Nox4 mediates the expression of plasminogen activator inhibitor-1 via p38 MAPK pathway in cultured human endothelial cells.

INTRODUCTION: Plasminogen Activator Inhibitor-1 (PAI-1) is the most potent endogenous inhibitor of fibrinolysis which is implicated in the pathogenesis of myocardial infarction and metabolic syndrome. The formation of reactive oxygen species (ROS) plays an important role in the pathology of vascular disorders and has been shown to increase PAI-1 expression by endothelial cells. Growing evidence indicates that NADPH oxidase and in particular the constitutively active Nox4-p22(phox) complexes are major sources of ROS in endothelial cells. The aim of the present study was to characterize the role of NADPH oxidase and in particular Nox4 in the regulation of PAI-1 expression in cultured Human Umbilical Venous Endothelial Cells (HUVECs). METHODS AND RESULTS: N-acetylcysteine (NAC, scavenger of ROS), diphenylene iodonium chloride (DPI, inhibitor of flavoproteins), M40403 (superoxyde dismutase mimic) and S17834 (inhibitor of NADPH oxidase) inhibited PAI-1 release and promoter activity in HUVECs. Specific knock down of Nox4 mRNA by siRNA caused a decrease in ROS production and NADPH oxidase activity. Moreover, Nox4 silencing decreased PAI-1 expression, release and activity as well as p38 MAPK pathways and NFkappaB activation. These signalling pathways are also involved in PAI-1 release. CONCLUSIONS: The NADPH oxidase inhibitors DPI and S 17834 as well as Nox4 silencing decreased PAI-1 synthesis in human cultured endothelial cells demonstrating the involvement of the constitutively active Nox4-containing NADPH oxidase in ROS-mediated PAI-1 transcription via p38 MAPK pathways. NADPH oxidase targeting with inhibitors such as S17834 could be an interesting strategy to decrease both oxidative stress and PAI-1 synthesis.

High-mobility group box 1 protein induces HIV-1 expression from persistently infected cells.

BACKGROUND: Necrosis is a frequent condition during AIDS, notably in organs targetted by opportunistic infections. Soluble factors released by necrotic cells are important for signalling cell damage, but little is known concerning their effect on HIV-1 replication. We focused on HMGB1, an abundant component of the chromatin that is released from necrotic cells and can act as a pro-inflammatory mediator. MATERIALS AND METHODS: A native form of HMGB1 was obtained from necrotic Hela cells, whereas a purified recombinant HMGB1 was generated in Escherichia coli. ACH-2 and U1 cells were used as models of persistent HIV-1 infection in lymphocytes and monocytes. Reactivation from latency was also investigated ex vivo using peripheral blood mononuclear cells (PBMC) collected from HIV-1-infected patients controlled by HAART. HIV-1 expression was quantified by enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction and branched DNA techniques. Flow cytometry and blocking experiments were used to identify the receptor used by HMGB1. Chromatin immunoprecipitation was used to investigate long-terminal repeat activation upon stimulation by HMGB1. RESULTS: HMGB1 increased HIV-1 transcription in chronically infected cells, a process that did not require de-novo protein synthesis. HIV-1 induction relied on HMGB1 interaction with the receptor for advanced glycation end-products. The activation pathway involved p38 and extracellular signal-related kinase as well as nuclear factor kappa B binding to the HIV-1 promoter. Finally, HMGB1 reactivated HIV-1 from latently infected PBMC collected in aviraemic HIV-infected patients. CONCLUSION: This work establishes for the first time a link between necrosis and HIV-1 replication, which involves HMGB1, a soluble mediator released by damaged cells.

Activation of sPLA2-IIA and PGE2 production by high mobility group protein B1 in vascular smooth muscle cells sensitized by IL-1beta.

Lipid mediators such as prostaglandin E2 (PGE2) play a central role during atherogenesis as a consequence of inflammation. PGE2 is produced from phospholipids by a cascade of enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E synthase (PGES). It is released by several cell types, including vascular smooth muscle cells (VSMCs). Recent work has shown that the secretory PLA2-IIA (sPLA2-IIA), the most abundant isoform of secreted PLA2 in VSMCs, acts as a potent cytokine and activates VSMCs through a positive feedback loop. High mobility group protein 1 (HMGB1), also known as amphoterin, is a ubiquitous protein that plays various roles in the nucleus. HMGB1 is released by necrotic cells and by immune cells in response to various inflammatory mediators and acts as a potent proinflammatory cytokine. The present study investigates the role of HMGB1 in the activation of sPLA2-IIA expression and PGE2 production in VSMCs. Recombinant HMGB1 slightly activated the sPLA2-IIA, COX-2, and mPGES-1 genes but dramatically stimulated these genes in VSMCs that had been incubated with the proinflammatory cytokine IL-1beta for 24 h. This effect was accompanied by significantly increased PGE2 release. Induction of the three known receptors of HMGB1, namely RAGE, TLR-2, and TLR-4, by IL-1beta suggests that proinflammatory cytokines sensitize VSMCs to HMGB1. This provides new insights into the role of HMGB1 in VSMCs, suggesting it may be essential for the progression of atherosclerosis.

Cell-specific posttranscriptional regulation of CFTR gene expression via influence of MAPK cascades on 3'UTR part of transcripts.

Expression of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, which contains the mutations responsible for CF, is regulated by cytokines (TNF-alpha and IL-1beta) in a cell-specific manner. TNF-alpha decreases CFTR mRNA in human colon cell lines (HT-29), but not in pulmonary cell lines (Calu-3), and IL-1beta increases it only in Calu-3 cells. We looked for the cytokine-induced posttranscriptional regulation of CFTR gene expression and studied the modulation of CFTR mRNA stability linked to its 3' untranslated sequence (3'UTR) in HT-29 and Calu-3 cells. The stability of CFTR mRNA was analyzed by Northern blot after in vitro incubation of total RNAs from CFTR-expressing cells with cytosolic proteins extracted from control or cytokine-treated HT-29 and Calu-3 cells. CFTR mRNA was degraded only by extracts of TNF-alpha-treated HT-29 cells and not by cytosolic proteins from untreated or IL-1beta-treated HT-29 cells. In contrast, extracts of untreated Calu-3 cells enhanced CFTR mRNA degradation, and IL-1beta treatment inhibited this; TNF-alpha had no significant effect. The 3'UTR part of CFTR mRNA was found to be required for this posttranscriptional regulation. The 5' part of the 3'UTR (the 217 first bases), which contains two AUUUA sequences, was implicated in CFTR mRNA destabilization and the following 136 bases, containing several C-repeats in U-rich environment, in its protection. The proteins, which reacted with the U- and C-repeats of CFTR mRNA 3'UTR, were mainly controlled by stimulation of the p42/p44 and p38 MAP kinase cascades with interaction between these pathways. This posttranscriptional control of gene expression is a common feature of CFTR and many proteins of inflammation.

Autocrine and paracrine transcriptional regulation of type IIA secretory phospholipase A2 gene in vascular smooth muscle cells.

OBJECTIVE: The inflammation that occurs during the development of atherosclerosis is characterized by a massive release of sPLA2-IIA (group IIA secretory phospholipase A2) from vascular smooth muscle cells (VSMCs). We have investigated the autocrine function of sPLA2-IIA in rat aortic and human VSMCs. METHODS AND RESULTS: We found that the transcription of the endogenous sPLA2-IIA gene increased by adding a cell supernatant containing human sPLA2-IIA proteins. We show that this effect was independent of the sPLA2 activity using sPLA2-IIA proteins lacking enzyme activity. Transient transfections with various sPLA2-IIA rat promoter-luciferase constructs demonstrated that the C/EBP, NK-kappaB, and Ets transcription factors are involved in the increase in sPLA2-IIA gene transcription. We also found the M-type sPLA2 receptor mRNA in VSMCs, and we showed that the sPLA2-luciferase reporter gene was induced by the specific agonist of the sPLA2 receptor, aminophenylmannopyranoside (APMP), and that this induction was mediated by the same transcription factor-binding sites. Finally, we used a sPLA2-IIA mutant unable to bind heparan-sulfate proteoglycans to show that the binding of wild-type sPLA2-IIA to proteoglycans is essential for the induction of an autocrine loop. CONCLUSIONS: We have thus identified new autocrine and paracrine pathways activating sPLA2-IIA gene expression in rat and human VSMCs.