Transcription factor NF-κB participates in regulation of epithelial cell turnover in the colon

2000 ◽  
Vol 279 (6) ◽  
pp. G1282-G1291 ◽  
Author(s):  
Mehmet Sait Inan ◽  
Veronica Tolmacheva ◽  
Qiang-Shu Wang ◽  
Daniel W. Rosenberg ◽  
Charles Giardina
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Transcription Factor ◽  
Epithelial Cell ◽  
Dna Binding ◽  
Epithelial Cells ◽  
Knockout Mice ◽  
Nuclear Antigen ◽  
Cell Turnover ◽  
Tnf Α

The transcription factor nuclear factor (NF)-κB regulates the expression of genes that can influence cell proliferation and death. Here we analyze the contribution of NF-κB to the regulation of epithelial cell turnover in the colon. Immunohistochemical, immunoblot, and DNA binding analyses indicate that NF-κB complexes change as colonocytes mature: p65-p50 complexes predominate in proliferating epithelial cells of the colon, whereas the p50-p50 dimer is prevalent in mature epithelial cells. NF-κB1 (p50) knockout mice were used to study the role of NF-κB in regulating epithelial cell turnover. Knockout animals lacked detectable NF-κB DNA binding activity in isolated epithelial cells and had significantly longer crypts with a more extensive proliferative zone than their wild-type counterparts (as determined by proliferating cell nuclear antigen staining and in vivo bromodeoxyuridine labeling). Gene expression profiling reveals that the NF-κB1 knockout mice express the potentially growth-enhancing tumor necrosis factor (TNF)-α and nerve growth factor-α genes at elevated levels, with in situ hybridization localizing some of the TNF-α expression to epithelial cells. TNF-α is NF-κB regulated, and its upregulation in NF-κB1 knockouts may result from an alleviation of p50-p50 repression. NF-κB complexes may therefore influence cell proliferation in the colon through their ability to selectively activate and/or repress gene expression.

Changes in human bladder epithelial cell gene expression associated with interstitial cystitis or antiproliferative factor treatment

2003 ◽  
Vol 14 (2) ◽  
pp. 107-115 ◽  
Author(s):  
Susan Keay ◽  
Francoise Seillier-Moiseiwitsch ◽  
Chen-Ou Zhang ◽  
Toby C. Chai ◽  
Jialu Zhang
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Interstitial Cystitis ◽  
Expression Patterns ◽  
Epithelial Cell Proliferation ◽  
Normal Cells ◽  
Bladder Epithelial Cell ◽  
Normal Bladder

Explanted bladder epithelial cells from patients with interstitial cystitis (IC) have been shown to differ from explanted control cells in several ways, including production of an antiproliferative factor (APF), altered production of certain epithelial growth factors, and rate of proliferation. To better understand the role of the APF in abnormal bladder epithelial cell proliferation in IC, we studied gene expression patterns in normal bladder epithelial cells treated with APF vs. mock APF and compared them to expression patterns in IC vs. normal cells using microarray analysis. Oligo-dT-primed total cellular RNA was labeled with [33P]dCTP and hybridized to GeneFilter GF211 microarray membranes (Research Genetics) containing cDNA for 3,964 human genes. Thirteen genes that function in epithelial cell proliferation or differentiation were consistently differentially expressed in both IC (compared with control) and APF-treated (compared with mock APF-treated) normal bladder epithelial cells. The general pattern of gene expression in IC and APF-treated cells suggested a less proliferative phenotype, with increased expression of E-cadherin, phosphoribosylpyrophosphate synthetase-associated protein 39, and SWI/SNF complex 170-kDa subunit, and decreased expression of vimentin, α2-integrin, α1-catenin, cyclin D1, and jun N-terminal kinase 1; these findings were confirmed for the structural gene products (E-cadherin, vimentin, α2-integrin, and α-catenin) by immunohistochemistry. These results are compatible with the previously noted decreased proliferation rate of IC and APF-treated normal cells, and indicate that the mechanism whereby APF inhibits cell proliferation may involve both downregulation of genes that stimulate cell proliferation along with upregulation of genes that inhibit cell growth.

Estrogen-dependent responses of the mammary fat pad in prepubertal dairy heifers

2006 ◽  
Vol 190 (3) ◽  
pp. 819-827 ◽  
Author(s):  
M J Meyer ◽  
A V Capuco ◽  
Y R Boisclair ◽  
M E Van Amburgh
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Epithelial Cell Proliferation ◽  
Fat Pad ◽  
Dairy Heifers ◽  
Ovarian Status ◽  
Mammary Fat Pad ◽  
Igf I

Ovaries are absolutely required for development of the mammary parenchyma (PAR) in cattle, reflecting estrogen-dependent epithelial cell proliferation. However, the estrogen receptor (ER) that mediates the mammary estrogen effects, ERα, is absent in proliferating epithelial cells. In the mouse, this discrepancy is explained in part by the ability of the mammary fat pad (MFP) to synthesize epithelial cell mitogens such as IGF-I in response to estrogen. Consistent with a similar role for the bovine MFP, 30% of its fibroblasts and adipocytes were immunoreactive for ERα in prepubertal dairy heifers. To assess estrogen-dependent gene expression in the MFP, 16 prepubertal dairy heifers were randomly assigned to a 2×2 factorial. The first factor was ovarian status, with heifers undergoing bilateral ovariectomy or left intact at 4.6 months of age. The second factor was applied 30 days after surgery and consisted of injection of estrogen or excipient. After 3 days of injection, heifers were administered an intrajugular bolus of bromodeoxyuridine (BrdU) and slaughtered 2 h later. The estrogen injection, but not ovarian status, caused significant increases in the fraction of epithelial cells labeled with BrdU and produced tissue-specific effects on gene expression. In the PAR, estrogen injection increased IGF-I gene expression by twofold despite reductions of 50% or more in ERα mRNA abundance and the fraction of epithelial cells immunoreactive for ERα. The estrogen-dependent increase in IGF-I mRNA was greater in the MFP, presumably because estrogen failed to downregulate ERα expression in this mammary compartment. Finally, estrogen-responsiveness of the MFP appears unique among the bovine fat depots as estrogen injection did not induce IGF-I expression in its s.c. counterpart. Our data demonstrate that the bovine MFP is highly responsive to exogenous estrogen, consistent with a role for this tissue compartment in communicating its effects on epithelial cell proliferation.

Spatial repression of PCNA by p53 during kidney development

2002 ◽  
Vol 283 (4) ◽  
pp. F727-F733 ◽  
Author(s):  
Zubaida Saifudeen ◽  
Jessica Marks ◽  
Hong Du ◽  
Samir S. El-Dahr
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Epithelial Cells ◽  
Transcriptional Repression ◽  
Kidney Development ◽  
Terminal Differentiation ◽  
Nuclear Antigen ◽  
Renal Epithelial Cells ◽  
Transcription Factor P53 ◽  
The Impact

Transcriptional repression is a key mechanism for the spatial specification of gene expression and cell fate determination. During kidney development, proliferating cell nuclear antigen (PCNA) is expressed in the nephrogenic zone and is downregulated rapidly as renal epithelial cells enter terminal differentiation and acquire functional characteristics. Our laboratory reported that the transcription factor p53 stimulates the terminal differentiation of renal epithelial cells by means of transcriptional activation of renal function genes (Saifudeen Z, Dipp S, and El-Dahr SS. J Clin Invest 109: 1021–1030, 2002). Because p53-induced growth arrest correlates with downregulation of PCNA gene expression, we examined the impact of p53 inactivation on PCNA expression in mice and evaluated the effect of p53 on PCNA transcription. Immunohistochemistry revealed that the transition from nephrogenesis to terminal epithelial cell differentiation correlates with accumulation of the transcription factor p53. Importantly, the spatially restricted pattern of PCNA expression is disrupted in kidneys of p53-deficient pups, in which there was a redistribution of PCNA expression into the differentiation zone (without a change in total kidney PCNA content) and distortion of the tubular architecture. Electrophoretic mobility shift assays revealed that the binding of kidney nuclear extracts to the p53 response elements in human and rat PCNA promoters is developmentally regulated. Transient transfection assays performed in p53-deficient HeLa cells revealed that exogenous p53 strongly represses transcription from human PCNA promoter-reporter constructs. Interestingly, deletion of the p53-binding site confers enhanced responsiveness to p53-mediated repression, suggesting that transcriptional repression of PCNA by p53 is achieved by a mechanism other than direct DNA binding. On the basis of these results, we propose the hypothesis that p53-mediated transcriptional repression plays a role in the spatial restriction of PCNA gene expression during normal renal development.

scholarly journals Pseudorabies Virus Infection of Epithelial Cells Leads to Persistent but Aberrant Activation of the NF-κB Pathway, Inhibiting Hallmark NF-κB-Induced Proinflammatory Gene Expression

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Nicolás Romero ◽  
Cliff Van Waesberghe ◽  
Herman W. Favoreel
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Epithelial Cells ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Pseudorabies Virus ◽  
Negative Feedback Loop ◽  
Tnf Α ◽  
Proinflammatory Gene ◽  
Proinflammatory Gene Expression

ABSTRACT The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes, like the gene for IκBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κB signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-κB signaling, which may aid the virus in modulating early proinflammatory responses in the infected host. IMPORTANCE The NF-κB transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-κB, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-κB activation shares some mechanistic features with canonical NF-κB activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IκB kinase (IKK) and even renders infected cells resistant to canonical NF-κB activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation.

Ceramide decreases surfactant protein B gene expression via downregulation of TTF-1 DNA binding activity

2006 ◽  
Vol 290 (2) ◽  
pp. L351-L358 ◽  
Author(s):  
Loretta Sparkman ◽  
Hemakumar Chandru ◽  
Vijayakumar Boggaram
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Promoter Activity ◽  
Surfactant Protein ◽  
Binding Activity ◽  
Mrna Levels ◽  
Dna Binding Activity ◽  
Surfactant Protein B ◽  
Tnf Α

Ceramide, a sphingolipid, is an important signaling molecule in the inflammatory response. Mediators of acute lung injury such as TNF-α, platelet-activating factor, and Fas/Apo ligand stimulate sphingomyelin hydrolysis to increase intracellular ceramide levels. Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for surfactant function and lung stability. In this study we investigated the effects of ceramide on SP-B gene expression in H441 lung epithelial cells. Ceramide decreased SP-B mRNA levels in control and dexamethasone-treated cells after 24-h incubation and inhibition of SP-B mRNA was associated with inhibition of immunoreactive SP-B. In transient transfections assays, ceramide inhibited SP-B promoter activity, indicating that the inhibitory effects are exerted at the transcriptional level. Deletion mapping experiments showed that the ceramide-responsive region is located within the −233/−80-bp region of human SP-B promoter. Electrophoretic mobility shift and reporter assays showed that ceramide reduced the DNA binding activity and transactivation capability of thyroid transcription factor 1 (TTF-1/Nkx2.1), a key factor for SP-B promoter activity. Collectively these data showed that ceramide inhibits SP-B gene expression by reducing the DNA biding activity of TTF-1/Nkx2.1 transcription factor. Protein kinase C inhibitor bisindolylmaleimide and the protein tyrosine kinase inhibitor genistein partially reversed ceramide inhibition, indicating that protein kinases play important roles in the ceramide inhibition of SP-B gene expression. Chemical inhibitors of de novo ceramide synthesis and sphingomyelin hydrolysis had no effect on TNF-α inhibition of SP-B promoter activity and mRNA levels, suggesting that ceramide does not play a role in the inhibition.

An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro

1998 ◽  
Vol 275 (2) ◽  
pp. F278-F284 ◽  
Author(s):  
Naruya Tomita ◽  
Masatsugu Horiuchi ◽  
Sawako Tomita ◽  
Gary H. Gibbons ◽  
John Y. S. Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Transcription Factor ◽  
Nuclear Antigen ◽  
Luciferase Reporter ◽  
High Affinity ◽  
Serum Stimulation ◽  
Transcription Factor E2f ◽  
Proliferation In Vitro

The transcription factor E2F controls expression of several genes involved in cell proliferation including c- myc, c- myb, proliferating cell nuclear antigen (PCNA), and cdk2 kinase. Having established that both PCNA and cdk2 kinase are induced in rat mesangial cells (MC) by serum stimulation, we attempted to inhibit MC proliferation in vitro by transfecting these cells with cationic liposomes containing a synthetic double-stranded oligodeoxynucleotide (ODN) with high affinity for E2F. Using a gel mobility shift assay, we detected increased specific binding of E2F in MC following serum stimulation. This binding was completely inhibited by preincubation of MC nuclear extracts with the double-stranded ODN with high affinity for E2F but not by preincubation with a missense ODN containing two point mutations. MC were also transfected with a luciferase reporter gene construct containing three E2F binding sites. Luciferase activity was enhanced by serum stimulation of MC, and this effect was specifically abolished by cotransfection of MC with E2F decoy ODN. Furthermore, RT-PCR analysis revealed that serum-induced upregulation of PCNA and cdk2 kinase gene expression was inhibited by E2F decoy ODN transfection but not by transfection of missense ODN. These changes in gene expression were paralleled by a reduction in PCNA and cdk2 kinase protein expression in E2F decoy ODN transfected cells. MC number increased following serum stimulation. This effect was blunted by transfection with E2F decoy ODN but not by transfection of missense ODN. These data suggest that the transcription factor E2F plays a crucial role in the regulation of MC proliferation and that this factor can be successfully targeted to inhibit MC cell cycle progression.

scholarly journals c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

2011 ◽  
Vol 301 (2) ◽  
pp. C522-C529 ◽  
Author(s):  
Justine Elliott ◽  
Nadezhda N. Zheleznova ◽  
Patricia D. Wilson
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Collecting Duct ◽  
Specific Inhibitor ◽  
Cell Phenotype ◽  
Epithelial Cell Proliferation ◽  
Matrix Adhesion ◽  
Cyst Lining

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY418)-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY418-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α2β1-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.

scholarly journals A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

2008 ◽  
Vol 22 (12) ◽  
pp. 2677-2688 ◽  
Author(s):  
Paul G. Tiffen ◽  
Nader Omidvar ◽  
Nuria Marquez-Almuina ◽  
Dawn Croston ◽  
Christine J. Watson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Oncostatin M ◽  
Specific Gene ◽  
Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

Concurrence between the gene expression pattern of Actinobacillus actinomycetemcomitans in localized aggressive periodontitis and in human epithelial cells

2005 ◽  
Vol 54 (5) ◽  
pp. 497-504 ◽  
Author(s):  
Joseph Richardson ◽  
Justin Corey Craighead ◽  
Sam Linsen Cao ◽  
Martin Handfield
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Aggressive Periodontitis ◽  
Actinobacillus Actinomycetemcomitans ◽  
Bacterial Gene ◽  
Bacterial Gene Expression

Actinobacillus actinomycetemcomitans is a facultatively intracellular pathogen and the aetiological agent of localized aggressive periodontitis. Screening of the genome of A. actinomycetemcomitans for in vivo-induced antigen determinants previously demonstrated that the proteome of this organism differs in laboratory culture compared with conditions found during active infection. The aim of the present study was to determine whether the bacterial gene expression pattern inferred with in vivo-induced antigen technology (IVIAT) in human infections was consistent with the gene expression pattern occurring upon epithelial cell association. To this end, a real-time PCR method was developed and used to quantify absolute and relative bacterial gene expression of A. actinomycetemcomitans grown extra- and intracellularly in two human epithelial cell lines (HeLa and IHGK). The amount of template used in the assay was normalized using the total count of viable bacteria (c.f.u.) as a reference point and performed in duplicate in at least two independent experiments. Controls for this experiment included 16S rRNA and gapdh. Transcription of all eight ORFs tested increased significantly (P < 0.05) in HeLa and IHGK cells compared with bacteria grown extracellularly. The concurrence of gene expression patterns found in the two models suggests that these epithelial cells are valid in vitro models of infection for the genes tested. IVIAT is an experimental platform that can be used as a validation tool to assess the reliability of animal and other models of infection and is applicable to most pathogens.

scholarly journals Glucocorticoid Receptor Activation Signals through Forkhead Transcription Factor 3a in Breast Cancer Cells

2006 ◽  
Vol 20 (10) ◽  
pp. 2304-2314 ◽  
Author(s):  
Wei Wu ◽  
Min Zou ◽  
Deanna R. Brickley ◽  
Travis Pew ◽  
Suzanne D. Conzen
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Transcription Factor ◽  
Epithelial Cell ◽  
Mammary Epithelial Cell ◽  
Breast Cancer Cells ◽  
Receptor Activation ◽  
Mammary Epithelial ◽  
Forkhead Transcription Factor ◽  
Igfbp 3

Abstract Activation of the glucocorticoid receptor (GR) plays a critical role in the stress response of virtually all cell types. Despite recent advances in large-scale genomic and proteomic data acquisition, identification of physiologically relevant molecular events downstream of nuclear hormone receptor activation remains challenging. By analyzing gene expression changes 30 min after dexamethasone (Dex) treatment, we previously found that immediate induction of serum and glucocorticoid-regulated kinase-1 (SGK-1) expression is required for GR-mediated mammary epithelial cell survival signaling. We now report that activation of the GR mediates Forkhead transcription factor 3a (FOXO3a) phosphorylation and inactivation in mammary epithelial cells. GR-mediated induction of SGK-1 expression is required for FOXO3a inactivation; additional growth factor stimulation is not required. To further explore the gene expression changes that occur downstream of GR-mediated FOXO3a inactivation, we analyzed temporal gene expression data and selected GR-down-regulated genes containing core FOXO3a binding motifs in their proximal promoters. This approach revealed several previously unrecognized transcriptional target genes of FOXO3a, including IGF binding protein-3 (IGFBP-3). Endogenous IGFBP-3 expression was confirmed to be dependent on the GR-SGK-1-FOXO3a signaling pathway. Moreover, GR activation decreased FOXO3a-induced apoptosis in SK-BR-3 breast cancer cells. Collectively, our data suggest that GR-mediated FOXO3a inactivation is an important mechanism contributing to glucocorticoid-mediated mammary epithelial cell survival.

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