Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1

2000 ◽  
Vol 279 (6) ◽  
pp. G1169-G1176 ◽  
Author(s):  
Shigetada Teshima ◽  
Hiromu Kutsumi ◽  
Tsukasa Kawahara ◽  
Kyoichi Kishi ◽  
Kazuhito Rokutan
Keyword(s):  
Guinea Pig ◽  
Chromatin Condensation ◽  
Primary Cultures ◽  
Nuclear Factor Κb ◽  
Thymidine Uptake ◽  
Nuclear Factor ◽  
Spontaneous Apoptosis ◽  
Oxygen Intermediates ◽  
Diphenylene Iodonium ◽  
Pit Cells

We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40- phox) and could spontaneously release superoxide anion (O2−). We demonstrate here that pit cells express a nonphagocyte-specific gp91- phox homolog (Mox1) but not gp91- phox. Inclusion of catalase significantly inhibited [3H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O2−and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH2F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-κB, and inhibition of this activity by nuclear factor-κB decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O2−produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.

Bafilomycin A1inhibits rhinovirus infection in human airway epithelium: effects on endosome and ICAM-1

2001 ◽  
Vol 280 (6) ◽  
pp. L1115-L1127 ◽  
Author(s):  
Tomoko Suzuki ◽  
Mutsuo Yamaya ◽  
Kiyohisa Sekizawa ◽  
Masayoshi Hosoda ◽  
Norihiro Yamada ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelium ◽  
Primary Cultures ◽  
Nuclear Factor Κb ◽  
Intercellular Adhesion Molecule ◽  
Nuclear Factor ◽  
Tracheal Epithelial Cells ◽  
Before And After ◽  
Infected Cells ◽  
Viral Titers

To examine the effects of bafilomycin A1, a blocker of vacuolar H+-ATPase, on rhinovirus (RV) infection in the airway epithelium, primary cultures of human tracheal epithelial cells were infected with RV14. Viral infection was confirmed by showing that viral RNA in the infected cells and the viral titers in the supernatants of infected cells increased with time. RV14 infection upregulated the production of cytokines and mRNA of intercellular adhesion molecule (ICAM)-1 in epithelial cells. Bafilomycin A1reduced the viral titers of RV14 and inhibited the production of cytokines and ICAM-1 before and after RV14 infection. Bafilomycin A1reduced susceptibility of epithelial cells to RV14 infection. RV14 increased activated nuclear factor-κB in the cells, and bafilomycin A1reduced the activated nuclear factor-κB. Bafilomycin A1decreased the number of acidic endosomes in the epithelial cells. These results suggest that bafilomycin A1may inhibit infection by RV14 by not only blocking RV RNA entry into the endosomes but also reducing ICAM-1 expression in the epithelial cells. Bafilomycin A1may therefore modulate airway inflammation after RV infection.

CGRP inhibits osteoprotegerin production in human osteoblast-like cells via cAMP/PKA-dependent pathway

2006 ◽  
Vol 291 (3) ◽  
pp. C529-C537 ◽  
Author(s):  
I. Villa ◽  
E. Mrak ◽  
A. Rubinacci ◽  
F. Ravasi ◽  
F. Guidobono
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Primary Cultures ◽  
Nuclear Factor Κb ◽  
Conditioned Media ◽  
Fine Tuning ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nuclear Factor ◽  
Human Osteoblast ◽  
Receptor Activator

The osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK) system was evaluated as a potential target of CGRP anabolic activity on bone. Primary cultures of human osteoblast-like cells (hOB) express calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1, and, because CGRP stimulates cAMP (one of the modulators of OPG production in osteoblasts), it was investigated whether it affects OPG secretion and expression in hOB. CGRP treatment of hOB (10−11 M–10−7 M) dose-dependently inhibited OPG secretion with an EC50 of 1.08 × 10−10 M, and also decreased its expression. This action was blocked by the antagonist CGRP8–37. Forskolin, a stimulator of cAMP production, and dibutyryl cAMP also reduced the production of OPG. CGRP (10−8 M) enhanced protein kinase A (PKA) activity in hOB, and hOB exposure to the PKA inhibitor, H89 (2 × 10−6 M), abolished the inhibitory effect of CGRP on OPG secretion. Conditioned media from CGRP-treated hOB increased the number of multinucleated tartrate-resistant acid phosphatase-positive cells and the secretion of cathepsin K in human peripheral blood mononuclear cells compared with the conditioned media of untreated hOB. These results show that the cAMP/PKA pathway is involved in the CGRP inhibition of OPG mRNA and protein secretion in hOB and that this effect favors osteoclastogenesis. CGRP could thus modulate the balance between osteoblast and osteoclast activity, participating in the fine tuning of all of the bone remodeling phases necessary for the subsequent anabolic effect.

TGF-alpha is a potent mitogen for primary cultures of guinea pig gastric mucous epithelial cells

1993 ◽  
Vol 265 (2) ◽  
pp. G361-G369 ◽  
Author(s):  
M. J. Rutten ◽  
P. J. Dempsey ◽  
T. E. Solomon ◽  
R. J. Coffey
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Guinea Pig ◽  
Primary Cultures ◽  
Fold Increase ◽  
Thymidine Uptake ◽  
Serum Free ◽  
Cell Counts ◽  
Gastric Mucous ◽  
Serum Free Conditions

Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are thought to be important in gastric epithelial proliferation and repair. It was therefore of interest to determine if TGF-alpha and EGF promoted the growth of an in vitro primary culture system of guinea pig gastric mucous epithelial cells (MEC). MEC were isolated from guinea pig stomachs and cultured in 24-well Primaria plates with DMEM with or without 10% fetal calf serum (FCS). Growth of MEC was determined by changes in [3H]thymidine uptake, cell counts, protein, and DNA. The sources of peptides were human recombinant TGF-alpha (recTGF-alpha) and human recombinant EGF (recEGF). Both recTGF-alpha and recEGF were used at equipotent doses as determined by competing activity in a 125I-labeled TGF-alpha radioreceptor binding assay using A-431 cells. Basal growth (no peptides) of MEC in 10% FCS was dependent on the initial plating density. Under serum-free conditions, [3H]thymidine uptake increased up to 17-fold at 24 h with recTGF-alpha (0.1-10.0 nM) compared with only a 4-fold increase using rec-EGF (0.1-10.0 nM) at this same time period. Under serum-free conditions, recTGF-alpha (0.01-10.0 nM) increased cell counts up to 4.9-fold over control cultures, whereas similar does of recEGF produced a 2.5-fold increase in cell counts. Administration of recEGF 1 ng/ml) resulted in a 1.9-fold increase in the 4.8-kb TGF-alpha mRNA transcript, and TGF-alpha protein immunoreactivity was found in both 24-h conditioned media and cell lysates.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Mechanical Stretch Up-Regulates the Human Oxytocin Receptor in Primary Human Uterine Myocytes

2005 ◽  
Vol 90 (1) ◽  
pp. 237-246 ◽  
Author(s):  
Vasso Terzidou ◽  
Suren R. Sooranna ◽  
Louise U. Kim ◽  
Steve Thornton ◽  
Phillip R. Bennett ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mrna Expression ◽  
Promoter Activity ◽  
Primary Cultures ◽  
Mechanical Stretch ◽  
Nuclear Factor Κb ◽  
Oxytocin Receptor ◽  
Nuclear Factor ◽  
L Cells

Abstract Oxytocin receptor (OTR) expression is increased before the onset of labor in all models of parturition. However, the mechanisms responsible for the increase in OTR expression are uncertain. Animal data suggest that uterine stretch increases OTR mRNA expression. In primary cultures of human uterine smooth muscle cells obtained from nonpregnant (NP) women and pregnant women before (NL) and after (L) the onset of labor, we investigated the effect of stretch on the expression of OTR mRNA and DNA binding of activator protein-1 (AP-1), CCAAT/enhancer binding protein (C/EBP)β, and nuclear factor-κB transcription factors. OTR expression was least in NL, intermediate in NP, and greatest in L cells. Stretch of NL cells resulted in up-regulation of OTR mRNA expression associated with increased OTR gene promoter activity. Stretch of NP and L cells did not affect OTR mRNA expression. The increased promoter activity was associated with increased DNA binding of C/EBP and AP-1 but not nuclear factor-κB transcription factors. Overexpression of C/EBP, but not AP-1, increased OTR promoter activity. We conclude that stretch of NL cells results in increased OTR mRNA expression probably through increased C/EBPβ DNA binding. These data suggest that stretch contributes to the massive increase in OTR expression before the onset of human labor.

scholarly journals 2-mercaptoethanol restores the ability of nuclear factor κB (NF κB) to bind DNA in nuclear extracts from interleukin 1-treated cells incubated with pyrollidine dithiocarbamate (PDTC). Evidence for oxidation of glutathione in the mechanism of inhibition of NFκB by PDTC

1996 ◽  
Vol 320 (3) ◽  
pp. 975-981 ◽  
Author(s):  
Paul BRENNAN ◽  
Luke A. J. O'NEILL
Keyword(s):  
Dna Binding ◽  
Interleukin 1 ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Oxygen Intermediates ◽  
Mechanism Of Inhibition ◽  
Nuclear Extracts ◽  
Anti Oxidant

The metal chelator and anti-oxidant pyrollidine dithiocarbamate (PDTC) has been used extensively in studies implicating reactive oxygen intermediates in the activation of nuclear factor κB (NFκB). In agreement with other studies, we have shown that PDTC inhibits NFκB activation in response to the pro-inflammatory cytokines interleukin 1 (IL1) and tumour necrosis factor (TNF). However, we have found that the inhibition was reversed by treatment of inhibited nuclear extracts with the reducing agent 2-mercaptoethanol. This was observed in extracts prepared from IL1-treated EL4.NOB-1 thymoma cells and TNF-treated Jurkat E6.1 lymphoma cells. These results suggested that the inhibition was caused by oxidation of NFκB on a sensitive thiol, possibly on the p50 subunit (which was detected in NFκB complexes in both cell types), and not by inhibition of the activation pathway. The possibility that PDTC was acting as a pro-oxidant was therefore investigated. PDTC caused an increase in oxidized glutathione, suggesting that it acts as an oxidizing agent in the cells tested rather than as an anti-oxidant. Similar results were obtained with diamide, a compound designed to oxidize glutathione. Finally, an increase in the ratio of oxidized to reduced glutathione was shown to inhibit NFκB–DNA binding in vitro. On the basis of these results we suggest that, while NFκB activation is unaffected by PDTC, DNA binding is inhibited through a mechanism involving a shift towards oxidizing conditions, and that this is the mechanism of action of both PDTC and diamide in the cells tested here.

Nuclear Factor-κB Activates Transcription of the Androgen Receptor Gene in Sertoli Cells Isolated from Testes of Adult Rats

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 781-789 ◽  
Author(s):  
Liying Zhang ◽  
Martin Charron ◽  
William W. Wright ◽  
Bandana Chatterjee ◽  
Chung S. Song ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Sertoli Cells ◽  
Receptor Gene ◽  
Primary Cultures ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Creb Binding Protein ◽  
Luciferase Reporter Gene ◽  
Adult Rats

Abstract The androgen receptor (AR) in Sertoli cells mediates the actions of testosterone on spermatogenesis. However, the transcription factors responsible for AR gene regulation in Sertoli cells remain unknown. In this study, we determined that nuclear factor-κB (NF-κB) regulates transcription of AR in primary cultures of Sertoli cells isolated from testes of adult rats. Electrophoretic mobility shift and antibody supershift assays with nuclear extracts prepared from Sertoli cells identified two binding sites, termed κB1 at −491/−482 bp and κB2 at −574/−565 bp, upstream of the transcription start site of the AR gene that bind the NF-κB subunits, p50 and p65. DNAse I footprint analyses showed that binding of the p50 NF-κB subunit protected the same regions on the rat AR promoter. Analyses of AR promoter-luciferase reporter gene activity after transfection of primary cultures of Sertoli cells demonstrated that mutation of the κB2 site or combined mutation of the κB1 and κB2 sites reduced activity by 40%. Preferential binding of the transcriptionally active p65/p50 heterodimer to the κB2 site rather than to the κB1 site supported these observations. Overexpression of the NF-κB p65 and p50 subunits in Sertoli cells increased activity from the wild-type AR promoter and the promoter with mutation of the κB1 site, but not the κB2 site. Activity was further stimulated by CBP (CREB binding protein), a coactivator of p65 transcriptional activity. Taken together, our data show that NF-κB is an activator of AR gene transcription in Sertoli cells and may be an important determinant of androgen activity during spermatogenesis.

Transforming Growth Factor-β1 is Responsible for Maturation-Dependent Spontaneous Apoptosis of Cultured Gastric Pit Cells

2002 ◽  
Vol 227 (6) ◽  
pp. 402-411 ◽  
Author(s):  
Shinji Tsutsumi ◽  
Wataru Tomisato ◽  
Tatsuya Hoshino ◽  
Tomofusa Tsuchiya ◽  
Tohru Mizushima
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Guinea Pig ◽  
Fetal Calf Serum ◽  
Transforming Growth Factor ◽  
Calf Serum ◽  
Transforming Growth Factor Β1 ◽  
Spontaneous Apoptosis ◽  
Pit Cells ◽  
Tgf Β1

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-β1 (TGF-β1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-β1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-β1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-β1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.

scholarly journals Preadipocytes Mediate Lipopolysaccharide-Induced Inflammation and Insulin Resistance in Primary Cultures of Newly Differentiated Human Adipocytes

Endocrinology ◽  
2006 ◽  
Vol 147 (11) ◽  
pp. 5340-5351 ◽  
Author(s):  
Soonkyu Chung ◽  
Kathleen LaPoint ◽  
Kristina Martinez ◽  
Arion Kennedy ◽  
Maria Boysen Sandberg ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Primary Cultures ◽  
Nuclear Factor Κb ◽  
Mapk Signaling ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Human Adipocytes ◽  
Chemokine Expression

Recent data suggest that proinflammatory cytokines secreted from adipose tissue contribute to the morbidity associated with obesity. However, characterization of the cell types involved in inflammation and how these cells promote insulin resistance in human adipocytes are unclear. We simulated acute inflammation using the endotoxin lipopolysaccharide (LPS) to define the roles of nonadipocytes in primary cultures of human adipocytes. LPS induction of the mRNA levels of proinflammatory cytokines (e.g. IL-6, TNF-α, and IL-1β) and chemokines (e.g. IL-8, monocyte chemoattractant protein-1) occurred primarily in the nonadipocyte fraction of newly differentiated human adipocytes. Nonadipocytes were characterized as preadipocytes based on their abundant mRNA levels of preadipocyte markers preadipocyte factor-1 and adipocyte enhancer protein-1 and only trace levels of markers for macrophages and myocytes. The essential role of preadipocytes in inflammation was confirmed by modulating the degree of differentiation in the cultures from approximately 0–90%. LPS-induced proinflammatory cytokine/chemokine expression and nuclear factor-κB and MAPK signaling decreased as differentiation increased. LPS-induced cytokine/chemokine expression in preadipocytes was associated with: 1) decreased adipogenic gene expression, 2) decreased ligand-induced activation of a peroxisome proliferator activated receptor (PPAR)-γ reporter construct and increased phosphorylation of PPARγ, and 3) decreased insulin-stimulated glucose uptake. Collectively, these data demonstrate that LPS induces nuclear factor-κB- and MAPK-dependent proinflammatory cytokine/chemokine expression primarily in preadipocytes, which triggers the suppression of PPARγ activity and insulin responsiveness in human adipocytes.

Constitutive nuclear factor–κB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes by controlling the expression of distinct Bcl-2 family proteins

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3683-3691 ◽  
Author(s):  
Fabrice Bureau ◽  
Alain Vanderplasschen ◽  
Fabrice Jaspar ◽  
Frédéric Minner ◽  
Paul-Pierre Pastoret ◽  
...  
Keyword(s):  
Immune Cells ◽  
Antisense Oligonucleotide ◽  
Nuclear Factor Κb ◽  
Mrna Splicing ◽  
Nuclear Factor ◽  
Spontaneous Apoptosis ◽  
T And B Cells ◽  
Cellular Accumulation ◽  
Monocyte Apoptosis ◽  
Pharmacologic Inhibitors

Constitutive nuclear factor kappaB (NF-κB) activity protects quiescent mature immune cells from spontaneous apoptosis. Here, we examined whether NF-κB exerts its antiapoptotic function in these cells through the control of Bcl-2 family proteins. Specific pharmacologic inhibitors of NF-κB were used to achieve total NF-κB inactivation in quiescent human blood lymphocytes, granulocytes, and monocytes. NF-κB inhibition induced drastic lymphocyte and granulocyte apoptosis, but only moderate monocyte apoptosis. T- and B-cell apoptosis was slow and associated with a gradual down-regulation of the prosurvival Bcl-2 family proteins Bcl-xL and Bcl-2, respectively. By contrast, granulocyte apoptosis was fast and accompanied by a rapid cellular accumulation of Bcl-xS, the proapoptotic Bcl-x isoform that is generated from alternative splicing of the bcl-x pre-mRNA. Finally, antisense bcl-xL and bcl-2knockdown in T and B cells, respectively, and induction of Bcl-xS expression in granulocytes through antisense oligonucleotide-mediated redirection of bcl-x pre-mRNA splicing were sufficient to induce significant apoptosis in these cells. Taken together, these results reveal that basal NF-κB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes through regulation of distinct members of the Bcl-2 family. This study sheds light on the constitutive mechanisms by which NF-κB maintains defense integrity.

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