Tumor necrosis factor-alpha induction of endothelial ephrin A1 expression is mediated by a p38 MAPK- and SAPK/JNK-dependent but nuclear factor-kappa B-independent mechanism

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that induces a broad spectrum of responses including angiogenesis. Angiogenesis promoted by TNF-alpha is mediated, at least in part, by ephrin A1, a member of the ligand family for Eph receptor tyrosine kinases. Although TNF-alpha induces ephrin A1 expression in endothelial cells, the signaling pathways mediating ephrin A1 induction remain unknown. In this study, we investigated the signaling mechanisms of TNF-alpha-dependent induction of ephrin A1 in endothelial cells. Both TNFR1 and TNFR2 appear to be involved in regulating ephrin A1 expression in endothelial cells, because neutralizing antibodies to either TNFR1 or TNFR2 inhibited TNF-alpha-induced ephrin A1 expression. Inhibition of nuclear factor-kappaB (NF-kappaB) activation by a trans-dominant inhibitory isoform of mutant IkappaBalpha did not affect ephrin A1 induction, suggesting that NF-kappaB proteins are not major regulators of ephrin A1 expression. In contrast, ephrin A1 induction was blocked by inhibition of p38 mitogen-activated protein kinase (MAPK) or SAPK/JNK, but not p42/44 MAPK, using either selective chemical inhibitors or dominant-negative forms of p38 MAPK or TNF receptor-associated factor 2. These findings indicate that TNF-alpha-induced ephrin A1 expression is mediated through JNK and p38 MAPK signaling pathways. Taken together, the results of our study demonstrated that induction of ephrin A1 in endothelial cells by TNF-alpha is mediated through both p38 MAPK and SAPK/JNK, but not p42/44 MAPK or NF-kappaB, pathways.     

NF-kappaB activation is required for human endothelial survival during exposure to tumor necrosis factor-alpha but not to interleukin-1beta or lipopolysaccharide.

In the presence of a protein synthesis inhibitor, cycloheximide, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), or lipopolysaccharide (LPS) induces human umbilical vein endothelial cells (HUVECs) to undergo apoptosis, suggesting that constitutive or inducible cytoprotective pathways are required for cell survival. We studied the correlation between nuclear factor-kappaB (NF-kappaB) activation and cell death induced by TNF-alpha, IL-1beta, or LPS. Adenovirus-mediated overexpression of a dominant-negative IkappaBalpha (inhibitor of kappaB) mutant blocked NF-kappaB activation by gel shift assay and blocked induction of vascular cell adhesion molecule-1 protein by TNF-alpha, IL-1beta, and LPS, a NF-kappaB-dependent response. In cells overexpressing the IkappaBalpha mutant, TNF-alpha induced cell death, whereas IL-1beta or LPS did not. We conclude that cell survival following TNF-alpha stimulation is NF-kappaB-dependent but that a constitutive or inducible NF-kappaB-independent pathway(s) protects IL-1beta- or LPS-treated HUVECs from cell death.     

A novel role of the interferon-inducible protein IFI16 as inducer of proinflammatory molecules in endothelial cells

The human IFI16 gene is an interferon-inducible gene implicated in the regulation of endothelial cell proliferation and tube morphogenesis. Immunohistochemical analysis has demonstrated that this gene is highly expressed in endothelial cells in addition to hematopoietic tissues. In this study, gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 revealed an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. Consistent with these observations, IFI16 triggered expression of adhesion molecules such as ICAM-1 and E-selectin or chemokines such as interleukin-8 or MCP-1. Treatment of cells with short hairpin RNA targeting IFI16 significantly inhibited ICAM-1 induction by interferon (IFN)-gamma demonstrating that IFI16 is required for proinflammatory gene stimulation. Moreover, functional analysis of the ICAM-1 promoter by deletion- or site-specific mutation demonstrated that NF-kappaB is the main mediator of IFI16-driven gene induction. NF-kappaB activation appears to be triggered by IFI16 through a novel mechanism involving suppression of IkappaBalpha mRNA and protein expression. Support for this finding comes from the observation that IFI16 targeting with specific short hairpin RNA down-regulates NF-kappaB binding activity to its cognate DNA and inhibits ICAM-1 expression induced by IFN-gamma. Using transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrate indeed that activation of the NF-kappaB response is mediated by IFI16-induced block of Sp1-like factor recruitment to the promoter of the IkappaBalpha gene, encoding the main NF-kappaB inhibitor. Activation of NF-kappaB accompanied by induction of proinflammatory molecules was also observed when IkappaBalpha expression was down-regulated by specific small interfering RNA, resulting in an outcome similar to that observed with IFI16 overexpression. Taken together, these data implicate IFI16 as a novel regulator of endothelial proinflammatory activity and provide new insights into the physiological functions of the IFN-inducible gene IFI16.     

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

High concentration of antioxidants N-acetylcysteine and mitoquinone-Q induces intercellular adhesion molecule 1 and oxidative stress by increasing intracellular glutathione

In endothelial cells, the intracellular level of glutathione is depleted during offering protection against proinflammatory cytokine TNF-alpha-induced oxidative stress. Administration of anti-inflammatory drugs, i.e., N-acetylcysteine (NAC) or mitoquinone-Q (mito-Q) in low concentrations in the human pulmonary aortic endothelial cells offered protection against depletion of reduced glutathione and oxidative stress mediated by TNF-alpha. However, this study addressed that administration of NAC or mito-Q in high concentrations resulted in a biphasic response by initiating an enhanced generation of both reduced glutathione and oxidized glutathione and enhanced production of reactive oxygen species, along with carbonylation and glutathionylation of the cellular proteins. This study further addressed that IkappaB kinase (IKK), a phosphorylation-dependent regulator of NF-kappaB, plays an important regulatory role in the TNF-alpha-mediated induction of the inflammatory cell surface molecule ICAM-1. Of the two catalytic subunits of IKK (IKKalpha and IKKbeta), low concentrations of NAC and mito-Q activated IKKalpha activity, thereby inhibiting the downstream NF-kappaB and ICAM-1 induction by TNF-alpha. High concentrations of NAC and mito-Q instead caused glutathionylation of IKKalpha, thereby inhibiting its activity that in turn enhanced the downstream NF-kappaB activation and ICAM-1 expression by TNF-alpha. Thus, establishing IKKalpha as an anti-inflammatory molecule in endothelial cells is another focus of this study. This is the first report that describes a stressful situation in the endothelial cells created by excess of antioxidative and anti-inflammatory agents NAC and mito-Q, resulting in the generation of reactive oxygen species, carbonylation and glutathionylation of cellular proteins, inhibition of IKKalpha activity, and up-regulation of ICAM-1expression.     

Identification of promoter elements responsible for transcriptional inhibition of polo-like kinase 1 and topoisomerase IIalpha genes by p21(WAF1/CIP1/SDI1)

Induction of p21 (WAF1/CIP1/SDI1), a physiological mediator of cell cycle arrest, inhibits multiple genes involved in cell division. We have investigated the determinants of p21- mediated inhibition of two of these genes, polo-like kinase 1 (PLK1) and topoisomerase IIalpha (TOPO IIalpha) p21 expression from an inducible promoter in human HT1080 cells rapidly decreases cellular levels of PLK1 and TOPO IIalpha promoters in transient and stable transfection assays. Promoter mutagenesis studies show that inhibition of the PLK1 promoter by p21 is mediated in part by tandem sequences CDE (cell cycle-dependent element) and CHR (cell cycle genes homology region). p21 response of the TOPO IIalpha promoter inhibition and the effects of promoter mutations differ under the conditions of growth arrest produced by p21 induction or by mimosine, a cell cycle inhibitor that increases p21 RNA but not protein expression in HT1080 cells. These results indicate that inhibition of cell division-associated genes by p21 is mediated by different but overlapping mechanisms, which are not a general con-sequence of cell cycle arrest.     

Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.     

Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.