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PubMed2005

CB1 cannabinoid receptors are involved in neuroprotection via NF-kappa B inhibition.

We reported earlier that closed head injury (CHI) in mice causes a sharp elevation of brain 2-arachidonoylglycerol (2-AG) levels, and that exogenous 2-AG reduces brain edema, infarct volume and hippocampal death and improved clinical recovery after CHI. The beneficial effect of 2-AG was attenuated by SR141716A, a CB1 cannabinoid receptor antagonist, albeit at relatively high doses. In the present study, we further explored the role of CB1 receptors in mediating 2-AG neuroprotection. CB1 receptor knockout mice (CB1-/-) showed minor spontaneous recovery at 24 h after CHI, in contrast to the significant improvement in neurobehavioral function seen in wild-type (WT) mice. Moreover, administration of 2-AG did not improve neurological performance and edema formation in the CB1-/- mice. In addition, 2-AG abolished the three- to four-fold increase of nuclear factor kappaB (NF-kappa B) transactivation, at 24 h after CHI in the WT mice, while it had no effect on NF-kappaB in the CB1-/- mice, which was as high as in the WT vehicle-treated mice. We thus propose that 2-AG exerts its neuroprotection after CHI, at least in part, via CB1 receptor-mediated mechanisms that involve inhibition of intracellular inflammatory signaling pathways.

Animals↗
PubMed2002

Activation of the specific neurotrophin receptors TrkA, TrkB and TrkC influences the function of eosinophils.

BACKGROUND: Recent studies have shown that nerve growth factor (NGF) can act on several immune cells as well as residential cells. But little is known about their role in modulating eosinophil function via activation of high-affinity receptors. OBJECTIVES: The aim of this study was to assess whether eosinophils express functional receptors and if their function is influenced by NGF, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). METHODS: Eosinophils were purified by negative immunoselection (purity > 96%). High-affinity neurotrophin receptors were demonstrated by reverse transcription polymerase chain reaction, western blotting and flow-cytometry analysis. Functionality of receptors was demonstrated by receptor phosphorylation after ligand binding. Eosinophils were incubated with NGF, BDNF and NT-3, and cells and supernatants were collected for measurement of the mediators IL-4, IL-5, IL-8, transforming growth factor (TGF)-beta1, eosinophil cationic protein (ECP), eosinophil protein X (EPX) as well as eosinophil viability. RESULTS: Eosinophils expressed mRNA for neurotrophin receptors. Proteins were detectable by western blot and fluorescent-activated cell sorter analysis. The receptors were phosphorylated after stimulation with neurotrophins. After NGF stimulation, a significant increase in IL-4 was detectable. BDNF and NT-3 stimulation led to a significant increase in EPX. Eosinophil viability was not influenced. CONCLUSIONS: Eosinophils express the functionally active receptors TrkA, TrkB and TrkC. Receptor activation stimulates eosinophils. This might be an additional pathway regulating inflammatory responses in allergic reactions.

Blotting, Western↗
PubMed1999

Th1 and Th2 T helper cell subsets affect patterns of injury and outcomes in glomerulonephritis.

The recognition that human immune responses can be directed by two different subsets of T helper cells (Th1 and Th2) has been an important development in modern immunology. Immune responses polarized by either the Th1 or Th2 subset predominance result in different inflammatory effector pathways and disease outcomes. Many autoimmune diseases are associated with either Th1- or Th2- polarized immune responses. Although these different immune response patterns are relevant to glomerulonephritis (GN), little attention has been paid to the consequences of Th1 or Th2 predominance of nephritogenic immune responses for the pattern and outcome of GN. Unlike other autoimmune conditions, GN results from a variety of different immune responses and has a range of histologic features and immune effectors in glomeruli. This review assesses the data available from studies of experimental and human GN that address the Th1 or Th2 predominance of nephritogenic immune responses and their relevance to the different histopathological patterns and outcomes of GN. In particular, the evidence that Th1-predominant nephritogenic immune responses are associated with severe proliferative and crescentic GN is presented.

Animals↗
PubMed2006

Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis.

p38 mitogen-activated protein kinase (MAPK) regulates cytokines in arthritis and is, in turn, regulated by MAPK kinase (MKK) 3 and MKK6. To modulate p38 function but potentially minimize toxicity, we evaluated the utility of targeting MKK3 by using MKK3(-/-) mice. These studies showed that TNF-alpha increased phosphorylation of p38 in WT cultured synoviocytes but that p38 activation, IL-1beta, and IL-6 expression were markedly lower in MKK3(-/-) synoviocytes. In contrast, IL-1beta or LPS-stimulated p38 phosphorylation and IL-6 production by MKK3(-/-) synoviocytes were normal. Detailed signaling studies showed that NF-kappaB also contributes to IL-6 production and that TNF-alpha-induced NF-kappaB activation is MKK3-dependent. In contrast, LPS-mediated activation of NF-kappaB does not require MKK3. To determine whether this dichotomy occurs in vivo, two inflammation models were studied. In K/BxN passive arthritis, the severity of arthritis was dramatically lower in MKK3(-/-) mice. Phospho-p38, phospho-MAPK activator protein kinase 2, IL-1beta, CXC ligand 1, IL-6, and matrix metalloproteinase (MMP) 3 levels in the joints of MKK3(-/-) mice were significantly lower than in controls. Exogenous IL-1beta administered during the first 4 days of the passive model restored arthritis to the same severity as in WT mice. In the second model, IL-6 production after systemic LPS administration was similar in WT and MKK3(-/-) mice. Therefore, selective MKK3 deficiency can suppress inflammatory arthritis and cytokine production while Toll-like receptor 4-mediated host defense remains intact.

Animals↗
PubMed2002

A CD36-initiated signaling cascade mediates inflammatory effects of beta-amyloid.

beta-Amyloid accumulation is associated with pathologic changes in the brain in Alzheimer's disease and has recently been identified in plaques of another chronic inflammatory disorder, atherosclerosis. The class B scavenger receptor, CD36, mediates binding of fibrillar beta-amyloid to cells of the monocyte/macrophage lineage, including brain macrophages (microglia). In this study, we demonstrate that in microglia and other tissue macrophages, beta-amyloid initiates a CD36-dependent signaling cascade involving the Src kinase family members, Lyn and Fyn, and the mitogen-activated protein kinase, p44/42. Interruption of this signaling cascade, through targeted disruption of Src kinases downstream of CD36, inhibits macrophage inflammatory responses to beta-amyloid, including reactive oxygen and chemokine production, and results in decreased recruitment of microglia to sites of amyloid deposition in vivo. The finding that engagement of CD36 by beta-amyloid initiates a Src kinase-dependent production of inflammatory mediators in cells of the macrophage lineage reveals a novel receptor-mediated pro-inflammatory signaling pathway of potential therapeutic importance.

Alleles↗
PubMed2006

The inflammatory mediator leukotriene D4 induces beta-catenin signaling and its association with antiapoptotic Bcl-2 in intestinal epithelial cells.

Increased levels of the inflammatory mediator leukotriene D4 (LTD4) are present at sites of inflammatory bowel disease, and such areas also exhibit an increased risk for subsequent cancer development. It is known that LTD4 affects the expression of many proteins that influence survival and proliferation of intestinal epithelial cells. We demonstrate here that after LTD4 exposure, beta-catenin translocates to the nucleus where it signals activation of the TCF/LEF family of transcription factors. These events are mediated via a phosphatidylinositol 3-kinase-dependent phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase 3beta. We also show that in the presence of LTD4, free beta-catenin translocates to the mitochondria where it associates with the cell survival protein Bcl-2. We hypothesize that LTD4 may enhance cell survival via activation of beta-catenin signaling, in particular, by promoting the association of beta-catenin with Bcl-2 in the mitochondria. Similar to Wnt-1 signaling, LTD4 signals an increased level of free beta-catenin and elevated TCF/LEF promotor activity. This work in intestinal epithelial cells further lends credence to the idea that inflammatory signaling pathways are intrinsically linked with potential oncogenic signals involved in cell survival and apoptosis.

Apoptosis↗
PubMed1999

Ozone effects on airway responsiveness, lung injury, and inflammation. Comparative rat strain and in vivo/in vitro investigations.

Asthmatic individuals appear to be particularly sensitive to the effects of certain air pollutants-including ozone (O(3)), an oxidant ambient air pollutant-for reasons that are poorly understood. The general purpose of these studies, therefore, was to expand and improve upon toxicologic methods for assessing ozone-induced effects on the airways of the rat by (1) developing an in vivo testing procedure that allows detection of airway responsiveness changes in rats exposed to ozone; (2) identifying a strain of rat that may be inherently more sensitive to the effects of ozone; and (3) validation of an in vitro epithelial culture system to more directly assess airway cellular/subcellular effects of ozone. Using methacholine inhalation challenges, we detected increased airway responsiveness in senescent F344 rats acutely after ozone exposure (2 ppm x 2 h). We also determined that acutely after ozone exposure (0.5 ppm x 8 h), Wistar rats developed significantly greater lung injury, neutrophilic inflammation, and bronchoalveolar lavage (BAL) fluid concentrations of IL-6 than either Sprague-Dawley (SD) or F344 rats. SD rats had greater BAL fluid concentrations of prostaglandin E(2) (PGE(2)), while F344 rats consistently exhibited the least effect. Wistar rat-derived tracheal epithelial (RTE) cultures were exposed in vitro to air or ozone (0.1-1.0 ppm x 1 h), and examined for analogous effects. In a concentration-dependent manner, ozone exposure resulted in acute but minor cytotoxicity. RT polymerase chain reaction (PCR) analysis of RNA isolated from ozone-exposed cells demonstrated variable increases in steady-state gene expression of IL-6 at 4 h postexposure, while at 24 h cellular fibronectin expression (EIIIA domain) was decreased. Exposure was without effect on macrophage inflammatory protein 2 (MIP-2) or gamma-glutamyl cysteine synthetase expression. At 6 h postexposure, IL-6 synthesis and apical release appeared increased in ozone-exposed cells (1 ppm x 1 h). MIP-2 release was not significantly increased in ozone-exposed cells. At 2 h postexposure, ozone exposure resulted in minor increases in apical fibronectin, but exposure was without effect on basolateral accumulation of fibronectin. Exposure to 1.0, but not 0.1 ppm (x 1 h), increased production of cyclooxygenase (i.e., PGE(2)) and noncyclooxygenase products of arachidonic acid. Results demonstrate that multiple inflammatory mediator pathways are affected by ozone exposure. Such effects could exacerbate morbidity in individuals with preexisting airway inflammation such as asthmatics.

Animals↗
PubMed1994

Arrangement of domains, and amino acid residues required for binding of vascular cell adhesion molecule-1 to its counter-receptor VLA-4 (alpha 4 beta 1).

Interaction of the vascular cell adhesion molecule (VCAM-1) with its counter-receptor very late antigen-4 (VLA-4) (integrin alpha 4 beta 1) is important for a number of developmental pathways and inflammatory functions. We are investigating the molecular mechanism of this binding, in the interest of developing new anti-inflammatory drugs that block it. In a previous report, we showed that the predominant form of VCAM-1 on stimulated endothelial cells, seven-domain VCAM (VCAM-7D), is a functionally bivalent molecule. One binding site requires the first and the other requires the homologous immunoglobulin-like domain. Rotary shadowing and electron microscopy of recombinant soluble VCAM-7D molecules suggests that the seven Ig-like domains are extended in a slightly bent linear array, rather than compactly folded together. We have systematically mutagenized the first domain of VCAM-6D (a monovalent, alternately spliced version mission domain 4) by replacing 3-4 amino acids of the VCAM sequence with corresponding portions of the related ICAM-1 molecule. Specific amino acids, important for binding VLA-4 include aspartate 40 (D40), which corresponds to the acidic ICAM-1 residue glutamate 34 (E34) previously reported to be essential for binding of ICAM-1 to its integrin counter-receptor LFA-1. A small region of VCAM including D40, QIDS, can be replaced by the similar ICAM-1 sequence, GIET, without affecting function or epitopes, indicating that this region is part of a general integrin-binding structure rather than a determinant of binding specificity for a particular integrin. The VCAM-1 sequence G65NEH also appears to be involved in binding VLA-4.

Amino Acid Sequence↗
PubMed2006

Cytokine expression patterns associated with systemic adverse events following smallpox immunization.

Vaccinia virus is reactogenic in a significant number of vaccinees, with the most common adverse events being fever, lymphadenopathy, and rash. Although the inoculation is given in the skin, these adverse events suggest a robust systemic inflammatory response. To elucidate the cytokine response signature of systemic adverse events, we used a protein microarray technique to precisely quantitate 108 serum cytokines and chemokines in vaccine recipients before and 1 week after primary immunization with Aventis Pasteur smallpox vaccine. We studied 74 individuals after vaccination, of whom 22 experienced a systemic adverse event and 52 did not. The soluble factors most associated with adverse events were selected on the basis of voting among a committee of machine-learning methods and statistical procedures, and the selected cytokines were used to build a final decision-tree model. On the basis of changes in protein expression, we identified 6 cytokines that accurately discriminate between individuals on the basis of adverse event status: granulocyte colony-stimulating factor, stem cell factor, monokine induced by interferon-gamma (CXCL9), intercellular adhesion molecule-1, eotaxin, and tissue inhibitor of metalloproteinases-2. This cytokine signature is characteristic of particular inflammatory response pathways and suggests that the secretion of cytokines by fibroblasts plays a central role in systemic adverse events.

Adolescent↗
PubMed2002

Matrix metalloproteinase(s) mediate(s) NO-induced dissociation of beta-catenin from membrane bound E-cadherin and formation of nuclear beta-catenin/LEF-1 complex.

Modulation of the adenomatous polyposis coli (APC)-beta-catenin pathway by inflammatory mediators and extracellular matrix may be important in colon carcinogenesis. We have recently shown that nitric oxide (NO) induces the accumulation of cytosolic beta-catenin and subsequent formation of the nuclear beta-catenin/lymphocyte enhancing factor (LEF)-1 complex in conditionally immortalized young mouse colonic epithelial (YAMC) cells. In the present study, we explored the mechanism(s) through which NO exerts its effect on cytosolic beta-catenin accumulation and nuclear beta-catenin/LEF-1 complex formation. We found that NO-induced degradation of the membrane bound E-cadherin at tight junctions. Using an anti-E-cadherin antibody specific for its extracellular domain, we detected a 50kDa degradation fragment of E-cadherin (120 kDa) from the culture medium conditioned by YAMC cells exposed to the NO-releasing drug, NOR-1, for 4 and 24 h. As beta-catenin is normally bound to transmembrane E-cadherin and thus anchored to the cytoskeleton structure, the degradation of E-cadherin induced by NO may cause dissociation of beta-catenin from membrane bound E-cadherin. This was demonstrated by the detection of beta-catenin accumulation in the soluble cytosolic fractions in YAMC after exposure to NO-releasing drugs. Furthermore, the degradation of E-cadherin and the release of beta-catenin to cytosol were accompanied by the formation of nuclear beta-catenin/LEF-1 complex, demonstrating the dissociation of beta-catenin from E-cadherin may be responsible for the activation of beta-catenin/LEF-1 transcription complex. Co-treatment with NO donors and broad-spectrum matrix metalloproteinase (MMP) inhibitors TIMP-1 (100 ng/ml), GM6001 (10 micro M) and GM1489 (10 micro M) abolished the degradation of E-cadherin induced by NO as demonstrated by western blot analysis. These MMP inhibitors also blocked the cytosolic accumulation of beta-catenin and nuclear formation of beta-catenin/LEF-1 complex. The sum effect of MMP inhibitors demonstrated that NO-induced activation of MMP may cause the degradation of E-cadherin and the subsequent dissociation of beta-catenin, thereby contributing to the cytosolic accumulation of beta-catenin and nuclear formation of beta-catenin/LEF-1 complex.

Animals↗
PubMed2004

Up-regulated interleukin-4 production by peripheral T-helper cells in idiopathic membranous nephropathy.

BACKGROUND: Helper T (Th) cells are classified into Th1 and Th2 subsets based on cytokine production and the Th1/Th2 paradigm explains differences in inflammatory effector pathways in various human diseases. Membranous nephropathy (MN) is an immune complex disease associated with Th2 nephritogenic immune response. However, overproduction of interleukin (IL)-4, a principal Th2 cytokine, has not been demonstrated. We investigated Th1/Th2 cytokine production by peripheral Th cells and its association with the degree of proteinuria in MN. METHODS: We analysed production of Th1/Th2 cytokines, interferon (IFN)-gamma and IL-4 by peripheral Th cells, using an intracellular cytokine detection method with flow cytometry in patients with MN (n = 24). The data were compared with data from healthy subjects (n = 51), subjects with minimal change nephrotic syndrome (MCNS; n = 13) and subjects with focal segmental glomerulosclerosis (FSGS; n = 12). We compared the percentages of IFN-gamma+ and IL-4+ Th cells and the peripheral Th1/Th2 ratio (IFN-gamma/IL-4 ratio) among the four groups. We also examined the association of IFN-gamma and IL-4 production with clinical parameters of MN. RESULTS: The mean percentage of IL-4+ cells in MN (3.9+/-1.2%) was significantly higher than in the control (2.4+/-1.0%), MCNS (2.3+/-1.4%) and FSGS (2.3+/-1.2%) groups (P<0.001, respectively). The Th1/Th2 ratio was significantly lower in MN (5.3+/-2.0) than in the control (8.2+/-4.2, P<0.05), MCNS (10.0+/-5.3, P<0.01) and FSGS (10.2+/-5.3, P<0.01) groups. Moreover, the percentage of IL-4+ cells correlated significantly with the amount of proteinuria in MN (r = 0.57, P<0.01). CONCLUSIONS: IL-4 production by peripheral Th cells is up-regulated in patients with MN and correlated with the severity of proteinuria. Intracellular cytokine analysis could be a useful index in idiopathic MN.

Adult↗
PubMed2000

Comparative studies of the colonic in situ expression of intercellular adhesion molecules (ICAM-1, -2, and -3), beta2 integrins (LFA-1, Mac-1, and p150,95), and PECAM-1 in ulcerative colitis and Crohn's disease.

A dysregulated local immune defense with a constant influx of leukocytes provides a basis for continuous intestinal inflammation in ulcerative colitis (UC) and Crohn's disease (CD). Cell adhesion molecules are pivotal for the migration of leukocytes from the circulation toward the colonic epithelium. A study quantifying the cells expressing intercellular adhesion molecules (ICAMs), beta2 integrins, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the colon was performed to illustrate the leukocyte migration pathway in inflammatory bowel disease. Serial colonic sections (10 UC, 10 CD, and 10 controls) were stained immunohistochemically for ICAM-1, ICAM-2, ICAM-3, CD11a, CD11b, CD18, and PECAM-1. Cell adhesion molecule expression was evaluated quantitatively with reference to topographic localization. In UC, polymorphonuclear leukocytes (PMNs) in contact with the crypt epithelium and in crypt abscesses expressed CD11b. CD tissue was characterized by CD11a-, CD11c-, and ICAM-1-expressing cells. ICAM-1 was detected on endothelial cells, leukocytes, and apical parts of epithelial membranes, whereas ICAM-2 was expressed on basal epithelial membranes. Most infiltrating leukocytes expressed ICAM-3, whereas perivascular mononuclear cells expressed PECAM-1. Interestingly, the epithelial basement membrane in UC stained for CD18. In conclusion, CD11b, CD18, and ICAM-2 seem to be important for PMN transepithelial migration in UC, whereas CD11a, CD11c, ICAM-1, and ICAM-3 seem central in leukocyte locomotion and aggregation in CD. Differentiated upregulation of cell adhesion molecules is suggested to be essential for the diversities between UC and CD.

CD18 Antigens↗
PubMed2001

Endothelial function and coronary artery disease.

The endothelium produces a number of vasodilator and vasoconstrictor substances that not only regulate vasomotor tone, but also the recruitment and activity of inflammatory cells and the propensity towards thrombosis. Endothelial vasomotor function is a convenient way to assess these other functions, and is related to the long-term risk of cardiovascular disease. Lipids (particularly low density lipoprotein cholesterol) and oxidant stress play a major role in impairing these functions, by reducing the bioavailability of nitric oxide and activating pro-inflammatory signalling pathways such as nuclear factor kappa B. Biomechanical forces on the endothelium, including low shear stress from disturbed blood flow, also activate the endothelium increasing vasomotor dysfunction and promoting inflammation by upregulating pro-atherogenic genes. In contrast, normal laminar shear stress promotes the expression of genes that may protect against atherosclerosis. The sub-cellular structure of endothelial cells includes caveolae that play an integral part in regulating the activity of endothelial nitric oxide synthase. Low density lipoprotein cholesterol and oxidant stress impair caveolae structure and function and adversely affect endothelial function. Lipid-independent pathways of endothelial cell activation are increasingly recognized, and may provide new therapeutic targets. Endothelial vasoconstrictors, such as endothelin, antagonize endothelium-derived vasodilators and contribute to endothelial dysfunction. Some but not all studies have linked certain genetic polymorphisms of the nitric oxide synthase enzyme to vascular disease and impaired endothelial function. Such genetic heterogeneity may nonetheless offer new insights into the variability of endothelial function.

Animals↗
PubMed2007

Intertwining of thrombosis and inflammation in atherosclerosis.

PURPOSE OF REVIEW: The aim of this article is to highlight the importance of thrombotic processes in the development and complications of atherosclerotic vascular disease. RECENT FINDINGS: Thrombin generated at sites of vascular inflammation activates major atheroma-associated cells including endothelial cells, platelets, smooth muscle cells, monocytes, and macrophages. Thrombin-activated cells produce a plethora of inflammatory mediators, such as regulated upon activation normal T cell expressed presumed secreted, macrophage migration inhibitory factor, and CD40 ligand, that promote atherosclerotic lesion formation and atherothrombotic complications of vascular disease. Additionally, thrombin-induced inflammatory mediators stimulate tissue factor procoagulant activity within atheroma to initiate a positive feedback loop where thrombin activation launches inflammatory signals that lead to further thrombin activation. Platelets, the main cellular effectors of the thrombotic system, also play a central role in the biology of atherosclerosis by producing inflammatory mediators and directing leukocyte incorporation into plaques through platelet-mediated leukocyte adhesion. SUMMARY: New research has identified signaling pathways that intertwine thrombotic and inflammatory pathways with the development and progression of atherosclerosis. These signaling pathways contain positive feedback loops that propagate atherogenesis. Targeting molecular regulators at the interface of thrombosis and inflammation simultaneously may reduce thrombosis and inflammation, thus breaking pathological cycles that promote atherosclerosis and associated thrombotic complications.

Atherosclerosis↗
PubMed2007

Innate immune mucin production via epithelial cell surface signaling: relationship to allergic disease.

PURPOSE OF REVIEW: Airway epithelial surface signaling is provided by epidermal growth factor receptor (EGFR) phosphorylation, resulting in innate immune responses. Here the focus is the EGFR cascades leading to immune mucin responses. The review is timely because recent discoveries implicate these pathways in multiple innate immune defenses in addition to mucin production. RECENT FINDINGS: EGFR activation causes mucin production and inhibition prevents mucin production by multiple stimuli. The receptors and their epithelial-bound proligands are examined. Proteases cleave and release soluble ligand, which then activates EGFR. A surface metalloprotease, tumor necrosis factor alpha-converting enzyme (TACE), modulates proligand release (and thus EGFR activation). TACE is activated by reactive oxygen species, which can be produced by a novel molecule, dual oxidase-1, which provides reactive oxygen species for TACE cleavage. Upstream of dual oxidase-1 are epithelial receptors that receive messages from inhaled irritants and stimulate the dual oxidase-1-TACE-ligand-EGFR cascade. SUMMARY: The EGFR surface signaling pathways are reviewed, with the focus on mucin production, involving human airway epithelial cultures and animal studies, including relevant studies of asthma in humans. Future studies may broaden the innate defenses and utilize these surface signaling pathways in various epithelia, with a variety of pathophysiologic stimuli, with the ultimate aim of examining these pathways in inflammatory diseases.

Animals↗
PubMed2001

The PYRIN domain: a member of the death domain-fold superfamily.

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.

Amino Acid Sequence↗
PubMed2006

TNF-alpha mediated suppression of tissue type plasminogen activator expression in vascular endothelial cells is NF-kappaB- and p38 MAPK-dependent.

BACKGROUND: Several proatherothrombotic conditions are associated with enhanced levels of circulating proinflammatory cytokines, which are believed to impair endothelial fibrinolytic capacity. OBJECTIVE: This study aims at investigating how tumor necrosis factor (TNF)-alpha regulates endothelial gene expression of the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA). METHODS: Cultured human umbilical vein endothelial cells were pretreated with selective inhibitors of the three major inflammatory signaling pathways activated by TNF-alpha; the nuclear factor kappa-B (NF-kappaB), the p38 mitogen-activated protein kinase (p38 MAPK), and the c-jun N-terminal kinase (JNK) pathways. Following TNF-alpha stimulation, effects on t-PA gene expression were evaluated with real-time reverse transcriptase polymerase chain reaction and interactions of nuclear proteins with potential gene regulatory elements were studied with electrophoretic mobility shift assays. RESULTS: Approximately 50% suppression of t-PA gene expression was observed after prolonged stimulation with TNF-alpha (> or =24 h). The repression was shown to be preferentially dependent on NF-kappaB activation, but also on p38 MAPK signaling. Further, we provide evidence for a TNF-alpha induced binding of NF-kappaB to the recently described kappaB site in the t-PA gene and of cyclic adenosine monophosphate response element binding protein (CREB) to the t-PA CRE-like site. CONCLUSIONS: We conclude that TNF-alpha impairs fibrinolytic capacity in vascular endothelial cells by a NF-kappaB and p38 MAPK-dependent suppression of t-PA. This mechanism sheds a light on how inflammation contributes to the pathogenesis of cardiovascular diseases.

Binding Sites↗