pubmed > NFKB > ap-1

CD40 Cross-Linking Induces Migration of Renal Tumor Cell through Nuclear Factor of Activated T Cells (NFAT) Activation.
CD40 crosslinking plays an important role in regulating cell migration, adhesion and proliferation in renal cell carcinoma (RCC). CD40/CD40L interaction on RCC cells activates different intracellular pathways but the molecular mechanisms leading to cell scattering are not yet clearly defined. Aim of our study was to investigate the main intracellular pathways activated by CD40 ligation and their specific involvement in RCC cell migration. CD40 ligation increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH (2)-terminal kinase (JNK) and p38 MAPK. Furthermore, CD40 crosslinking activated different transcriptional factors on RCC cell lines: AP-1, NFkB and some members of the Nuclear Factor of Activated T cells (NFAT) family. Interestingly, the specific inhibition of NFAT factors by cyclosporine A, completely blocked RCC cell motility induced by CD40 ligation. In tumor tissue, we observed a higher expression of NFAT factors and in particular an increased activation and nuclear migration of NFATc4 on RCC tumor tissues belonging to patients that developed metastases when compared to those who did not. Moreover, CD40-CD40L interaction induced a cytoskeleton reorganization and increased the expression of integrin β1 on RCC cell lines, and this effect was reversed by cyclosporine A and NFAT inhibition. These data suggest that CD40 ligation induces the activation of different intracellular signaling pathways, in particular the NFATs factors, that could represent a potential therapeutic target in the setting of patients with metastatic RCC.
Publication Date: 2021-08-28
Journal: International journal of molecular sciences

Monensin inhibits cell proliferation and tumor growth of chemo-resistant pancreatic cancer cells by targeting the EGFR signaling pathway.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies with <5% five-year survival rate due to late diagnosis, limited treatment options and chemoresistance. There is thus an urgent unmet clinical need to develop effective anticancer drugs to treat pancreatic cancer. Here, we study the potential of repurposing monensin as an anticancer drug for chemo-resistant pancreatic cancer. Using the two commonly-used chemo-resistant pancreatic cancer cell lines PANC-1 and MiaPaCa-2, we show that monensin suppresses cell proliferation and migration, and cell cycle progression, while solicits apoptosis in pancreatic cancer lines at a low micromole range. Moreover, monensin functions synergistically with gemcitabine or EGFR inhibitor erlotinib in suppressing cell growth and inducing cell death of pancreatic cancer cells. Mechanistically, monensin suppresses numerous cancer-associated pathways, such as E2F/DP1, STAT1/2, NFkB, AP-1, Elk-1/SRF, and represses EGFR expression in pancreatic cancer lines. Furthermore, the in vivo study shows that monensin blunts PDAC xenograft tumor growth by suppressing cell proliferation via targeting EGFR pathway. Therefore, our findings demonstrate that monensin can be repurposed as an effective anti-pancreatic cancer drug even though more investigations are needed to validate its safety and anticancer efficacy in pre-clinical and clinical models.
Publication Date: 2018-12-19
Journal: Scientific reports

KPNB1-mediated nuclear import is required for motility and inflammatory transcription factor activity in cervical cancer cells.
Karyopherin β1 is a nuclear import protein involved in the transport of proteins containing a nuclear localisation sequence. Elevated Karyopherin β1 expression has been reported in cancer and transformed cells and is essential for cancer cell proliferation and survival. Transcription factors such as NFĸB and AP-1 contain a nuclear localisation sequence and initiate the expression of multiple factors associated with inflammation and cancer cell biology. Our study investigated the effect of inhibiting nuclear import via Karyopherin β1 on cancer cell motility and inflammatory signaling using siRNA and the novel small molecule, Inhibitor of Nuclear Import-43, INI-43. Inhibition of Karyopherin β1 led to reduced migration and invasion of cervical cancer cells. Karyopherin β1 is essential for the translocation of NFĸB into the nucleus as nuclear import inhibition caused its cytoplasmic retention and decreased transcriptional activity. A similar decrease was seen in AP-1 transcriptional activity upon Karyopherin β1 inhibition. Consequently reduced interleukin-6, interleukin-1 beta, tumour necrosis factor alpha and granulocyte macrophage colony stimulating factor expression, target genes of NFkB and AP-1, was observed. Migration studies inhibiting individual transcription factors suggested that INI-43 may affect a combination of signaling events. Our study provides further evidence that inhibiting KPNB1 has anti-cancer effects and shows promise as a chemotherapeutic target.
Publication Date: 2017-04-22
Journal: Oncotarget

Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction.
Bladder outlet obstruction (BOO) induces significant organ remodeling, leading to lower urinary tract symptoms accompanied by urodynamic changes in bladder function. Here, we report mRNA and miRNA transcriptome sequencing of bladder samples from human patients with different urodynamically defined states of BOO. Patients' miRNA and mRNA expression profiles correlated with urodynamic findings. Validation of RNA sequencing results in an independent patient cohort identified combinations of 3 mRNAs (NRXN3, BMP7, UPK1A) and 3 miRNAs (miR-103a-3p, miR-10a-5p, miR-199a-3p) sufficient to discriminate between bladder functional states. All BOO patients shared cytokine and immune response pathways, TGF-β and NO signaling pathways, and hypertrophic PI3K/AKT signaling pathways. AP-1 and NFkB were dominant transcription factors, and TNF-α was the top upstream regulator. Integrated miRNA-mRNA expression analysis identified pathways and molecules targeted by differentially expressed miRNAs. Molecular changes in BOO suggest an increasing involvement of miRNAs in the control of bladder function from the overactive to underactive/acontractile states.
Publication Date: 2017-02-01
Journal: JCI insight

Tweak up-regulates endothelin-1 system in mouse and human endothelial cells.
To analyse the ability of TWEAK to modify the endothelin system, particularly endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1), studying the intracellular mechanisms implied. TNF-like weak inducer of apoptosis (TWEAK) is a member of TNF superfamily; it has different biological functions such as inflammation, angiogenesis, proliferation, and apoptosis. TWEAK and fibroblast growth-factor-inducible 14 are expressed in different cell types, including endothelial and smooth muscle cells. Despite their presence in endothelial cells, the effect of TWEAK on endothelial function is incompletely defined. In cells, TWEAK induced protein (Western blot) and mRNA (quantitative polymerase chain reaction) expression of ECE-1. Results were related to transcriptional changes, as ECE-1 promoter activity (transfection assays) was also increased. Transfections with serial deletions of ECE-1 promoter suggest a potential role for AP-1 and NFkB, which were confirmed by electrophoretic mobility shift assays. When AP-1 or NFkB activations were inhibited by specific inhibitors of AP-1, PD-98059 (Erk1/2 inhibitor), or SP-600125 (JNK inhibitor), and also with an inhibitor of NFKB and PDTC, TWEAK effect was partially blocked in both cases, suggesting that both transcription factors are implied in ECE-1 regulation. Moreover, the endothelial changes induced by TWEAK were also tested in vivo, using 3-month-old male CD-1 mice treated with TWEAK 10 µg/kg body weight for 24 h, finding similar effects, a rise in ET-1 production (enzyme-linked immunosorbent assay), and ECE-1 expression in aorta and lung tissues. Mice showed slight hypertension after 4 h of treatment, which disappeared at 24 h. In pathological situations such as chronic inflammation, TWEAK could be more harmful through this effect at endothelial level. Pharmacological blockade of this cytokine could prevent the haemodynamic and structural changes related to an increased ET-1 synthesis.
Publication Date: 2016-12-28
Journal: Cardiovascular research

A Blockade of IGF Signaling Sensitizes Human Ovarian Cancer Cells to the Anthelmintic Niclosamide-Induced Anti-Proliferative and Anticancer Activities.
Ovarian cancer is the most lethal gynecologic malignancy, and there is an unmet clinical need to develop new therapies. Although showing promising anticancer activity, Niclosamide may not be used as a monotherapy. We seek to investigate whether inhibiting IGF signaling potentiates Niclosamide's anticancer efficacy in human ovarian cancer cells. Cell proliferation and migration are assessed. Cell cycle progression and apoptosis are analyzed by flow cytometry. Inhibition of IGF signaling is accomplished by adenovirus-mediated expression of siRNAs targeting IGF-1R. Cancer-associated pathways are assessed using pathway-specific reporters. Subcutaneous xenograft model is used to determine anticancer activity. We find that Niclosamide is highly effective on inhibiting cell proliferation, cell migration, and cell cycle progression, and inducing apoptosis in human ovarian cancer cells, possibly by targeting multiple signaling pathways involved in ELK1/SRF, AP-1, MYC/MAX and NFkB. Silencing IGF-1R exert a similar but weaker effect than that of Niclosamide's. However, silencing IGF-1R significantly sensitizes ovarian cancer cells to Niclosamide-induced anti-proliferative and anticancer activities both in vitro and in vivo. Niclosamide as a repurposed anticancer agent may be more efficacious when combined with agents that target other signaling pathways such as IGF signaling in the treatment of human cancers including ovarian cancer.
Publication Date: 2016-08-09
Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Increased sphingosine 1-phosphate mediates inflammation and fibrosis in tubular injury in diabetic nephropathy.
Hyperglycemia induces all isoforms of transforming growth factor β (TGFβ), which in turn play key roles in inflammation and fibrosis that characterize diabetic nephropathy. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid, derived from sphingosine by the action of sphingosine kinase (SK). S1P mediates many biological processes, which mimic TGFβ signaling. To determine the role of SK1 and S1P in inducing fibrosis and inflammation, and the interaction with TGFβ-1, 2 and 3 signalling in diabetic nephropathy, human proximal tubular cells (HK2 cells) were exposed to normal (5 mmol/L) or high (30 mmol/L) glucose or TGFβ-1, -2, -3 ± an SK inhibitor (SKI-II) or SK1 siRNA. Control and diabetic wild type (WT) and SK1(-/-) mice were studied. Fibrotic and inflammatory markers, and relevant downstream signalling pathways were assessed. SK1 mRNA and protein expression was increased in HK2 cells exposed to high glucose or TGFβ1,-2,-3. All TGFβ isoforms induced fibronectin, collagen IV and macrophage chemoattractant protein 1 (MCP1), which were reversed by both SKI-II and SK1 siRNA. Exposure to S1P increased phospho-p44/42 expression, AP-1 binding and NFkB phosphorylation. WT diabetic mice exhibited increased renal cortical S1P, fibronectin, collagen IV and MCP1 mRNA and protein expression compared to SK1(-/-) diabetic mice. In summary, this study demonstrates that inhibiting the formation of S1P reduces tubulointerstitial renal inflammation and fibrosis in diabetic nephropathy.
Publication Date: 2015-09-29
Journal: Clinical and experimental pharmacology & physiology

The Oncogenic Properties Of The Redox Inflammatory Protein Inducible Nitric Oxide Synthase In ER(-) Breast Cancer.
Inflammation generates reactive chemical species that induce conditions of oxidative nitrosative stress as emerged as factor in poor outcome of many cancers. Our recent findings show that in the inflammatory protein inducible nitric oxide synthase (iNOS) is a strong predictor of poor outcome in ER(-) patients (Glynn et al. JCI 2010). Furthermore 46 genes, of which 23 were associated with basal like breast cancer, were elevated when iNOS high. In vitro studies using ER(-) cell lines showed that fluxes of nitric oxide (NO) delivered by NO donors surprising mimic this relationship in the patient cohort. Using this model, we show that NO at different specific concentrations stimulate pro-oncogenic mechanisms such as AKT, ERK, NFkB, AP-1, and HIF-1α that lead to increase of metastatic and cancer stem cells proteins. In addition, we show that tumor suppressor gene BRCA1 and PP2A are inhibited by these NO levels. Similarly other studies show that these concentrations of NO increase immunosuppressive proteins TGF-β and IL-10 in leukocytes to decrease efficacy of some anticancer therapies further contributing to pro-tumorigenic environment. Using this model we have identified several new compounds that have efficacy in xenographic models. These finding have provided a model that shows how NO can affect numerous mechanism that leads to a more aggressive phenotype.
Publication Date: 2015-08-01
Journal: Redox biology

Adiponectin secreted by tubular renal cells during LPS exposure worsens the cellular inflammatory damage.
The pathogenetic role of adiponectin (ADPN) in kidney failure is not yet elucidated, since in vitro and in vivo studies have demonstrated that ADPN exerts both anti-inflammatory and pro-inflammatory effects. Starting from our previous findings demonstrating that HK-2 cells express and secrete ADPN, in this study we investigated the autocrine role of ADPN in tubular inflammatory damage induced by lipopolysaccharide (LPS) and the underlying molecular mechanisms. Firstly, we observed that short-term exposure to LPS enhanced ADPN protein expression as well as the adiponectin receptor ADIPOR1 mRNA content together with its signaling pathway downstream, pAMPK/pERK/pJNK, whose up-regulation status was reversed when ADPN gene knockdown occurred. Interestingly, in the same experimental conditions, we observed that ADPN mediated the nuclear translocation of the transcription factors nuclear factor kappa B (NFkB) and pcFos/pcJun (activator protein 1, AP-1), both induced by the pJNK pathway and involved in tumor necrosis factor (TNF)-α transactivation. Indeed, by transient transfection assay, we observed that the LPS-induced increase of TNF-α promoter activity was abrogated in cells pretreated with the inhibitors of NFkB and AP-1. Collectively our results suggest that in HK-2 cells, ADPN produced upon LPS stimulus could worsen the inflammatory damage in an autocrine-dependent manner.
Publication Date: 2015-07-29
Journal: Journal of nephrology

Activator protein 1 is a key terminal mediator of inflammation-induced preterm labor in mice.
Activation of uterine inflammatory pathways leads to preterm labor (PTL), associated with high rates of neonatal mortality and morbidity. The transcription factors nuclear factor κB (NFκB) and activator protein 1 (AP-1) regulate key proinflammatory and procontractile genes involved in normal labor and PTL. Here we show that NFκB activation normally occurs in the mouse myometrium at gestation day E18, prior to labor, whereas AP-1 and JNK activation occurs at labor onset. Where labor was induced using the progesterone receptor antagonist RU486, NFkB and AP-1/JNK activation both occurred at the time of labor (20 h compared to 60 h in DMSO-treated controls). Using an LPS (Escherichia coli: serotype O111)-induced PTL model that selectively activates AP-1 but not NFkB, we show that myometrial AP-1 activation drives production of cytokines (Il-6, Il-8, and Il-1β), metalloproteinases (Mmp3 and Mmp10), and procontractile proteins (Cox-2 and Cx43) resulting in PTL after 7 h. Protein levels of CX43 and IL-1β, and IL-1β cleavage, were increased following LPS-induced activation of AP-1. Inhibition of JNK by SP600125 (30 mg/kg) delayed PTL by 6 h (7.5 vs. 13.5 h P<0.05). Our data reveal that NFκB activation is not a functional requirement for infection/inflammation-induced preterm labor and that AP-1 activation is sufficient to drive inflammatory pathways that cause PTL.
Publication Date: 2014-02-06
Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Postprandial VLDL lipolysis products increase monocyte adhesion and lipid droplet formation via activation of ERK2 and NFκB.
Postprandial lipemia is characterized by a transient increase in circulating triglyceride-rich lipoproteins such as very low-density lipoprotein (VLDL) and has been shown to activate monocytes in vivo. Lipolysis of VLDL releases remnant particles, phospholipids, monoglycerides, diglycerides, and fatty acids in close proximity to endothelial cells and monocytes. We hypothesized that postprandial VLDL lipolysis products could activate and recruit monocytes by increasing monocyte expression of proinflammatory cytokines and adhesion molecules, and that such activation is related to the development of lipid droplets. Freshly isolated human monocytes were treated with VLDL lipolysis products (2.28 mmol/l triglycerides + 2 U/ml lipoprotein lipase), and monocyte adhesion to a primed endothelial monolayer was observed using a parallel plate flow chamber coupled with a CCD camera. Treated monocytes showed more rolling and adhesion than controls, and an increase in transmigration between endothelial cells. The increased adhesive events were related to elevated expression of key integrin complexes including Mac-1 [α(m)-integrin (CD11b)/β2-integrin (CD18)], CR4 [α(x)-integrin (CD11c)/CD18] and VLA-4 [α4-integrin (CD49d)/β1-integrin (CD29)] on treated monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) and THP-1 monocytes with VLDL lipolysis products increased expression of TNFα, IL-1β, and IL-8 over controls, with concurrent activation of NFkB and AP-1. NFκB and AP-1-induced cytokine and integrin expression was dependent on ERK and Akt phosphorylation. Additionally, fatty acids from VLDL lipolysis products induced ERK2-dependent lipid droplet formation in monocytes, suggesting a link to inflammatory signaling pathways. These results provide novel mechanisms for postprandial monocyte activation by VLDL lipolysis products, suggesting new pathways and biomarkers for chronic, intermittent vascular injury.
Publication Date: 2013-10-29
Journal: American journal of physiology. Heart and circulatory physiology

A comprehensive metabolic modeling of thyroid pathway in relation to thyroid pathophysiology and therapeutics.
The thyroid pathway represents a complex interaction of different glands for thyroid hormone synthesis. Thyrotropin releasing hormone is synthesized in the hypothalamus and regulates thyrotropin stimulating hormone gene expression in the pituitary gland. In order to understand the complexity of the thyroid pathways, and using experimental data retrieved from the biomedical literature (e.g., NCBI, HuGE Navigator, Protein Data Bank, and KEGG), we constructed a metabolic map of the thyroid hormone pathway at a molecular level and analyzed it topologically. A total of five hub nodes were predicted in regards to the transcription thyroid receptor (TR), cAMP response element-binding protein (CREB), signal transducer and activator of transcription 3 (STAT 3), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), and activator protein 1 (AP-1) as being potentially important in study of thyroid disorders and as novel putative therapeutic drug targets. Notably, the thyroid receptor is a highly connected hub node and currently used as a therapeutic target in hypothyroidism. Our analysis represents the first comprehensive description of the thyroid pathway, which pertains to understanding the function of the protein and gene interaction networks. The findings from this study are therefore informative for pathophysiology and rational therapeutics of thyroid disorders.
Publication Date: 2013-09-21
Journal: Omics : a journal of integrative biology

Effect of estrogen and tamoxifen on the expression pattern of AP-1 factors in MCF-7 cells: role of c-Jun, c-Fos, and Fra-1 in cell cycle regulation.
The activated transcription factor ERα plays an important role in the breast development and progression of cancer. In a non-classical pathway ER interacts with other transcription factors AP-1, NFkB, SP1, etc. AP-1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. AP-1 factors are leucine zipper proteins belonging to members of the Jun family (c-Jun, JunB, and JunD) and Fos family (c-Fos, FosB, Fra-1, and Fra-2) proteins. Although AP-1 factors are well characterized, not much is known about the expression pattern of the AP-1 factors in breast cancer cells. Hence to determine which AP-1 factors are expressed and regulated by estrogen, we used human breast cancer MCF-7 cells as in vitro model system. The MCF-7 cells were treated with or without estradiol-17β (E2) or antiestrogen tamoxifen (TMX) and the cell proliferation and viability was assessed by MTT assay. The expression of different AP-1 factors was analyzed by semi-quantitative RT-PCR. The cells treated with E2 found to increase the cell proliferation by more than 35 % and TMX an antiestrogen decreased by 29 % compared to control. The E2 found to induce the expression of c-Jun, Fra-1, and c-Fos, while TMX decreased the expression. In addition TMX also decreased the mRNA levels of Jun-D and Fra-2. These results suggest that the AP-1 factors c-Jun, c-Fos, and Fra-1 may be involved in the proliferation and transformation of MCF-7 cells. E2 also found to induce cyclin D1 and cyclin E1 mRNA transcripts of cell cycle regulators while TMX significantly decreased compared to control. Further E2 induced the anti-apoptotic Bcl-2 and TMX decreased mRNA transcripts. The data presented here support the E2-ERα-mediated MCF-7 cell proliferation and confirms the role of AP-1 factors in cell cycle regulation.
Publication Date: 2013-04-30
Journal: Molecular and cellular biochemistry

Activation of diverse eicosanoid pathways in osteoarthritic cartilage: a lipidomic and genomic analysis.
Non-steroidal anti-inflammatory drugs (NSAIDs) that are prescribed for treatment of osteoarthritis (OA) symptoms including pain and inflammation target the production eicosanoids which exhibit numerous functions in various cell types. In these studies, we have (a) identified the diverse eicosanoid pathways that are activated in human chondrocytes of normal and OA cartilage, (b) delineated the modulation of eicosanoids in the presence of NSAIDS and selective COX-2 inhibitors, and (c) characterized eicosanoid products and various transcripts modulated by various inhibitors of eicosanoids in human OA cartilage by gene expression arrays. Immunoassay analysis of culture supernatants were utilized to determine the spectrum of eicosanoids derived from both the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of normal and human OA cartilage in ex-vivo conditions. Human OA cartilage was incubated in ex-vivo conditions to examine spontaneous or IL-1 induced production of eicosanoids in the presence of various COX inhibitors. Gene expression analysis was performed to analyze the expression of mRNA in the presence and absence of COX-2 inhibitors in OA cartilage in ex-vivo conditions. Normal and OA human cartilage explants produced multiple eicosanoids of the COX and LOX pathways. PGF1α, PGF2α, PGE2 > TXB2, PGD2, and LTB4 were spontaneously generated by normal and OA cartilage. Among these, elevated levels of PGE2 and LTB4 were generated in OA as compared to normal cartilage. IL-1 treatment further enhanced these eicosanoids production. Treatment of OA cartilage explants with cyclooxygenase inhibitors (celecoxib & indomethacin) augmented LTB4 accumulation by 2- to 4-fold. A follow-up pharmacogenomic analysis identified approximately 90 cytokine and growth factor related transcripts that were modulated following selective COX-2 inhibition. These studies for the first time demonstrate that normal and OA cartilage generates multiple and differential eicosanoid products. Inhibition of the COX- pathway in human OA cartilage caused accumulation of end products (LTB4) of the 5LO pathway. Furthermore, celecoxib, a selective COX-2 inhibitor, regulated numerous genes in cartilage, which are linked to the NFkB and AP-1 pathways at the mRNA level. In conclusion, these experiments demonstrate the complex and pleotropic role of eicosanoids in human cartilage homeostasis and pathophysiology of OA.
Publication Date: 2012-08-16
Journal: Bulletin of the NYU hospital for joint diseases

[Oxidative stress and endothelial dysfunction].
Endothelial dysfunction and oxidative stress are the main pathophysiological mechanisms of several diseases such as hypertension, atherosclerosis, dyslipidemia, diabetes mellitus, cardiovascular disease, renal failure and ischemia-reperfusion injury. Reactive oxygen species (ROS) can modulate cellular function, receptor signals and immune responses in physiological conditions, but when present in excess, they mediate progressive endothelial damage through growth and migration of vascular smooth muscle and inflammatory cells, alteration of extracellular matrix, apoptosis of endothelial cells, activation of transcription factors (NFkB, AP-1), over-expression of inflammatory cytokines and adhesion molecules (ICAM-1, VCAM-1 , E-selectin). Recent evidences suggest that the major source of ROS is the NADPH-oxidase, especially activated by angiotensin II, shear stress and hyperglycemia. The unbalance between production of free radicals and the ability to neutralize them by antioxidant systems causes a condition of "oxidative stress". ROS alter vascular tone by increasing concentration of cytosolic calcium and especially causing a decreased availability of nitric oxide, the principal agent of endothelial function with vasodilating action. The data emerged from experimental and clinical studies confirm that endothelium-dependent vasodilation is altered in many diseases.
Publication Date: 2011-02-15
Journal: Minerva medica

Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators.
Many pathogens exist in multiple physiological niches within the host. Differences between aerobic and anaerobic conditions are known to alter the expression of bacterial virulence factors, typically through the conditional activity of transactivators that modulate their expression. More recently, changes in physiological niches have been shown to affect the expression of viral genes. For many viruses, differences in oxygen tension between hypoxia and normoxia alter gene expression or function. Oxygen tension also affects many mammalian transactivators including AP-1, NFkB, and p53 by affecting the reduced state of critical cysteines in these proteins. We have recently determined that an essential cys-x-x-cys motif in the EBNA1 transactivator of Epstein-Barr virus is redox-regulated, such that transactivation is favoured under reducing conditions. The crucial Tat transactivator of human immunodeficiency virus (HIV) has an essential cysteine-rich region, and is also regulated by redox. Contrary to EBNA1, it is reported that Tat's activity is increased by oxidative stress. Here we have compared the effects of hypoxia, oxidative stress, and cellular redox modulators on EBNA1 and Tat. Our results indicate that unlike EBNA1, Tat is less active during hypoxia. Agents that generate hydroxyl and superoxide radicals reduce EBNA1's activity but increase transactivation by Tat. The cellular redox modulator, APE1/Ref-1, increases EBNA1's activity, without any effect on Tat. Conversely, thioredoxin reductase 1 (TRR1) reduces Tat's function without any effect on EBNA1. We conclude that oxygen partial pressure and oxidative stress affects the functions of EBNA1 and Tat in a dramatically opposed fashion. Tat is more active during oxidative stress, whereas EBNA1's activity is compromised under these conditions. The two proteins respond to differing cellular redox modulators, suggesting that the oxidized cysteine adduct is a disulfide bond(s) in Tat, but sulfenic acid in EBNA1. The effect of oxygen partial pressure on transactivator function suggests that changes in redox may underlie differences in virus-infected cells dependent upon the physiological niches they traffic to.
Publication Date: 2010-05-13
Journal: Virology journal

Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles.
Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.
Publication Date: 2009-11-17
Journal: Toxicology and applied pharmacology

[Regulation of PAI-1 expression].
PAI-1 (plasminogen activator inhibitor-1) is a member of plasminogen cascade with an inhibitory role in plasmin activation. Plasmin is a protease capable of acting on wide range of substrates and, together with metaloproteinases, is a main proteolytic enzyme. Except its role in plasminogen cascade, PAI-1 has an affinity to vitronectin and uPA/uPAR what involves PAI-1 in cell's motility. PAI-1 gene is regulated in response to cytokines, hormones and many growth factors among which TGFbeta is the most important one. The PAI-1 promoter contains SBE, CAGA box, HRE, ERE, NFkB - binding sites, Sp-1, AP-1 and other. Cooperation between transcription factors bound to promoter and cross-talks between kinases and other upstream proteins decide about gene expression. This work describes the present knowledge in this field.
Publication Date: 2009-06-12
Journal: Postepy biochemii

TNF-alpha production in the skin.
Upregulation of TNF-alpha is a key early response to ultraviolet B (UVB) by keratinocytes (KCs), and represents an important component of the inflammatory cascade in skin. UVB irradiation induces TNF-alpha expression in both KCs and dermal fibroblasts, with TNF-alpha mRNA induction seen as early as 1.5 h after UVB. We previously reported that the effects are wavelength-specific: TNF-alpha expression and secretion are induced by UVB (290-320 nm), but not by UVA (320-400 nm). Moreover, we found that IL-1alpha, a cytokine also present in irradiated skin, substantially and synergistically enhances the induction of TNF-alpha by UVB, and the induction of TNF-alpha by this combination of UVB with IL-1alpha is mediated through increased TNF-alpha gene transcription. We investigated the molecular mechanism for UVB-induction of the TNF-alpha gene with a series of TNF-alpha promoter constructs, ranging from 1.2 kbp (from -1179 to +1 with respect to the TNF-alpha transcription initiation site) down to 0.1 kbp (-109 to +1), each driving expression of a CAT reporter. Our results showed a persistent nine to tenfold increase of CAT activity in all TNF-alpha promoter/reporter constructs in response to UVB (30 mJ/cm(2)) exposure. These results indicate the presence of UVB-responsive cis-element(s) located between -109 and +1 of the TNF-alpha promoter, a region that contains a putative AP-1 site and a putative NFkB site. UVB-induction was abolished when the TNF-alpha promoter was mutated by one base pair at the AP-1 binding site. Cells treated with SP600125, an AP-1 inhibitor that inhibits JNK (c-Jun N-terminal kinase), also showed suppression of the 0.1 kbp TNF-alpha promoter/reporter construct. The authentic endogenous gene in untransfected cells was also blocked by the inhibitor. Electrophoretic Mobility Shift Assay indicated new complexes from UVB-treated nuclear extracts and anti-phospho-c-Jun, a regulatory component of the AP-1 transcription factor, creating a supershift indicating increased phosphorylation of c-Jun and hence higher AP-1 activity. Keratinocyte-derived TNF-alpha is a component of the early induction phase of the inflammatory cascade.
Publication Date: 2008-10-01
Journal: Archives of dermatological research

Diesel exhaust increases EGFR and phosphorylated C-terminal Tyr 1173 in the bronchial epithelium.
Epidemiological studies have demonstrated adverse health effects of environmental pollution. Diesel exhaust (DE) is a major contributor to particulate matter pollution. DE exposure has been shown to induce a pronounced inflammatory response in the airways, together with an enhanced epithelial expression of cytokines such as IL-8, Gro-alpha, IL-13 and activation of redox sensitive transcription factors (NFkappaB, AP-1), and MAP kinases (p38, JNK). The aim of the present investigation was to elucidate the involvement of the epidermal growth factor receptor (EGFR) signalling pathway in the epithelial response to DE in-vivo. Immunohistochemical staining was used to quantify the expression of the EGFR, phosphorylated Tyrosine residues, MEK and ERK in the bronchial epithelium of archived biopsies from 15 healthy subjects following exposure to DE (PM10, 300 mug/m3) and air. DE induced a significant increases in the expression of EGFR (p = 0.004) and phosphorylated C-terminal Tyr 1173 (p = 0.02). Other investigated EGFR tyrosine residues, Src related tyrosine (Tyr 416), MEK and ERK pathway were not changed significantly by DE. Exposure to DE (PM10, 300 mug/m3) caused enhanced EGFR expression and phosphorylation of the tyrosine residue (Tyr 1173) which is in accordance with the previously demonstrated activation of the JNK, AP-1, p38 MAPK and NFkB pathways and associated downstream signalling and cytokine production. No effects were seen on the MEK and ERK pathway suggesting that at the investigated time point (6 hours post exposure) there was no proliferative/differentiation signalling in the bronchial epithelium. The present findings suggest a key role for EGFR in the bronchial response to diesel exhaust.
Publication Date: 2008-05-08
Journal: Particle and fibre toxicology