant factor directing vascular cells into different biological fates. Based on the adhesion molecule expression patterns responding to different TNF-α concentrations, we adopted the lower TNF-α (0.2 ng/ml) to rule out the possible involvement of other TNF-α-induced biological effects. Inhibition of glutathione synthesis by l-buthionine-( S, R)-sulfoximine (BSO) resulted in down-regulations of the TNF-α-induced adhesion molecule expression and monocyte–endothelial monolayer binding. BSO attenuated the TNF-α-induced nuclear factor-kappaB (NF-κB) activation, however, with no detectable effect on AP-1 and its related mitogen-activated protein kinases (MAPKs). Deletion of an AP-1 binding site in intercellular adhesion molecule-1 (ICAM-1) promoter totally abolished its constitutive promoter activity and its responsiveness to TNF-α. Inhibition of ERK, JNK, or NF-κB attenuates TNF-α-induced ICAM-1 promoter activation and monocyte–endothelial monolayer binding. Our study indicates that TNF-α induces adhesion molecule expression and monocyte–endothelial monolayer binding mainly via activation of NF-κB in a glutathione-sensitive manner. We also demonstrated that intracellular glutathione does not modulate the activation of MAPKs and/or their downstream AP-1 induced by lower TNF-α. Although AP-1 activation by the lower TNF-α was not detected in our systems, we could not rule out the possible involvement of transiently activated MAPKs/AP-1 in the regulation of TNF-α-induced adhesion molecule expression.

Role of the JNK pathway in thrombin-induced ICAM-1 expression in endothelial cells

Thrombin induces leukocyte adherence to endothelial cells via increased expression of intercellular adhesion molecule-1 (ICAM-1). Although ICAM-1 expression is regulated by NF-kappaB, recent studies have suggested that additional signaling mechanisms may also be involved. The goal of this study was to determine whether mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38), mediate thrombin-induced ICAM-1 expression in endothelial cells. Western blot analysis using anti-ICAM-1 antibody and luciferase assays were performed in cultured endothelial cells after addition of signal transduction inhibitors or transfection of various gene constructs. JNK kinase activity was determined by a kinase assay using c-Jun as a substrate or by Western blot analysis with anti-phospho-JNK antibody. Treatment of endothelial cells with the JNK-specific inhibitors, SP600125 or JNK inhibitory peptide 1 (JNKI1), resulted in a significant decrease in thrombin-induced ICAM-1 expression as demonstrated by Western blot analysis (67 +/- 3% and 72 +/- 7%, respectively). In contrast, inhibitors of MEK and p38 had only minimal effect. The combination of SP600125 and the NF-kappaB inhibitor, BAY11-7082, resulted in complete inhibition of thrombin-induced ICAM-1 expression. The Galpha(q) inhibitor, YM-254890, inhibited thrombin-induced JNK activation and ICAM-1 expression. Dominant-negative Ras and Rac1, but not Rho, inhibited thrombin-induced JNK activation and ICAM-1 promoter activity. Finally, thrombin-induced JNK activation and ICAM-1 promoter activity were inhibited by betaARK1ct (a Gbetagamma subunit scavenger) and Csk. These data suggest that, in concert with NF-kappaB, JNK regulates thrombin-induced ICAM-1 expression by a mechanism that is dependent on Galpha(q), Gbetagamma, Ras, Rac1 and the Src kinase family.

Synergistic induction of intercellular adhesion molecule-1 by the human cytomegalovirus transactivators IE2p86 and pp71 is mediated via an Sp1-binding site.

Human cytomegalovirus (HCMV) infection of transplant recipients is frequently associated with allograft vasculopathy and rejection. One potential mechanism is vascular injury from HCMV-triggered, immunologically mediated processes. HCMV infection has been shown to increase the expression of intercellular adhesion molecule-1 (ICAM-1). The objective of this study was to determine the molecular basis of HCMV-enhanced ICAM-1 gene expression. Transient transfection experiments identified the IE2p86 protein as a potent activator of the ICAM-1 promoter. The tegument protein pp71 showed a strong synergistic effect on IE2p86-mediated ICAM-1 promoter activation. Mutagenesis experiments defined a DNA element from -110 to -42 relative to the transcription start site as responsive for IE2p86. Further point mutations within this DNA element identified an Sp1-binding site that was essential for strong synergistic activation by IE2p86 and pp71. To confirm the activation of ICAM-1 gene expression, human fibroblasts (HFF) as well as endothelial cells (HUVEC) were infected with recombinant IE2p86- and pp71-expressing baculoviruses, respectively. In FACS analysis, a synergistic induction of ICAM-1 was detectable when cells were co-infected with the two recombinant baculoviruses. These findings clearly demonstrate that IE2p86 and pp71 are crucial regulatory factors for HCMV-induced ICAM-1 upregulation.

Reducing agents inhibit rhinovirus-induced up-regulation of the rhinovirus receptor intercellular adhesion molecule-1 (ICAM-1) in respiratory epithelial cells.

Rhinoviruses are the major cause of common colds and of asthma exacerbations. Intercellular adhesion molecule-1 (ICAM-1) has a central role in airway inflammation and is the receptor for 90% of rhinoviruses. Rhinovirus infection of airway epithelium induces ICAM-1. Because redox state is directly implicated in inflammatory responses via molecular signaling mechanisms, here we studied the effects of reducing agents on rhinovirus-induced ICAM-1 expression, mRNA up-regulation, promoter activation, and nuclear factor activation. To investigate the effects of rhinovirus infection on the intracellular redox balance, we also studied whether rhinovirus infection triggers the production of reactive oxygen species. We found that reduced (GSH) but not oxidized (GSSG) glutathione (1-100 microM) inhibited in a dose-dependent manner rhinovirus-induced ICAM-1 up-regulation and mRNA induction in primary bronchial and A549 respiratory epithelial cells. GSH but not GSSG also inhibited rhinovirus-induced ICAM-1 promoter activation and rhinovirus-induced NF-kB activation. In parallel, we found that rhinovirus infection induced a rapid increase of intracellular superoxide anion that was maximal at the time of NF-kB activation. This oxidant generation was completely inhibited by GSH. We conclude that redox-mediated intracellular pathways represent an important target for the therapeutic control of rhinovirus-induced diseases.

Galpha(q) and Gbetagamma regulate PAR-1 signaling of thrombin-induced NF-kappaB activation and ICAM-1 transcription in endothelial cells.

As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-delta mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(2). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(2) were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-delta. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-delta and Gbetagamma/PI3-kinase pathways that converge at Akt.

Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

Nitric Oxide Inhibits Endothelial IL-1β-induced ICAM-1 Gene Expression at the Transcriptional Level Decreasing Sp1 and AP-1 Activity

Nitric oxide (NO) has frequently been shown to inhibit leukocyte adherence to activated endothelium thus displaying anti-adhesive and immunosuppressive activities. A molecular mechanism contributing to this effect is described. Primary murine aortic endothelial cells were activated with interleukin (IL)-1beta to express intercellular adhesion molecule-1 (ICAM-1) mRNA in the presence or absence of the physiological spontaneous NO-donor S-nitrosocysteine. Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyse the effects of NO on ICAM-1 mRNA expression and on the activity of transcription factors involved in ICAM-1 transcription. In addition, luciferase reporter gene activity of cytokine-activated cells transiently transfected with an ICAM-1 promoter-luciferase construct and cultured in the presence of the slow-releasing NO-donor DETA/NO was determined. NO at subtoxic concentrations decreases IL-1beta-induced endothelial ICAM-1 mRNA expression. This inhibition occurs at the transcriptional level, as NO affects IL-1b-induced ICAM-1 promoter activity in transiently transfected cells. Using gel-shift assays and double-stranded oligonucleotide consensus sequences of the known transcription factor binding sites of the ICAM-1 promoter, Sp1 and AP-1 were identified as transcriptional activators of IL-1beta-driven ICAM-1 expression. The DNA binding of both of these transcription factors to specific binding sites of the ICAM-1 promoter was decreased in MAEC exposed to NO. Our studies indicate that the anti-adhesive effect of NO concentrations equivalent to high-output NO synthesis is mediated, at least in part, by inhibition of ICAM-1 expression via a concerted action of NO on the redox-sensitive transcriptional activators Sp1 and AP-1. This molecular mechanism may contribute to the anti-inflammatory actions of NO synthesized by the inducible NO synthase.

Effect of desloratadine and loratadine on rhinovirus-induced intercellular adhesion molecule 1 upregulation and promoter activation in respiratory epithelial cells

Background: Rhinoviruses have been recently associated with the majority of asthma exacerbations for which current therapy is inadequate. Intercellular adhesion molecule 1 (ICAM-1) has a central role in airway inflammation in asthma, and it is the receptor for 90% of rhinoviruses. Rhinovirus infection of airway epithelium induces ICAM-1. Desloratadine and loratadine are compounds belonging to the new class of H1-receptor blockers. Anti-inflammatory properties of antihistamines have been recently documented, although the underlying molecular mechanisms are not completely defined. Objective: We have investigated the effects of desloratadine and loratadine on rhinovirus-induced ICAM-1 expression, mRNA upregulation, and promoter activation. Methods: Cultured primary bronchial or transformed (A549) respiratory epithelial cells were pretreated with desloratadine and loratadine for 16 hours and infected with rhinovirus type 16 for 8 hours. ICAM-1 surface expression was evaluated with flow cytometry, and ICAM-1 mRNA was evaluated with specific RT-PCR. In A549 cells promoter activation was evaluated with a chloramphenicol acetyltransferase assay, and binding activity of nuclear factor κB in nuclear extracts was evaluated with an electrophoretic mobility shift assay. Results: Desloratadine and loratadine (0.1-10 μmol/L) inhibited rhinovirus-induced ICAM-1 upregulation in both primary bronchial or transformed (A549) respiratory epithelial cells. In A549 cells the 2 compounds showed a dose-dependent inhibition with similar efficacy (inhibitory concentration of 50%, 1 μmol/L). Desloratadine and loratadine also inhibited ICAM-1 mRNA induction caused by rhinovirus infection in a dose-dependent manner, and they completely inhibited rhinovirus-induced ICAM-1 promoter activation. Desloratadine also inhibited rhinovirus-induced nuclear factor κB activation. Desloratadine and loratadine had no direct effect on rhinovirus infectivity and replication in cultured epithelial cells. Conclusion: These effects are unlikely to be mediated by H1-receptor antagonism and suggest a novel mechanism of action that may be important for the therapeutic control of virus-induced asthma exacerbations. (J Allergy Clin Immunol 2001;108:221-8.)

Tumor necrosis factor α-induced activation of downstream NF-κB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion: Involvement of PKCα, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-α induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-α-induced ICAM-1 expression. TNF-α produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-α-induced response. 12- O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-α resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-α-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C 2 ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-κB DNA–protein binding and ICAM-1 promoter activity were enhanced by TNF-α and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-κB DNA–protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-α- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCα or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-α and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-α activates PC-PLC to induce activation of PKCα and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-κB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.

Protein kinase C-zeta mediates TNF-alpha-induced ICAM-1 gene transcription in endothelial cells.

We addressed the role of protein kinase C (PKC) isozymes in mediating tumor necrosis factor-alpha (TNF-alpha)-induced oxidant generation in endothelial cells, a requirement for nuclear factor-kappaB (NF-kappaB) activation and intercellular adhesion molecule-1 (ICAM-1) gene transcription. Depletion of the conventional (c) and novel (n) PKC isozymes following 24 h exposure of human pulmonary artery endothelial (HPAE) cells with the phorbol ester, phorbol 12-myristate 13-acetate (500 nM), failed to prevent TNF-alpha-induced oxidant generation. In contrast, inhibition of PKC-zeta synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-alpha stimulation. We observed that PKC-zeta also induced the TNF-alpha-induced NF-kappaB binding to the ICAM-1 promoter and the resultant ICAM-1 gene transcription. We showed that expression of the dominant negative mutant of PKC-zeta prevented the TNF-alpha-induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-zeta augmented the response. These data imply a critical role for the PKC-zeta isozyme in regulating TNF-alpha-induced oxidant generation and in signaling the activation of NF-kappaB and ICAM-1 transcription in endothelial cells.

Corticosteroids inhibit rhinovirus-induced intercellular adhesion molecule-1 up-regulation and promoter activation on respiratory epithelial cells

Background: Rhinoviruses are associated with the majority of asthma exacerbations. To date, the pathogenesis of virus-induced asthma exacerbations is still unclear, and no safe effective therapy is available. Intercellular adhesion molecule-1 (ICAM-1) has a central role in inflammatory cell recruitment to the airways in asthma and is the receptor for 90% of rhinoviruses. We have previously shown that rhinovirus infection of lower airway epithelium induces ICAM-1 expression by a transcriptional mechanism that is critically nuclear factor-κB-dependent. Objective: The purpose of this study was to investigate the effect of systemic (hydrocortisone [HC], dexamethasone [DM]) and topical (mometasone furoate [MF]) corticosteroids on rhinovirus-induced ICAM-1 up-regulation. Methods: Cultured primary bronchial or transformed (A549) respiratory epithelial cells were pretreated with corticosteroids for 16 hours and infected with rhinovirus type 16 for 8 hours. ICAM-1 surface expression was evaluated by flow cytometry. In A549 cells ICAM-1 messenger RNA was evaluated by specific reverse transcription–PCR and promoter activation by chloramphenicol acetyltransferase assay. Results: We observed inhibition of rhinovirus-induced ICAM-1 up-regulation with corticosteroid pretreatment in both primary bronchial epithelial and A549 cells. In A549 cells systemic and topical corticosteroids demonstrated a dose-dependent inhibition with similar efficacy (inhibitory concentration 50% 10–10 mol/L, 10–11 mol/L, and 10–11 mol/L for HC, DM, and MF respectively). MF also inhibited ICAM-1 messenger RNA induction by rhinovirus infection in a dose-dependent manner. MF completely inhibited rhinovirus-induced ICAM-1 promoter activation. HC, DM, and MF had no direct effect on rhinovirus infectivity and replication in cultured cells. Conclusion: Corticosteroids decrease rhinovirus-induced ICAM-1 up-regulation in respiratory epithelial cells and modulate pretranscriptional mechanisms. This effect may be important for the therapeutic control of virus-induced asthma exacerbations. (J Allergy Clin Immunol 2000;105:318-26.)

Rhinovirus Infection Induces Expression of Its Own Receptor Intercellular Adhesion Molecule 1 (ICAM-1) via Increased NF-κB-mediated Transcription

Virus infections, the majority of which are rhinovirus infections, are the major cause of asthma exacerbations. Treatment is unsatisfactory, and the pathogenesis unclear. Lower airway lymphocyte and eosinophil recruitment and activation are strongly implicated, but the mechanisms regulating these processes are unknown. Intercellular adhesion molecule-1 (ICAM-1) has a central role in inflammatory cell recruitment to the airways in asthma and is the cellular receptor for 90% of rhinoviruses. We hypothesized that rhinovirus infection of lower airway epithelium might induce ICAM-1 expression, promoting both inflammatory cell infiltration and rhinovirus infection. We therefore investigated the effect of rhinovirus infection on respiratory epithelial cell ICAM-1 expression and regulation to identify new targets for treatment of virus-induced asthma exacerbations. We observed that rhinovirus infection of primary bronchial epithelial cells and the A549 respiratory epithelial cell line increased ICAM-1 cell surface expression over 12- and 3-fold, respectively. We then investigated the mechanisms of this induction in A549 cells and observed rhinovirus-induction of ICAM-1 promoter activity and ICAM-1 mRNA transcription. Rhinovirus induction of ICAM-1 promoter activity was critically dependent upon up-regulation of NF-kappaB proteins binding to the -187/-178 NF-kappaB binding site on the ICAM-1 promoter. The principal components of the rhinovirus-induced binding proteins were NF-kappaB p65 homo- or heterodimers. These studies identify ICAM-1 and NF-kappaB as new targets for the development of therapeutic interventions for virus-induced asthma exacerbations.

Specificities of CD40 signaling: involvement of TRAF2 in CD40-induced NF-kappaB activation and intercellular adhesion molecule-1 up-regulation.

Several tumor necrosis factor receptor-associated factor (TRAF) family proteins including TRAF2, TRAF3, TRAF5, and TRAF6, as well as Jak3, have been implicated as potential mediators of CD40 signaling. An extensive in vitro binding study indicated that TRAF2 and TRAF3 bind to the CD40 cytoplasmic tail (CD40ct) with much higher affinity than TRAF5 and TRAF6 and that TRAF2 and TRAF3 bind to different residues of the CD40ct. Using CD40 mutants incapable of binding TRAF2, TRAF3, or Jak3, we found that the TRAF2-binding site of the CD40ct is critical for NF-kappaB and stress-activated protein kinase activation, as well as the up-regulation of the intercellular adhesion molecule-1 (ICAM-1) gene, whereas binding of TRAF3 and Jak3 is dispensable for all of these functions. Overexpression of a dominantly active IkappaBalpha strongly inhibited CD40-induced NF-kappaB activation, ICAM-1 promoter activity, and cell-surface ICAM-1 up-regulation. These studies suggest a potential signal transduction pathway from the CD40 receptor to the transcriptional activation of the ICAM-1 gene.

Activation of ICAM-1 promoter by lysophosphatidylcholine: Possible involvement of protein tyrosine kinases

Lysophosphatidylcholine (lyso-PC) selectively upregulates the mRNA level of intercellular adhesion molecule-1 (ICAM-1) but not that of vascular cell adhesion molecule-1 (VCAM-1) in cultured human umbilical vein endothelial cells. Transfection studies show that lyso-PC activates the ICAM-1 promoter but not the VCAM-1 promoter. Gel mobility shift assays document an increase in NF- κB binding in cells treated with lyso-PC. The increases of ICAM-1 mRNA and NF- κB binding were inhibited by the protein tyrosine kinase inhibitors, genistein and lavendustin A, but not by inhibitors for cyclic AMP-dependent protein kinases or protein kinase C. Our results suggest that lyso-PC induces ICAM-1 expression most likely by activating NF- κB, and that the effect appears to be protein tyrosine kinase-dependent.

Hormonal regulation of ICAM-1 gene expression in thyroid cells, FRTL-5

Our previous works have shown that human thyroid follicular cells from Graves’ disease and FRTL-5 rat thyroid cells express the intercellular adhesion molecule-1 (ICAM-1) molecule and its expression is upregulated by several cytokines, interferon-γ, tumor necrosis factor-α, interleukin-1β and interleukin-6. We used FRTL-5 cells which show hormonal dependence of growth and function for the study of hormonal regulation of ICAM-1 gene. We studied ICAM-1 mRNA expression and promoter regulation after cloning of rat ICAM-1 promoter. We found very interesting findings that thyroid stimulating hormone (TSH) and forskolin downregulates steady state MHC class I and ICAM-1 mRNA levels in FRTL-5 cells; furthermore, TSH/cAMP inhibit cytokines (interferon-γ, tumor necrosis factor-α)-mediated maximal ICAM-1 mRNA expression. In addition, hydrocortisone and insulin differentially regulate the ICAM-1 mRNA levels; hydrocortisone markedly suppresses the mRNA level but insulin partially recovers hydrocortisone mediated ICAM-1 suppression. The interferon-γ and tumor necrosis factor-α increases full ICAM-1 promoter (pCAM-1822) activity and this cytokine mediated increase of the promoter activity is also inhibited by TSH and forskolin. Thus TSH/cAMP pathways play roles as a antagonistic action for maximal expression of ICAM-1 gene by these cytokines. We propose this TSH action is one of physiologic mechanisms to preserve self tolerance in face of abnormal cytokine challenges in systemic inflammatory condition or acute phase response.