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Critical Mediator (critical + mediator)
Selected AbstractsAngiotensin II Is a Critical Mediator of Prazosin-Induced Angiogenesis in Skeletal MuscleMICROCIRCULATION, Issue 6 2007Matthew C. Petersen ABSTRACT Objective: The purpose of this study was to determine whether a high-salt diet modulates physiological angiogenesis in skeletal muscle by altering angiotensin II (ANGII) and vascular endothelial growth factor (VEGF) levels. Methods: Sprague-Dawley rats were placed on a control diet (0.4% NaCl by weight) or high-salt diet (4.0% NaCl) prior to treatment with the vasodilator prazosin in the drinking water. In addition, a group of animals fed high salt were infused intravenously with ANGII at a low dose to prevent ANGII suppression by high salt, and a group of rats fed control diet were treated with the angiotensin II type I (AT1) receptor blocker losartan and prazosin. Results: Prazosin induced significant angiogenesis in the tibialis anterior muscle after 1 week of treatment. High-salt-fed rats demonstrated a complete inhibition of this angiogenic response. Maintenance of ANGII levels restored prazosin-induced angiogenesis in animals fed a high-salt diet. In addition, losartan treatment blocked prazosin-induced angiogenesis in animals on a control diet. Western blot analysis indicated that prazosin-induced angiogenesis was independent of changes in muscle levels of VEGF. Conclusions: This study demonstrates an inhibitory effect of high salt intake on prazosin-induced angiogenesis. Further, these results indicate that ANGII acting through the AT1 receptor is a critical pathway in this model of angiogenesis. [source] Real-time observation of Wnt ,-catenin signaling in the chick embryoDEVELOPMENTAL DYNAMICS, Issue 1 2010Anne C. Rios Abstract A critical mediator of cell,cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Reporter constructs containing multimerized TCF DNA binding sites have been used to detect Wnt ,-catenin dependent activity during animal development. In this report, we have constructed and compared several TCF green fluorescent protein (GFP) reporter constructs. They contained 3, 8, or 12 TCF binding sites upstream of a minimal promoter driving native or destabilized enhanced GFP (EGFP). We have used the electroporation of somites in the chick embryo as a paradigm to test them in vivo. We have verified that they all respond to Wnt signaling in vivo. We have then assessed their efficiency at reflecting the activity of the Wnt pathway. Using destabilized EGFP reporter constructs, we show that somite cells dynamically regulate Wnt/,-catenin,dependent signaling, a finding that was confirmed by performing time-lapse video confocal observation of electroporated embryos. Developmental Dynamics 239:346,353, 2010. © 2009 Wiley-Liss, Inc. [source] Localization of the mosaic transmembrane serine protease corin to heart myocytesFEBS JOURNAL, Issue 23 2000John D. Hooper Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125,135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure. [source] Platelet-activating factor-induced NF-,B activation and IL-8 production in intestinal epithelial cells are Bcl10-dependentINFLAMMATORY BOWEL DISEASES, Issue 4 2010Alip Borthakur PhD Abstract Background: Platelet-activating factor (PAF), a potent proinflammatory phospholipid mediator, has been implicated in inducing intestinal inflammation in diseases such as inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). However, its mechanisms of inducing inflammatory responses are not fully understood. Therefore, studies were designed to explore the mechanisms of PAF-induced inflammatory cascade in intestinal epithelial cells. Methods: Nuclear factor kappa B (NF-,B) activation was measured by luciferase assay and enzyme-linked immunosorbent assay (ELISA), and interleukin 8 (IL-8) production was determined by ELISA. B-cell lymphoma 10 (Bcl10), caspase recruitment domain-containing membrane-associated guanylate kinase protein 3 (CARMA3), and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) mRNA and protein levels were assessed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. siRNA silencing of Bcl10 was used to examine its role in PAF-induced NF-,B activation and IL-8 production. The promoter region of the Bcl10 gene was cloned with the PCR method and promoter activity measured by luciferase assay. Results: The adaptor protein Bcl10 appeared to play an important role in the PAF-induced inflammatory pathway in human intestinal epithelial cells. Bcl10 was required for PAF-induced I,B, phosphorylation, NF-,B activation, and IL-8 production in NCM460, a cell line derived from normal human colon, and Caco-2, a transformed human intestinal cell line. PAF also stimulated Bcl10 interactions with CARMA3 and MALT1, and upregulated Bcl10 expression in these cells via transcriptional regulation. Conclusions: These findings highlight a novel PAF-induced inflammatory pathway in intestinal epithelial cells, requiring Bcl10 as a critical mediator and involving CARMA3/Bcl10/MALT1 interactions. The proinflammatory effects of PAF play prominent roles in the pathogenesis of IBD and this pathway may present important targets for intervention in chronic inflammatory diseases of the intestine. (Inflamm Bowel Dis 2009;) [source] Aberrant p53 alters DNA damage checkpoints in response to cisplatin: Downregulation of CDK expression and activityINTERNATIONAL JOURNAL OF CANCER, Issue 5 2004Katharine H. Wrighton Abstract The p53 tumor suppressor protein is a critical mediator of cell cycle arrest and apoptosis in response to genotoxic stress. Abrogation of p53 function is a major feature of tumor development and may result in a compromised DNA-damage response. In our study, we examined the effect of expressing a human p53 cDNA, encoding a histidine to leucine amino acid substitution at codon 179 (H179L), on the ability of wild-type p53-containing NIH3T3 cells to respond to treatment with the chemotherapeutic cisplatin. After 72 hr of cisplatin treatment control cells underwent apoptosis preceded by a combination of S- and G2 arrest, as judged by flow cytometry of propidium iodide-stained cells, and TUNEL and caspase-3 assays. This correlated with increased expression of the pro-apoptotic protein Bax. In contrast, cells stably expressing H179L-p53 arrested in S-phase following cisplatin treatment, which correlated with a marked decrease in the expression of cdc2, cyclin B1 and cyclin A, and a decrease in CDK2 and cyclin A-associated kinase activity. Interestingly, H179L p53 expressing cells underwent apoptosis earlier than control cells, indicating that this aberrant p53 may enhance cisplatin chemosensitivity. These data suggest that dominant-negative p53 can influence the expression and activity of CDK complexes, thereby modifying cell behavior following cisplatin-induced genotoxicity. © 2004 Wiley-Liss, Inc. [source] PTHrP Signaling Targets Cyclin D1 and Induces Osteoblastic Cell Growth Arrest,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2005Nabanita S Datta PhD Abstract PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. Introduction: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. Materials and Methods: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. Results: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. Conclusion: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts. [source] Stat1-mediated cytoplasmic attenuation in osteoimmunologyJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Hiroshi Takayanagi Abstract Signal transducer and activator of transcription 1 (Stat1) is a critical mediator of gene transcription in type I interferon (IFN-,/,) signaling that is essential for host defense against viruses. In the skeletal system, type I IFNs (IFN-,/,) also play an important physiological role in the inhibition of receptor activator of NF-,B ligand (RANKL)-induced osteoclast differentiation and bone resorption: mice deficient in IFN signaling exhibit decreased bone mass accompanied by the activation of osteoclastogenesis. On the other hand, an unexpected increase in bone mass was observed in Stat1-deficient mice, indicating that Stat1 has a hitherto unknown function in the regulation of bone formation. Indeed, Stat1 was found to have a unique, non-canonical function as a cytoplasmic attenuator of Runx2, a key transcription factor for osteoblast differentiation. Thus, the loss of Stat1 results in excessive activation of Runx2 and osteoblast differentiation, thereby tipping the balance in favor of bone formation over bone resorption. This is an interesting example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a novel regulatory mechanism of bone remodeling by immunomodulatory molecules. Here, we summarize recent advances in the study of Stat1 and IFNs in the context of osteoimmunology, including latest reports that question whether the inhibitory function of Stat1 in chondrocytes is responsible for dwarfism in achondroplasia. © 2004 Wiley-Liss, Inc. [source] 6-Dehydrogingerdione, an active constituent of dietary ginger, induces cell cycle arrest and apoptosis through reactive oxygen species/c-Jun N-terminal kinase pathways in human breast cancer cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 9 2010Ya-Ling Hsu Abstract This study is the first to investigate the anticancer effect of 6-dehydrogingerdione (DGE), an active constituent of dietary ginger, in human breast cancer MDA-MB-231 and MCF-7 cells. DGE exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclin B1, cyclin A, Cdc2 and Cdc25C. DGE also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. DGE triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activation. We also found the generation of reactive oxygen species is a critical mediator in DGE-induced cell growth inhibition. DGE clearly increased the activation of apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2 (ERK1/2) and p38. In addition, antioxidants vitamin C and catalase significantly decreased DGE-mediated JNK activation and apoptosis. Moreover, blocking JNK by specific inhibitors suppressed DGE-triggered mitochondrial apoptotic pathway. Taken together, these findings suggest that a critical role for reactive oxygen species and JNK in DGE-mediated apoptosis of human breast cancer. [source] C-C chemokine receptor 2 (CCR2) deficiency improves bleomycin-induced pulmonary fibrosis by attenuation of both macrophage infiltration and production of macrophage-derived matrix metalloproteinasesTHE JOURNAL OF PATHOLOGY, Issue 5 2004Toshiyuki Okuma Abstract Macrophage infiltration is implicated in various types of pulmonary fibrosis. One important pathogenetic process associated with pulmonary fibrosis is injury to basement membranes by matrix metalloproteinases (MMPs) that are produced mainly by macrophages. In this study, C-C chemokine receptor 2-deficient (CCR2,/,) mice were used to explore the relationship between macrophage infiltration and MMP activity in the pathogenesis of pulmonary fibrosis, using the bleomycin-induced model of this disease process. CCR2 is the main (if not only) receptor for monocyte chemoattractant protein-1/C-C chemokine ligand 2 (MCP-1/CCL2), which is a critical mediator of macrophage trafficking, and CCR2 ,/, mice demonstrate defective macrophage migration. Pulmonary fibrosis was induced in CCR2,/, and wild-type (CCR2+/+) mice by intratracheal instillation of bleomycin. No significant differences in the total protein concentration in bronchoalveolar lavage (BAL) fluid, or in the degree of histological lung inflammation, were observed in the two groups until day 7. Between days 3 and 21, however, BAL fluid from CCR2,/, mice contained fewer macrophages than BAL fluid from CCR2+/+ mice. Gelatin zymography of BAL fluid and in situ zymography revealed reduced gelatinolytic activity in CCR2,/, mice. Immunocytochemical staining showed weaker expression of MMP-2 and MMP-9 in macrophages in BAL fluid from CCR2,/, mice at day 3. Gelatin zymography of protein extracted from alveolar macrophages showed reduced gelatinolytic activity of MMP-2 and MMP-9 in CCR2,/, mice. At days 14 and 21, lung remodelling and the hydroxyproline content of lung tissues were significantly reduced in CCR2,/, mice. These results suggest that the CCL2/CCR2 functional pathway is involved in the pathogenesis of bleomycin-induced pulmonary fibrosis and that CCR2 deficiency may improve the outcome of this disease by regulating macrophage infiltration and macrophage-derived MMP-2 and MMP-9 production. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Economic Well-Being and Children's Social Adjustment: The Role of Family Process in an Ethnically Diverse Low-Income SampleCHILD DEVELOPMENT, Issue 3 2002Rashmita S. Mistry Using latent variable structural equation modeling, a family economic stress model that links economic well-being to child well-being in an ethnically diverse, low-income sample of 419 elementary school-age children was evaluated. The sample was 57% African American and 28% Hispanic, and most families were headed by single mothers. The results provided support for the position that family process is a critical mediator of the effects of economic hardship on children's social adjustment. Lower levels of economic well-being, and the corollary elevated perceptions of economic pressure indirectly affected parenting behavior through an adverse impact on parental psychological well-being. Distressed parents reported feeling less effective and capable in disciplinary interactions with their child and were observed to be less affectionate in parent,child interactions. In turn, less than optimal parenting predicted lower teacher ratings of children's positive social behavior and higher ratings of behavior problems. Multiple-group analyses revealed that the pathways by which economic hardship influences children's behavior appear to operate similarly for boys and girls, and for African American and Hispanic families. [source] T-bet expression by dendritic cells is required for the repolarization of allergic airway inflammation,EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2008Karin L. Heckman Abstract By cross-linking B7-DC on dendritic cells (DC) the human IgM antibody (B7-DC XAb) shifts polarized immune responses from Th2 to Th1 in an antigen-specific manner. The molecular determinants governing the ability of DC to reprogram the polarity of T cell recall responses are not yet known. In addition to the expected role of T-bet expressed by T cells in regulating Th1 responses, we find using in vitro assays and an established in vivo model of allergic airway inflammation that T-bet expression by DC is also required for the polarity shift promoted by B7-DC XAb. T-bet expression by both T cells and DC is critically important for B7-DC XAb-induced down-regulation of IL-4, up-regulation of IFN-, and suppression of allergic airway inflammation. Moreover, retroviral reconstitution of T-bet expression in T-bet-deficient DC rescued their ability to modulate both naive and memory T-cell responses from Th2 to Th1. Our observations further our understanding of the critical mediators controlling the ability of DC to modify the responses of previously activated T cells and reveal the interesting use of the same transcription factor to regulate the inductive phenotype of DC and the inducible phenotype of T cells. [source] | |||||||||||||