Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-κB pathways

2003 ◽  
Vol 284 (6) ◽  
pp. C1397-C1404 ◽  
Author(s):  
Dezheng Zhao ◽  
Sabina Kuhnt-Moore ◽  
Huiyan Zeng ◽  
Jack S. Wu ◽  
Mary P. Moyer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Production ◽  
Rho Gtpase ◽  
Intestinal Inflammation ◽  
Binding Activity ◽  
Dominant Negative ◽  
Colonic Epithelial Cells ◽  
Mapk Phosphorylation ◽  
Dna Binding Activity ◽  
Rho Family

Neurotensin (NT), a neuropeptide highly expressed in the gastrointestinal tract, participates in the pathophysiology of intestinal inflammation. We recently showed that NT stimulates interleukin-8 (IL-8) expression in NCM460 nontransformed human colonic epithelial cells via both mitogen-activating protein kinase (MAPK)- and NF-κB-dependent pathways. However, the molecular mechanism by which NT induces expression of proinflammatory cytokines such as IL-8 has not been investigated. In this study we show that inhibition of endogenous Rho family proteins (RhoA, Rac1, and Cdc42) by their respective dominant negative mutants inhibits NT-induced IL-8 protein production and promoter activity. Western blot experiments demonstrated that NT strongly activated RhoA, Rac1, and Cdc42. Overexpression of the dominant negative mutants of RhoA, Rac1, and Cdc42 significantly inhibited NT-induced NF-κB-dependent reporter gene expression and NF-κB DNA binding activity. NT also stimulated p38 MAPK phosphorylation, and overexpression of dominant negative mutants of RhoA, Rac1, and Cdc42 did not significantly alter p38 and ERK1/2 phosphorylation in response to NT. Together, our findings indicate that NT-stimulated IL-8 expression is mediated via a Rho-dependent NF-κB-mediated pathway.

Differential effects of deoxycholic acid and taurodeoxycholic acid on NF-κB signal transduction and IL-8 gene expression in colonic epithelial cells

2004 ◽  
Vol 286 (6) ◽  
pp. G1000-G1008 ◽  
Author(s):  
M. Mühlbauer ◽  
B. Allard ◽  
A. K. Bosserhoff ◽  
S. Kiessling ◽  
H. Herfarth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Deoxycholic Acid ◽  
Nuclear Translocation ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Colonic Epithelial Cells

Several effects of bile acids (BAs) on colonic epithelial cells (CECs) have been described, including induction of proliferation and apoptosis. Some of these effects are mediated through activation of the NF-κB transcriptional system. In this study, we investigated the molecular mechanisms underlying the BA-induced gene expression in CECs. The human CEC line HT-29 and primary human CECs were treated with dilutions of salts of deoxycholic acid (DCA) and taurodeoxycholic acid (TDCA). NF-κB binding activity was analyzed with EMSA, RelA translocation with immunofluorescence, and IκBα- and RelA-phosphorylation with Western blot analysis. IL-8 mRNA and protein expression were assessed by quantitative PCR and ELISA. Functional impact of NF-κB activation was determined by blocking the proteasome activity with MG132 or by preventing IKK activity with a dominant-negative IKKβ delivered by adenoviral dominant-negative (dn) IKKβ (Ad5dnIKKβ). DCA and TDCA induced IL-8 expression in a dose- and time-dependent manner. It is interesting that DCA but not TDCA induced IκBα-phophorylation, RelA translocation, and NF-κB binding activity. Accordingly, the proteasome inhibitor MG132 blocked DCA- but not TDCA-induced IL-8 gene expression. In contrast, TDCA-induced IL-8 gene expression correlated with enhanced RelA phosphorylation, which was blocked by Ad5dnIKKβ. Our data suggest that DCA-induced signal transduction mainly utilized the IκB degradation and RelA nuclear translocation pathway, whereas TDCA primarily induced IL-8 gene expression through RelA phosphorylation. These differences may have implications for the understanding of the pathophysiology of inflammation and carcinogenesis in the gut.

IL-8 secretion and neutrophil activation by HT-29 colonic epithelial cells

1994 ◽  
Vol 267 (6) ◽  
pp. G991-G997 ◽  
Author(s):  
C. P. Kelly ◽  
S. Keates ◽  
D. Siegenberg ◽  
J. K. Linevsky ◽  
C. Pothoulakis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Inflammation ◽  
Neutrophil Migration ◽  
Intercellular Adhesion Molecule ◽  
Northern Hybridization ◽  
Intercellular Adhesion Molecule 1 ◽  
Colonic Epithelial Cells ◽  
Neutrophil Extravasation ◽  
Cytokine Stimulation ◽  
Ht 29

This study examines the ability of HT-29 human colonic epithelial cells to stimulate neutrophil migration and adhesion. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, was detected in conditioned media from both unstimulated (1.1 ng/ml) and IL-1 beta-stimulated (16.1 ng/ml) HT-29 cultures. Conditioned medium from IL-1 beta-exposed HT-29 cells stimulated neutrophil migration (395% of control, P < 0.01), and this effect was completely inhibited by anti-IL-8 antibody. HT-29 medium also induced shedding of neutrophil L-selectin and increased expression of neutrophil CD11/CD18 adhesion receptors. Coculture of HT-29 cells with human endothelial cell monolayers resulted in increased neutrophil transendothelial migration (169% of control, P < 0.01), which was blocked by both anti-IL-8 and anti-CD18 antibody. Northern hybridization analysis demonstrated increased levels of mRNA for IL-8 and intercellular adhesion molecule-1 (ICAM-1) in cytokine-treated HT-29 cells. Cytokine stimulation of HT-29 monolayers was also associated with increased neutrophil adhesion to these cells. Neutrophil-HT-29 cell adhesion was blocked by monoclonal antibodies to neutrophil CD18 or to ICAM-1 on the HT-29 cells (86% and 56% inhibition, respectively, P < 0.01 for both). These data suggest that IL-8 secretion by activated colonic epithelial cells may contribute to neutrophil extravasation and tissue infiltration in intestinal inflammation.

Characterization of a dominant negativeC. elegansTwist mutant protein with implications for human Saethre-Chotzen syndrome

Development ◽  
2002 ◽  
Vol 129 (11) ◽  
pp. 2761-2772
Author(s):  
Ann K. Corsi ◽  
Thomas M. Brodigan ◽  
Erik M. Jorgensen ◽  
Michael Krause
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Dna Binding Domain ◽  
Mesodermal Cell ◽  
C Elegans ◽  
Dna Binding Activity ◽  
A Charge

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.

Treatment of cultured myotubes with the proteasome inhibitor β-lactone increases the expression of the transcription factor C/EBPβ

2007 ◽  
Vol 292 (1) ◽  
pp. C216-C226 ◽  
Author(s):  
Wei Wei ◽  
Hongmei Yang ◽  
Michael Menconi ◽  
Peirang Cao ◽  
Chester E. Chamberlain ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Proteasome Inhibitor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dna Binding Activity ◽  
Cultured Myotubes ◽  
Nuclear Levels ◽  
Factor C

The role of the proteasome in the regulation of cellular levels of the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is poorly understood. We tested the hypothesis that C/EBPβ levels in cultured myotubes are regulated, at least in part, by proteasome activity. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with the specific proteasome inhibitor β-lactone resulted in increased nuclear levels of C/EBPβ as determined by Western blotting and immunofluorescent detection. This effect of β-lactone reflected inhibited degradation of C/EBPβ. Surprisingly, the increased C/EBPβ levels in β-lactone-treated myotubes did not result in increased DNA-binding activity. In additional experiments, treatment of the myotubes with β-lactone resulted in increased nuclear levels of growth arrest DNA damage/C/EBP homologous protein (Gadd153/CHOP), a dominant-negative member of the C/EBP family that can form heterodimers with other members of the C/EBP family and block DNA binding. Coimmunoprecipitation and immunofluorescent detection provided evidence that C/EBPβ and Gadd153/CHOP interacted and colocalized in the nuclei of the β-lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBPβ DNA-binding activity was restored in β-lactone-treated myotubes. The results suggest that C/EBPβ is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBPβ and block its DNA-binding activity. The observations are important because they increase the understanding of the complex regulation of the expression and activity of C/EBPβ in skeletal muscle.

Ozone-induced IL-8 expression and transcription factor binding in respiratory epithelial cells

1997 ◽  
Vol 272 (3) ◽  
pp. L504-L511 ◽  
Author(s):  
I. Jaspers ◽  
E. Flescher ◽  
L. C. Chen
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Epithelial Cells ◽  
Inflammatory Cells ◽  
Binding Activity ◽  
Ozone Exposure ◽  
Air Exposure ◽  
Dna Binding Activity ◽  
Nf Kappab

Ozone, one of the most reactive oxidant gases to which humans are routinely exposed, induces inflammation in the lower airways. The airway epithelium is one of the first targets that inhaled ozone will encounter, but its role in airway inflammation is not well understood. Expression of inducible genes involved in the inflammatory response, such as interleukin (IL)-8, is controlled by transcription factors. Expression of the IL-8 gene is regulated by the transcription factors nuclear factor (NF)-kappaB, NF-IL-6, and possibly activator protein-1 (AP-1). Type II-like epithelial cells (A549) were grown on a collagen-coated membrane and exposed in vitro to 0.1 ppm ozone or air. Exposure to ozone induced DNA-binding activity of NF-kappaB, NF-IL-6, and AP-1. IL-8 mRNA and IL-8 protein levels were also increased after ozone exposure. These results link ozone-induced DNA-binding activity of transcription factors and the production of IL-8 by epithelial cells thus demonstrating a potential cellular cascade resulting in the recruitment of inflammatory cells into the airway lumen.

scholarly journals Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription.

1996 ◽  
Vol 16 (11) ◽  
pp. 6372-6384 ◽  
Author(s):  
C Y Chen ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Actin Gene ◽  
Response Factor ◽  
Dna Binding Activity ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

We recently showed that the cardiogenic homeodomain factor Nkx-2.5 served as a positive acting accessory factor for serum response factor (SRF) and that together they provided strong transcriptional activation of the cardiac alpha-actin promoter, depending upon intact serum response elements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T. McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130, 1996). As shown here, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by a mechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter. Activation of a truncated promoter consisting of the proximal alpha-actin SRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by a point mutation in the third helix of its homeodomain. Investigation of protein-protein interactions showed that Nkx-2.5 was bound to SRF in the absence of DNA in soluble protein complexes retrieved from cardiac myocyte nuclei but could also be detected in coassociated binding complexes on the proximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRF DNA-binding activity and was blocked by the dominant negative SRFpm1 mutant, which allowed for dimerization of SRF monomers but prevented DNA binding. Interactive regions shared by Nkx-2.5 and SRF were mapped to N-terminal/helix I and helix II/helix III regions of the Nkx-2.5 homeodomain and to the N-terminal extension of the MADS box. Our study suggests that physical association between Nkx-2.5 and SRF is one way that cardiac specified genes are activated in cardiac cell lineages.

Pneumococci induced TLR- and Rac1-dependent NF-κB-recruitment to the IL-8 promoter in lung epithelial cells

2006 ◽  
Vol 290 (4) ◽  
pp. L730-L737 ◽  
Author(s):  
Bernd Schmeck ◽  
Sylvia Huber ◽  
Kerstin Moog ◽  
Janine Zahlten ◽  
Andreas C. Hocke ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Activation ◽  
Rho Gtpase ◽  
Dominant Negative ◽  
Lung Epithelial Cells ◽  
Dominant Negative Mutant ◽  
Epithelial Cell Line ◽  
Phosphatidylinositol 3 Kinase ◽  
Lung Epithelial ◽  
Phosphatidylinositol 3

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia. The respiratory epithelium constitutes the first line of defense against invading lung pathogens, including pneumococci. We analyzed the involvement of Toll-like receptors (TLR) and Rho-GTPase signaling in the activation of human lung epithelial cells by pneumococci. S. pneumoniae induced release of interleukin-8 (IL-8) by human bronchial epithelial cell line BEAS-2B. Specific inhibition of Rac1 by Nsc23766 or a dominant-negative mutant of Rac1 strongly reduced cytokine release. In addition, pneumococci-related cell activation (IL-8 release, NF-κB-activation) depended on MyD88, phosphatidylinositol 3-kinase, and Cdc42 but not on RhoA. Pneumococci enhanced TLR1 and TLR2 mRNA expression in BEAS-2B cells, whereas TLR4 and TLR6 expression was constitutively high. TLR1 and 2 synergistically recognized pneumococci in cotransfection experiments. TLR4, TLR6, LPS-binding protein, and CD14 seem not to be involved in pneumococci-dependent cell activation. At the IL-8 gene promoter, recruitment of phosphorylated NF-κB subunit p65 was blocked by inhibition of Rac1, whereas binding of the phosphorylated activator protein-1 subunit c-Jun to the promoter was not diminished. In summary, these results suggest that S. pneumoniae activate human epithelial cells by TLR1/2 and a phosphatidylinositol 3-kinase- and Rac1-dependent NF-κB-recruitment to the IL-8 promoter.

Neurotensin stimulates interleukin-8 expression in human colonic epithelial cells through Rho GTPase-mediated NFkappaB pathways

2003 ◽  
Vol 124 (4) ◽  
pp. A465-A466
Author(s):  
Dezheng Zhao ◽  
Sabina Kuhnt-Moore ◽  
Huiyan Zeng ◽  
Jack S. Wu ◽  
Mary P. Moyer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Rho Gtpase ◽  
Interleukin 8 ◽  
Colonic Epithelial Cells
Sign in / Sign up

Export Citation Format

Share Document