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1.
Biochem Pharmacol ; 168: 465-472, 2019 10.
Article in English | MEDLINE | ID: mdl-31325449

ABSTRACT

BACKGROUND AND PURPOSE: We have previously reported that endocannabinoids modulate permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion. EXPERIMENTAL APPROACH: Caco-2 cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O2), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists. KEY RESULTS: Complete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB1). Basolateral application of NADA (via TRPV1), OA (via CB1 and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability. CONCLUSIONS AND IMPLICATIONS: A variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-2 permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB1 antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability.


Subject(s)
Cell Membrane Permeability/drug effects , Endocannabinoids/metabolism , PPAR alpha/metabolism , Receptor, Cannabinoid, CB1/metabolism , TRPV Cation Channels/metabolism , Caco-2 Cells , Cell Hypoxia/drug effects , Cell Hypoxia/physiology , Cell Membrane Permeability/physiology , Endocannabinoids/pharmacology , Humans , Receptor, Cannabinoid, CB1/agonists , Transendothelial and Transepithelial Migration/drug effects , Transendothelial and Transepithelial Migration/physiology
2.
Br J Pharmacol ; 165(8): 2598-610, 2012 Apr.
Article in English | MEDLINE | ID: mdl-21745190

ABSTRACT

BACKGROUND AND PURPOSE: Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro. EXPERIMENTAL APPROACH: Confluent Caco-2 cell monolayers were treated for 24 h with IFNγ and TNFα (10 ng·mL(-1) ). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB(1) , CB(2) , TRPV1, PPARγ and PPARα. A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids. KEY RESULTS: Δ(9) -Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine-induced increased permeability; an effect sensitive to CB(1) receptor antagonism. Anandamide and 2-arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB(1) antagonism. No role for the CB(2) receptor was identified in these studies. Co-application of THC, cannabidiol or a CB(1) antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation. CONCLUSIONS AND IMPLICATIONS: These findings suggest that locally produced endocannabinoids, acting via CB(1) receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.


Subject(s)
Cannabinoids/pharmacology , Intestinal Mucosa/metabolism , Permeability/drug effects , Amidohydrolases/antagonists & inhibitors , Benzamides/pharmacology , Benzodioxoles/pharmacology , Caco-2 Cells , Carbamates/pharmacology , Cell Survival/drug effects , Enzyme Inhibitors/pharmacology , Humans , Indoles/pharmacology , Inflammation/metabolism , Interferon-gamma/pharmacology , Monoacylglycerol Lipases/antagonists & inhibitors , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/metabolism , Tumor Necrosis Factor-alpha/pharmacology
3.
Leukemia ; 25(1): 145-52, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20966935

ABSTRACT

B-cell lymphoma 6 (BCL6) and PR domain containing 1 (PRDM1) are considered as master regulators for germinal center (GC) formation and terminal B-cell differentiation. Dysregulation of BCL6 and PRDM1 has been associated with lymphomagenesis. Here, we show for the first time that direct cell-cell contact between follicular dendritic cells (FDC) and B-lymphocytes, by influencing the expression of a set of microRNAs (miRNAs), regulates the expression of BCL6 and PRDM1. We identify that, on cell adhesion to FDC, FDC induces upregulation of PRDM1 expression through downregulation of miR-9 and let-7 families and induces downregulation of BCL-6 through upregulation of miR-30 family in B-lymphocytes and lymphoma cells. We further demonstrate that the miR-30 family directly controls BCL-6 expression and miR-9-1 and let-7a directly control PRDM-1 expression through targeting their 3'UTR, mediating the FDC effect. Our studies define a novel regulatory mechanism in which the FDC, through induction of miRNAs in B-lymphocytes, orchestrates the regulation of transcription factors, promotes germinal center B-cell survival and differentiation. Dysregulation of miRNAs may interfere with B-cell survival and maturation, thus representing a novel molecular mechanism, as well as a potential therapeutic target in B-cell lymphomas.


Subject(s)
DNA-Binding Proteins/genetics , Dendritic Cells, Follicular/physiology , Lymphoma, B-Cell/metabolism , MicroRNAs/physiology , Repressor Proteins/genetics , 3' Untranslated Regions/physiology , Cell Communication , Cell Line, Tumor , Cell Survival , Down-Regulation , Humans , Positive Regulatory Domain I-Binding Factor 1 , Proto-Oncogene Proteins c-bcl-6 , Up-Regulation
4.
J Pharmacol Exp Ther ; 335(1): 92-102, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20592049

ABSTRACT

Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion, and intestinal motility. However, the effects of cannabinoids on intestinal permeability have not yet been established. The aim of the present study is to examine the effects of cannabinoids on intestinal permeability in an in vitro model. Caco-2 cells were grown until fully confluent on inserts in 12-well plates. Transepithelial electrical resistance (TEER) measurements were made as a measure of permeability. EDTA (50 µM) was applied to reversibly increase permeability (reduce TEER). The effects of cannabinoids on permeability in combination with EDTA, or alone, were assessed. Potential target sites of action were investigated using antagonists of the cannabinoid (CB)(1) receptor, CB(2) receptor, transient receptor potential vanilloid subtype 1 (TRPV1), peroxisome proliferator-activated receptor (PPAR)γ, PPARα, and a proposed cannabinoid receptor. When applied to the apical or basolateral membrane of Caco-2 cells, Δ(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD) enhanced the speed of recovery of EDTA-induced increased permeability. This effect was sensitive to cannabinoid CB(1) receptor antagonism only. Apical application of endocannabinoids caused increased permeability, sensitive to cannabinoid CB(1) receptor antagonism. By contrast, when endocannabinoids were applied basolaterally, they enhanced the recovery of EDTA-induced increased permeability, and this involved additional activation of TRPV1. All cannabinoids tested increased the mRNA of the tight junction protein zona occludens-1, but only endocannabinoids also decreased the mRNA of claudin-1. These findings suggest that endocannabinoids may play a role in modulating intestinal permeability and that plant-derived cannabinoids, such as THC and CBD, may have therapeutic potential in conditions associated with abnormally permeable intestinal epithelium.


Subject(s)
Cannabinoids/pharmacology , Intestinal Absorption/drug effects , Algorithms , Biological Transport, Active , Caco-2 Cells , Cannabidiol/pharmacology , Dronabinol/pharmacology , Electric Impedance , Electrophysiology , Enterocytes/drug effects , Enterocytes/ultrastructure , Humans , Microvilli/drug effects , PPAR alpha/drug effects , PPAR gamma/drug effects , Permeability/drug effects , Piperidines/pharmacology , Pyrazoles/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Cannabinoid/drug effects , TRPV Cation Channels/drug effects , Tight Junctions/drug effects
5.
Br J Pharmacol ; 153(2): 263-70, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17906675

ABSTRACT

The emerging potential for the cannabinoid (CB) system in modulating gastrointestinal inflammation has gained momentum over the last few years. Traditional and anecdotal use of marijuana for gastrointestinal disorders, such as diarrhoea and abdominal cramps is recognized, but the therapeutic benefit of cannabinoids in the 21st century is overshadowed by the psychoactive problems associated with CB1 receptor activation. However, the presence and function of the CB2 receptor in the GI tract, whilst not yet well characterized, holds great promise due to its immunomodulatory roles in inflammatory systems and its lack of psychotropic effects. This review of our current knowledge of CB2 receptors in the gastrointestinal tract highlights its role in regulating abnormal motility, modulating intestinal inflammation and limiting visceral sensitivity and pain. CB2 receptors represent a braking system and a pathophysiological mechanism for the resolution of inflammation and many of its symptoms. CB2 receptor activation therefore represents a very promising therapeutic target in gastrointestinal inflammatory states where there is immune activation and motility dysfunction.


Subject(s)
Gastrointestinal Tract/physiology , Inflammation/physiopathology , Receptor, Cannabinoid, CB2/physiology , Respiratory Physiological Phenomena , Animals , Cannabinoid Receptor Modulators/physiology , Humans , Immune System/physiology , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/physiopathology
6.
Oncogene ; 20(47): 6899-909, 2001 Oct 18.
Article in English | MEDLINE | ID: mdl-11687969

ABSTRACT

The transforming growth factor-beta (TGF beta) type II receptor (T beta R-II) is responsible for transducing the growth inhibitory signals of TGF beta. The T beta R-II gene promoter lacks both a TATA box and a CAAT box near the transcription initiation site, and has been shown to contain binding sequences for several transcription factors (Sp1, AP1, NF-Y, Cut and ERT) which are important for T beta R-II gene promoter activity in vitro. However, it is still not clear which interactions are important for the regulation of T beta R-II gene promoter activity in vivo. Using in vivo genomic DNA footprinting of normal human epithelial cells (HaCaT), we have identified two novel identical and strongly protected sites (ggggctgg) at positions -59 and -102 of the T beta R-II gene promoter. Mutation of either site significantly reduced promoter activity in transient transfections. Protein binding to these sites, as determined by electrophoretic mobility shift assays (EMSA), was specifically competed with consensus Sp1 oligonucleotides. Furthermore, anti-Sp1/3 antibodies produced band shifts when incubated with the T beta R-II -59 and -102 DNA probes. Importantly, Sp1 protein binding was influenced by the presence of an intact NF-Y binding site at position -83. Our data suggests that both Sp1 and NF-Y may play an important role in regulating T beta R-II gene promoter basal activity in vivo.


Subject(s)
Promoter Regions, Genetic , Receptors, Transforming Growth Factor beta/genetics , Response Elements , Sp1 Transcription Factor/physiology , Transcriptional Activation , Binding Sites , CCAAT-Binding Factor/physiology , Cell Line , DNA/metabolism , DNA Footprinting , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Humans , Protein Serine-Threonine Kinases , Receptor, Transforming Growth Factor-beta Type II , Sp1 Transcription Factor/genetics , Sp3 Transcription Factor , Transcription Factors/genetics , Transcription Factors/physiology , Transfection
7.
Cell Growth Differ ; 12(6): 327-35, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11432807

ABSTRACT

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.


Subject(s)
Histocompatibility Antigens Class II , Nuclear Proteins , Promoter Regions, Genetic/physiology , Trans-Activators/genetics , DNA-Binding Proteins/metabolism , Gene Expression , Host Cell Factor C1 , Humans , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Melanocytes/cytology , Melanocytes/metabolism , Melanoma , Octamer Transcription Factor-1 , Signal Transduction , Transcription Factors/metabolism , Tumor Cells, Cultured
8.
Gastroenterology ; 120(5): 1117-27, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11266376

ABSTRACT

BACKGROUND AND AIMS: Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. METHODS: In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. RESULTS: The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly. CONCLUSIONS: COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.


Subject(s)
Intestinal Mucosa/enzymology , Intestinal Mucosa/immunology , Isoenzymes/genetics , Phosphatidylinositol 3-Kinases/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Tumor Necrosis Factor-alpha/pharmacology , Androstadienes/pharmacology , Caco-2 Cells , Cyclooxygenase 2 , Dinoprostone/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/immunology , HT29 Cells , Homeostasis/physiology , Humans , Interleukin-1/pharmacology , Interleukin-10/pharmacology , Interleukin-13/pharmacology , Interleukin-4/pharmacology , Intestinal Mucosa/cytology , Membrane Proteins , NF-kappa B/metabolism , RNA, Messenger/analysis , Wortmannin
9.
J Biol Chem ; 276(18): 15264-8, 2001 May 04.
Article in English | MEDLINE | ID: mdl-11279146

ABSTRACT

The major histocompatibility complex (MHC) class II transactivator (CIITA) acts as a master switch to activate expression of the genes required for MHC-II antigen presentation. During B-cell to plasma cell differentiation, MHC-II expression is actively silenced, but the mechanism has been unknown. In plasma cell tumors such as multiple myeloma the repression of MHC-II is associated with the loss of CIITA. We have identified that positive regulatory domain I binding factor 1 (PRDI-BF1), a transcriptional repressor, inhibits CIITA expression in multiple myeloma cell lines. Repression of CIITA depends on the DNA binding activity of PRDI-BF1 and its specific binding site in the CIITA promoter. Deletion of a histone deacetylase recruitment domain in PRDI-BF1 does not inhibit repression of CIITA nor does blocking histone deacetylase activity. This is in contrast to PRDI-BF1 repression of the c-myc promoter. Repression of CIITA requires either the N-terminal acidic and conserved PR motif or the proline-rich domain. PRDI-BF1 has been shown to be a key regulator of B-cell and macrophage differentiation. These findings now indicate that PRDI-BF1 has at least two mechanisms of repression whose function is dependent on the nature of the target promoter. Importantly, PRDI-BF1 is defined as the key molecule in silencing CIITA and thus MHC-II in multiple myeloma cells.


Subject(s)
Genes, MHC Class II , Multiple Myeloma/genetics , Repressor Proteins/genetics , Trans-Activators/genetics , Transcription Factors , Histone Deacetylases/metabolism , Multiple Myeloma/pathology , Promoter Regions, Genetic , RNA, Messenger/genetics
10.
Am J Bot ; 88(9): 1545-57, 2001 Sep.
Article in English | MEDLINE | ID: mdl-21669687

ABSTRACT

An assemblage of 33 fossil pollen and spores, recovered from the 3600-m high Pislepampa locality of E. W. Berry, Eastern Cordillera, Bolivia, adds considerably to our knowledge of three aspects of the region in late Neogene time: (1) the paleovegetation, (2) the paleoclimate, and (3) the paleoelevation of the Central Andes. The plant microfossils recognized are Isoetes, Lycopodium (three types), Cnemidaria, Cyathea (three types), Grammitis, Hymenophyllum, Pteris, trilete fern spores (two types), Danaea, monolete fern spores (four types), Podocarpus, Gramineae, Palmae, Ilex, cf. Oreopanax, Cavanillesia, cf. Pereskia, Compositae (three types), Ericaceae, Tetrorchidium, and unknowns (three types). The diversity of the Compositae suggest that this flora has a maximum age around the Miocene-Pliocene boundary, that is, 6-7 million years. All members of the paleocommunity presently grow in the bosque montano húmedo (cloud forest) along the eastern slope of the Central Andes of Bolivia, which occurs between MATs (mean annual temperatures) of ∼10° and 20°C. The Pislepampa flora probably represents the lower limits of this forest because the fossil leaves collected by Berry from the same locality all have entire margins, suggesting that the flora grew near the cloud forest-tropical forest transition. Presently, the lower limit of the cloud forest forest has MATs of ∼20°C, a mean annual precipitation between 1000 and 1500 mm, and that part containing most of the identified genera of fossil pollen is found at elevations ∼1200-1400 m. These conditions are thus inferred for the Pislepampa flora; however, because of the uncertainty of the magnitude of global climate change and of possible changes in the ecological range of plant genera, we estimate an error of at least ±1000 m for the paleoelevation estimate. When the total uplift is corrected for probable amounts of erosionally driven isostatic rebound, the paleoelevation estimate suggests that from one-third to one-half of the uplift of the Eastern Cordillera had occurred by the beginning of the Pliocene. This estimate is consistent with other paleoelevation estimates from the Central Andes.

11.
Cancer Res ; 60(20): 5789-96, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11059775

ABSTRACT

The inadequate ability of cancer cells to present antigen on the cell surface via MHC class I molecules is one mechanism by which tumor cells evade antitumor-associated antigen immunity. In many cases, such as in renal cell carcinoma (RCC), the lack of MHC class I antigen presentation can be attributed to the down-regulation of genes needed for antigen processing, such as the transporters associated with antigen processing (TAP)1 and TAP2, and the proteasomal components low molecular weight proteins (LMP)2 and LMP7. The TAP1 and LMP2 genes are transcribed from a shared bidirectional promoter containing an IFN response factor element that confers IFN-gamma inducibility. Here, we investigate the differential responsiveness to IFN-gamma of RCC cell lines, Caki-1 and Caki-2, which have been reported to have abnormally low expressions of TAP1 and LMP2. We now demonstrate that the Caki-2 cell line is defective in the IFN-gamma signaling pathway. The effects of IFN-gamma on TAP1 and LMP2 expression revealed a loss of up-regulation in Caki-2 cells, but not in Caki-1 cells. In vivo DNA footprinting shows a specific loss of occupancy at the IFN response factor element site in Caki-2 cells, whereas Caki-1 cells show full promoter occupancy. Furthermore, in vitro DNA-binding studies indicated that Caki-2 cells do not have IFN-regulatory factor 1- or signal transducer and activator of transcription 1 (Stat1)-binding activity after IFN-gamma stimulation. Examination of Stat1, Jak1, and Jak2 proteins demonstrated that the proteins were expressed, however, not phosphorylated, upon IFN-gamma treatment in Caki-2 cells. Also, this cell line expressed both IFN-gamma receptor chains. IFN-gamma inducibility could not be rescued by introduction of normal Jak1 and/or Jak2 proteins. However, overexpression of Jak1 did increase TAP1 and LMP2 expression independent of IFN-gamma, indicating that the Stat1 and IFN-regulatory factor 1 proteins present in Caki-2 can be activated. These findings suggest that the loss of TAP1 and LMP2 induction is a defect in the earliest steps of the IFN-gamma signaling pathway resulting in the inability of Caki-2 cells to up-regulate the MHC class I antigen-processing pathway. Because immunotherapy may be one of the most promising approaches for treating RCC, understanding the mechanisms by which these tumors circumvent cytokine signaling, thereby evading antitumor-specific-antigen immunity, would greatly aid the efficacy of such therapy.


Subject(s)
Carcinoma, Renal Cell/metabolism , Exoribonucleases/biosynthesis , Interferon-gamma/pharmacology , Kidney Neoplasms/metabolism , Proto-Oncogene Proteins , Viral Matrix Proteins/biosynthesis , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/immunology , DNA, Neoplasm/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Exoribonucleases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , HeLa Cells , Humans , Interferon Regulatory Factor-1 , Interferon-gamma/immunology , Interferon-gamma/physiology , Janus Kinase 1 , Janus Kinase 2 , Kidney Neoplasms/genetics , Kidney Neoplasms/immunology , Phosphoproteins/biosynthesis , Phosphoproteins/metabolism , Phosphorylation , Promoter Regions, Genetic/genetics , Protein-Tyrosine Kinases/metabolism , Receptors, Interferon/biosynthesis , STAT1 Transcription Factor , Signal Transduction/drug effects , Signal Transduction/physiology , Trans-Activators/metabolism , Trans-Activators/physiology , Transcription Factors/metabolism , Tumor Cells, Cultured , Viral Matrix Proteins/genetics , Interferon gamma Receptor
12.
EMBO J ; 19(15): 4111-22, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10921891

ABSTRACT

Analysis of mRNA levels in cells that express or lack signal transducers and activators of transcription 1 (Stat1) reveals that Stat1 mediates the constitutive transcription of many genes. Expression of the low molecular mass polypeptide 2 (LMP2), which requires Stat1, has been studied in detail. The overlapping interferon consensus sequence 2/gamma-interferon-activated sequence (ICS-2/GAS) elements in the LMP2 promoter bind to interferon regulatory factor 1 (IRF1) and Stat1 and are occupied constitutively in vivo. The point mutant of Stat1, Y701F, which does not form dimers involving SH2-phosphotyrosine interactions, binds to the GAS element and supports LMP2 expression. Unphosphorylated Stat1 binds to IRF1 directly and we conclude that this complex uses the ICS-2/GAS element to mediate constitutive LMP2 transcription in vivo. The promoter of the IRF1 gene, which also contains a GAS site but not an adjacent ICS-2 site, is not activated by Stat1 Y701F. The promoters of other genes whose constitutive expression requires Stat1 may also utilize complexes of unphosphorylated Stat1 with IRF1 or other transcription factors.


Subject(s)
Cysteine Endopeptidases , DNA-Binding Proteins/metabolism , Phosphoproteins/metabolism , Proteins/genetics , Trans-Activators/metabolism , Base Sequence , Binding Sites , Dimerization , Gene Expression Profiling , Gene Expression Regulation , Humans , Interferon Regulatory Factor-1 , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Phosphorylation , Protein Binding , STAT1 Transcription Factor , Transcription, Genetic
13.
Hepatogastroenterology ; 47(33): 714-7, 2000.
Article in English | MEDLINE | ID: mdl-10919016

ABSTRACT

BACKGROUND/AIMS: Nitric oxide synthesis is increased in rectal biopsies from patients with ulcerative colitis and colonic epithelial cells are considered to be a major source of nitric oxide in intestinal inflammation. METHODOLOGY: Human colonic biopsies from normal bowel mucosa and colonic epithelial cell line HT-29 were cultured in the presence of the inflammatory cytokines IL-1 alpha + TNF-alpha + IFN-alpha added after 1 hour pretreatment with vehicle or Interleukin-13. Nitrite levels were determined at 30 hours in culture supernatants by a fluorometric assay. RESULTS: Unstimulated human colonic biopsies and HT-29 cells produced a basal amount of nitrite. Stimulation with IL-1 alpha + TNF-alpha + IFN-alpha induced a significant (P < 0.001) increase of nitrite generation by both human colonic biopsies and HT-29 cells. The presence of Interleukin-13 produced a significant (P < 0.001) suppression of the cytokine-induced nitrite generation from both colonic biopsies and HT-29 cells. CONCLUSIONS: Nitric oxide generation in human colonic mucosa is susceptible to manipulation by proinflammatory cytokines. Interleukin-13 has an inhibitory effect on cytokine induced nitrite production in colonic mucosa and could play an anti-inflammatory role in intestinal inflammation.


Subject(s)
Colon/metabolism , Interleukin-13/physiology , Intestinal Mucosa/metabolism , Nitric Oxide/biosynthesis , Adult , Aged , Cells, Cultured , Female , Humans , Male , Middle Aged , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II
14.
Ann Thorac Surg ; 69(2): 634-5, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10735721

ABSTRACT

We report the case of a child with hypoplastic left heart syndrome who developed pulmonary arteriovenous (AV) malformations after superior cavopulmonary anastomoses. Resolution of the pulmonary AV malformations occurred following a completion Fontan procedure. This phenomenon has been reported previously, but only in patients with heterotaxy and polysplenia.


Subject(s)
Arteriovenous Fistula/surgery , Fontan Procedure , Postoperative Complications , Pulmonary Artery/surgery , Pulmonary Circulation , Pulmonary Veins/surgery , Anastomosis, Surgical , Arteriovenous Fistula/etiology , Female , Humans , Hypoplastic Left Heart Syndrome/complications , Infant , Pulmonary Artery/abnormalities , Pulmonary Veins/abnormalities , Vena Cava, Superior/surgery
15.
Mol Cell Biol Res Commun ; 4(3): 137-43, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11281727

ABSTRACT

Nitric oxide (NO) and the many derivatives and reactive oxygen intermediates thereof are all molecules that are utilised by mammalian cells in the war against microbial pathogens and tumours. They are potentially toxic molecules and, with damage control being crucial, the production and metabolism of nitric oxide is a tightly regulated process. The duality of NO is well documented. On the one hand, beneficial effects include normal healing in the skin and intestinal mucosa, killing of certain bacteria, regulating T cell proliferation and differentiation (Th1 vs Th2), and regulating leukocyte recruitment, by affecting adhesion molecule expression. On the other hand, persistent high levels of NO can lead to the production of toxic metabolites (peroxynitrite and hydroxyls), which can have detrimental effects, such as increased microvascular and epithelial permeability, increased oxidative stress (which can damage DNA), and damage to iron-sulphur proteins in mitochondria. NO has been reported to modulate its own production and the mechanisms involved in this self-regulation are being hotly pursued. The purpose of this review is to update recent intriguing advances in our understanding of the interaction of the phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathway in regulating the activity of the enzymes that generate NO, namely, the nitric oxide synthases.


Subject(s)
Nitric Oxide Synthase/metabolism , Nitric Oxide/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases , Reactive Oxygen Species/metabolism , Animals , Models, Biological , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/genetics , Phosphatidylinositol 3-Kinases/genetics , Protein Isoforms , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Signal Transduction/genetics , Signal Transduction/physiology
16.
Focus ; 15(2): 5-6, 2000 Jan.
Article in English | MEDLINE | ID: mdl-11367304

ABSTRACT

AIDS: Adolescent pregnancy rates are dropping overall, but a significant number of adolescent pregnancies occur in women infected with HIV. Pregnancy and parenting issues are intensified and more complex in this group than in the uninfected. Newly diagnosed young women with HIV face important life decisions but they are poorly prepared to address them. The significance of pregnancy is so potent in impoverished communities that many women want to become pregnant at a very young age. A study of HIV-infected women with substance abuse histories shows that they identify childbearing with independence and hope. Young mothers and their children benefit from services that help them build on their strengths and encourage skills development. They need vocational support, health care, case management, and counseling. Providers need to be careful, however, to remain professional and not become overly involved.^ieng


Subject(s)
HIV Seropositivity/psychology , Mothers/psychology , Pregnancy in Adolescence , Adolescent , Counseling , Female , Humans , Pregnancy
17.
J Immunol ; 163(12): 6622-30, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10586057

ABSTRACT

MHC class II deficiency found in bare lymphocyte syndrome patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-gamma. Here, we show that the defect in G1B, one such cell line, is attributed to the lack of functional RFX5, the largest subunit of RFX. The RFX5 gene isolated from G1B cells contains two separate single-base pair mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neighboring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/immunology , Genes, MHC Class II/immunology , Interferon-gamma/physiology , Leucine/metabolism , Transcription Factors/metabolism , Amino Acid Motifs/immunology , Amino Acid Sequence , Base Pairing/genetics , Base Sequence , Cloning, Molecular , Cysteine/genetics , Cysteine/metabolism , DNA, Neoplasm/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Genetic Complementation Test , HLA-DR Antigens/biosynthesis , HLA-DR Antigens/metabolism , HLA-DR alpha-Chains , Humans , Leucine/genetics , Molecular Sequence Data , Neoplasm Proteins/metabolism , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Phenotype , Point Mutation , Protein Structure, Tertiary , Regulatory Factor X Transcription Factors , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcription Factors/isolation & purification , Transcriptional Activation/genetics , Tumor Cells, Cultured
18.
Arch Biochem Biophys ; 371(2): 290-300, 1999 Nov 15.
Article in English | MEDLINE | ID: mdl-10545217

ABSTRACT

Resistance to the growth inhibitory effects of transforming growth factor beta (TGFbeta) has been associated with decreased levels of the TGFbeta type II receptor (TbetaR-II) and has been correlated with tumorigenicity. Previously, we reported an A --> G mutation at position -364 in the TbetaR-II promoter in A431 tumor cells which results in reduced TbetaR-II promoter activity. In this study, we show that the CDP/Cut (CCAAT displacement protein) transcription factor, a transcriptional repressor, binds both the wild type and the mutant TbetaR-II promoter. We also demonstrate that the A --> G mutation increases CDP/Cut binding affinity, and that overexpression of CDP/Cut reduces transcription from TbetaR-II promoter reporter constructs. Increased binding of the CDP/Cut repressor protein, as a result of a mutation at position -364, represents a novel mechanism of regulation in a neoplastic cell of the promoter of a tumor suppressor gene, TbetaR-II.


Subject(s)
Gene Expression Regulation, Neoplastic , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Receptors, Transforming Growth Factor beta/genetics , Repressor Proteins/metabolism , Transforming Growth Factor beta/metabolism , Binding Sites , DNA Mutational Analysis , Female , Homeodomain Proteins , Humans , Neoplasms/etiology , Protein Binding , Protein Serine-Threonine Kinases , Receptor, Transforming Growth Factor-beta Type II , Transcription Factors , Tumor Cells, Cultured , Vulvar Neoplasms
19.
Oncogene ; 18(43): 5889-903, 1999 Oct 21.
Article in English | MEDLINE | ID: mdl-10557076

ABSTRACT

Class II transactivator (CIITA) activates the expression of major histocompatibility class II genes, which encode antigen-presenting molecules recognized by the T-cell receptor of CD4+ T cells. IFN-gamma induced CIITA transcription in many cell types is directed by the CIITA Type IV promoter. Here we report that the human CIITA Type IV promoter IRF-E binds IRF-1 and can be activated by exogenous expression of IRF-1. Surprisingly, the CIITA Type IV promoter IRF-E is also activated by IRF-2, another member of the IRF family that generally acts as a transcriptional repressor. In addition, we found that IRF-1 and IRF-2 synergistically activate the CIITA Type IV promoter. Electrophoretic mobility shift assays revealed that IRF-1 and IRF-2 can simultaneously occupy the IRF-E of the CIITA Type IV promoter, suggesting a novel mechanism for the role of these two proteins in promoter activation. Our results also indicate that IRF-1 and IRF-2 can cooperatively activate and co-occupy the IRF-E of the guanylate binding protein (GBP) promoter. Finally, CIITA induction by IFN-gamma does not occur in a pancreatic tumor cell line that expresses a mutated IRF-2, representing the first IRF-2 mutation identified in a human tumor cell line.


Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins , Phosphoproteins/metabolism , Promoter Regions, Genetic , Repressor Proteins , Trans-Activators/genetics , Transcription Factors , Gene Expression Regulation , Humans , Interferon Regulatory Factor-1 , Interferon Regulatory Factor-2 , Pancreatic Neoplasms , Regulatory Sequences, Nucleic Acid , Tumor Cells, Cultured
20.
J Biol Chem ; 274(45): 32342-50, 1999 Nov 05.
Article in English | MEDLINE | ID: mdl-10542275

ABSTRACT

Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly.


Subject(s)
B-Lymphocytes/metabolism , DNA-Binding Proteins/metabolism , Genes, MHC Class II , Nuclear Proteins , Trans-Activators/genetics , Trans-Activators/metabolism , Transcriptional Activation , Base Sequence , Cell Line , DNA Footprinting , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Zinc Fingers
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