The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 178: 8107-8116.
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stout, B. A.
Right arrow Articles by Tesfaigzi, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stout, B. A.
Right arrow Articles by Tesfaigzi, Y.

STAT1 Activation Causes Translocation of Bax to the Endoplasmic Reticulum during the Resolution of Airway Mucous Cell Hyperplasia by IFN-{gamma}1

Barbara A. Stout*, Karla Melendez*, JeanClare Seagrave*, Michael J. Holtzman{dagger}, Bridget Wilson{ddagger}, Jialing Xiang§ and Yohannes Tesfaigzi2,*

* Lovelace Respiratory Research Institute, Albuquerque, NM 87185; {dagger} Pulmonary and Critical Care Divisions, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110; {ddagger} Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131; and § Department of Biological, Chemical, and Physical Science, Illinois Institute of Technology, Chicago, IL 60616


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Disruption of the normal resolution process of inflammation-induced mucous cell hyperplasia may lead to sustained mucous hypersecretion in chronic diseases. During prolonged exposure of mice to allergen, IFN-{gamma} reduces mucous cell hyperplasia, but the signaling responsible for the cell death is largely unknown. A brief phosphorylation of STAT1 by IFN-{gamma} was required for cell death in airway epithelial cells (AEC), and during prolonged exposure to allergen, mucous cell hyperplasia remained elevated in STAT1–/– but was resolved in STAT1+/+ mice. Although IFN-{gamma} treatment of primary human AECs and other airway cell lines left Bax protein levels unchanged, it caused translocation of Bax from the cytosol to the endoplasmic reticulum (ER) but not to the mitochondria. Localization of Bax to the ER was observed in IFN-{gamma}-treated primary AECs isolated from STAT1+/+ mice but not in cells from STAT1–/– mice. In addition, ER Bax was detected in mucous cells of STAT1+/+ but not STAT1–/– airways of mice exposed to allergen for prolonged periods. IFN-{gamma} did not release cytochrome c from mitochondria but reduced ER calcium stores and dilated the ER, confirming that the IFN-{gamma}-induced cell death is mediated through changes localized in the ER. Collectively, these observations suggest that STAT1-dependent translocation of Bax to the ER is crucial for IFN-{gamma}-induced cell death of AECs and the resolution of allergen-induced mucous cell hyperplasia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Persistent mucous cell hyperplasia (MCH)3 is the basis for chronic mucous hypersecretions and airflow obstruction in cystic fibrosis, asthma, and chronic bronchitis. Many studies have focused on studying the mechanisms that underlie the development of such a remodeling of the airway epithelium with the attempt to block airway MCH. Very few studies have described the processes involved in the normal resolution process of MCH. Our previous studies suggest that aberrant repair processes could be the foundation for sustained MCH in chronic diseases associated with chronic mucous hypersecretion both in humans with cystic fibrosis and horses with reactive airway obstruction (1, 2). Understanding the molecular mechanisms of the resolution process could lead to better therapies that facilitate the restoration of the aberrant repair process and reduce epithelial cell metaplasia in general or MCH in particular.

The development of MCH in asthma poses a risk for reducing airflow and can be fatal when mucus hypersecretion occurs acutely. In the murine model of asthma, spontaneous resolution of MCH has been observed after cessation of allergen exposure in the absence of further allergen exposure (3, 4) or when mice are exposed to allergen continuously for prolonged periods (4). In this context, the experimental role of IFN-{gamma} signaling in the resolution process of MCH was identified when instillation of IFN-{gamma} caused enhanced resolution of allergen-induced MCH by causing cell death in airway mucous cells. We confirmed the importance of IFN-{gamma} in resolving MCH by the finding that mice deficient in IFN-{gamma} (our unpublished observation) and STAT1 (4), a protein that plays an obligated role in mediating IFN-{gamma}-dependent responses, are unable to resolve MCH during prolonged exposure to allergen.

Hyperplastic mucous cells express regulators of apoptosis from the Bcl-2 family of proteins (5, 6). The Bcl-2 family of cytoplasmic proteins can register diverse forms of intracellular damage, gauge whether other cells have provided a positive or negative death stimulus, and determine the inhibition or progression of the suicide program (7). These events lead to mitochondrial membrane permeabilization (8), or calcium release from the endoplasmic reticulum (ER) (9, 10) to initiate activation of caspases and DNases and the appearance of apoptotic morphology. The resolution of allergen-induced MCH by IFN-{gamma} is not mediated by the death signal Fas (4), but we have shown that Bax knockout (bax–/–) mice exhibit delayed resolution of MCH compared with their wild-type littermates, suggesting that Bax is involved in the resolution of allergen-induced MCH (11). However, the path linking IFN-{gamma} and STAT1 to cellular apoptosis in hyperplastic mucous cells is unclear. IFN-{gamma}-induced cellular apoptosis by activation of STAT1 has been attributed to the up-regulation of caspases 1, 2, 3, and 11 (12, 13, 14). However, the IFN-{gamma}-induced upstream signals that lead to caspase activation are largely unknown.

We found that a brief activation of STAT1 was sufficient to cause cell death in airway epithelial cells (AECs) and that STAT1 was required for this signaling. Resolution of MCH during prolonged exposure to allergen was completely suppressed in the absence of STAT1 although Bax expression was not affected by IFN-{gamma} signaling or the absence of STAT1. These findings pointed toward the importance of Bax localization and we found that IFN-{gamma} through STAT1 translocated Bax to the ER but not to the mitochondria. IFN-{gamma}-induced changes to the airway epithelial ER were confirmed by the detection of reduced ER calcium stores and dilated ER. These results provide a new paradigm for IFN-{gamma} signaling through STAT1 to elicit ER stress by affecting Bax localization, and that abrogation of this ER stress pathway may cause sustained MCH in chronic asthma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

The immortalized human bronchial epithelial cells, AALEB cells, were provided by Dr. S. Randell (University of North Carolina, Chapel Hill, NC) and they were characterized (15). Primary normal human bronchial epithelial cells (NHBECs) were procured from Cambrex. AALEB cells and NHBECs were cultured in bronchial epithelial cell growth medium (Cambrex Bio Science) at 37°C in an atmosphere of 5% CO2 and air. The MTT kit to measure viability by tetrazolium reduction was from Promega. Recombinant human or murine IFN-{gamma} was obtained from PeproTech. Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich.

Animals

STAT1+/+ and STAT1–/– mice on C57BL/6 background (4- to 6-wk-old) were procured from Taconic Farms. Sensitization and exposure of mice to allergen were as previously described (4). Briefly, mice were immunized by i.p. injection with 1 µg of OVA mixed with 100 µg of Al(OH)3 in a volume of 0.5 ml on days 1 and 7. On day 14, mice were exposed to OVA aerosol (2 mg/m3) in whole body exposure chambers for 6 h/day on four consecutive days.

All experiments were approved by the Institutional Animal Care and Use Committee and were conducted at Lovelace Respiratory Research Institute, a facility approved by the Association for the Assessment and Accreditation for Laboratory Animal Care International.

Primary epithelial cell isolation and culture

AECs were isolated from STAT1–/– and STAT1+/+ mice as described (16) except that the isolated epithelial cells were plated and cultured on coverslips coated with rat tail collagen (BD Biosciences) then processed for immunofluorescent staining.

Histopathological evaluation

Lung tissues from allergen-exposed mice were processed as previously described (17) and tissue sections (5 µm) were stained with Alcian blue/periodic acid-Schiff as previously described (6). The number of airway mucous cells and the volume of intraepithelial stored mucosubstances were quantified as described (18). In all cases, both methods of quantification for MCH showed similar results, indicating that increases were due to MCH and not only enlargement of existing mucous cells.

DNA constructs and transfections

Transient transfections of the pcDNA3m-STAT1 constructs into AALEB cells were performed using FuGENE 6 (Roche) as a transfecting agent. Cell treatments were initiated 24–48 h after transfection to allow for protein expression from the DNA constructs.

Cellular fractionation

Harvested AECs were resuspended in a hypotonic homogenization buffer (10 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1 mM MgCl2, 1 mM DTT, and protease inhibitor mixture). After swelling, cells were broken in a Dounce homogenizer and passed through 21- and 30-gauge needles. After adding sucrose (final concentration 250 mM), the homogenate was centrifuged at 800 x g for 10 min to remove the nuclear pellet. The supernatant was then centrifuged at 8,000 x g for 10 min to obtain the mitochondria-rich fraction followed by centrifugation of the resulting supernatant at 100,000 x g for 10 min to remove nonmitochondrial membranes from the cytosolic supernatant. The pellets from the centrifugation at 8,000 x g were solubilized in CHAPS-containing solubilization buffer (5 mM sodium phosphate (pH 7.4), 2.5 mM EDTA, 100 mM NaCl, 1 mM NaF, 1 mM Na3VO4, protease inhibitor mixture, and 1% CHAPS detergent).

SDS-PAGE and immunoblotting

Cells were processed for SDS-PAGE as described (19). Protein concentration was determined using the BCA assay (Pierce), and equal amounts of protein were electrophoresed unless otherwise stated. Relative protein amounts were determined by densitometry using the GS800 imaging system (Bio-Rad). Detection of adenoviral-expressed Bax or Flag-tagged STAT1 was using anti-hemagglutinin (StressGen Biotechnologies) and anti-Flag (Sigma-Aldrich) Abs, respectively. Abs to cytochrome c and Bax were purchased from BD Pharmingen.

Quantitative real-time PCR

RNA isolated using the RNeasy Micro kit (Qiagen) eluted from the columns subjected to quantitative real-time PCR on the ABI PRISM 7900HT Real-Time PCR System using the One-Step RT-PCR Master Mix (Applied Biosystems). The relative standard curve method was used for linear regression analysis of unknown samples and data are presented as fold change after averaging the threshold cycle ({Delta}Ct) values for the untreated samples. All results were derived from the linear amplification curve, and IFN-{gamma}-treated samples showed lower {Delta}Ct values compared with controls. Because all results were derived from the linear amplification curve the use of {Delta}{Delta}Ct method ensures that only mRNA amplification within the linear range is compared. Normalizing mRNA levels using 18S rRNA, cyclophilin, and CDKN1B showed similar results.

Immunostaining

Bax protein detection by immunohistochemistry was performed as previously described (11). Unless otherwise specified, all fluorescent reagents used in immunofluorescent assays were procured from Molecular Probes. For immunofluorescent staining, AECs were grown on coverslips coated with poly-D-lysine (Discovery Labware; BD Biosciences) and treated with 50 ng/ml IFN-{gamma} or vehicle for 12 or 24 h followed by further processing 12, 24, 48, or 72 h after addition of IFN-{gamma}. In mitochondrial tracking experiments, samples were incubated in fresh medium containing 100 nM MitoTracker Orange, fixed in 3% paraformaldehyde with 3% sucrose in PBS, then permeabilized in 0.2% Triton X-100 in PBS. Samples were blocked in 3% BSA in PBS, incubated with 2 µg/ml rabbit polyclonal anti-human Bax N-20 (sc-493; Santa Cruz Biotechnology) or control rabbit IgG followed by 2 µg/ml secondary Ab donkey anti-rabbit IgG conjugated to Cy5 (Jackson ImmunoResearch Laboratories) or to Alexa Fluor 647 and counterstained with 100 µg/ml Hoechst 33342. In ER tracking experiments, fixed samples were incubated with 5 µg/ml mouse monoclonal anti-calnexin (BD Transduction Laboratory) or isotype control mouse IgG1 (Abcam), followed by incubation with 2 µg/ml secondary Ab donkey anti-mouse conjugated to Alexa Fluor 555 and mounted onto slides with mounting medium (50% glycerol in 0.1 M Tris-HCl (pH 8)) or Prolong Gold (Molecular Probes). Murine epithelial cells isolated from the airways of STAT1+/+ and STAT1–/– mice were cultured on coverslips coated with rat tail collagen (BD Biosciences), treated with 50 ng/ml murine IFN-{gamma} for 24 or 48 h, and processed for Bax immunofluorescent staining similar to the human cells, except a 1/500 dilution of antiserum from rabbits immunized with murine Bax (catalog no. 554106; BD Pharmingen) or control serum from unimmunized rabbits was used. Paraffin-embedded lung tissue sections (3 µm) from STAT1–/– and STAT1+/+ mice were treated with four washes of 1 mg/ml sodium borohydride (pH 8.0) to reduce tissue autofluorescence, followed by tissue permeabilization in ice-cold 100% acetone. Tissue sections were blocked in a 1% normal goat serum, 2% BSA, and 0.1% Triton X-100 solution, incubated with a 1/500 dilution of antiserum from rabbits immunized with murine Bax (catalog no. 554106; BD Pharmingen) or control serum from unimmunized rabbits, incubated with 2 µg/ml secondary Ab donkey anti-rabbit IgG conjugated to Alexa Fluor 647 and 100 µg/ml Hoechst 33342. Sections were then incubated in 5 µg/ml mouse monoclonal anti-calnexin (BD Transduction Laboratory), followed by the secondary Ab donkey anti-mouse conjugated to Alexa Fluor 555 and mounted with Prolong Gold (Molecular Probes).

Colocalization images of the cells and tissues were taken using the x63 objective of a Zeiss Axioplan 2 Imaging microscope as 0.3-µm increment z-stacks with multiple images and deconvoluted using a nearest neighbors algorithm with the Everest Imaging System with SlideBook software (Intelligent Imaging Innovation) to remove out-of-focus light. Phase-contrast images were obtained using the x10 objective of a phase-contrast microscope (Westover Scientific) and Motic Images Plus software (Motic China Group). Quantification for ER Bax was conducted by counting 60 cells each by a person unaware of treatment groups.

Transmission electron microscopy

AALEB cells were grown as monolayers on tissue-grade plastic dishes, treated with 50 ng/ml IFN-{gamma} for 72 h with fresh medium and IFN-{gamma} replacement daily. Cell monolayers were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), then lifted from plastic dishes by the addition of propylene oxide (20) and processed as previously described (21).

ER calcium measurements

Following various lengths of time of IFN-{gamma} treatment, cells were loaded in the dark with 4 µM Fura 2-AM (Molecular Probes) in 0.4% Pluronic F-127 (Molecular Probes) in HBSS to facilitate entry of Fura 2-AM into the cells, followed by a postloading incubation in the dark with HBSS as recommended by the manufacturer. The buffer was removed and thapsigargin (Tocris Bioscience) in HBSS or HBSS alone was added to the wells (two at a time) and immediately processed by the Fluoroskan Ascent plate reader (Thermo Electron Corporation) using excitation of Fura 2 at 340 and 390 nm. The average emission for 10 time points from both these excitation wavelengths was determined from 32 spots in each well over 7 min. The pre-Fura 2 loading autofluorescence values were subtracted from each measurement and the net 340/390 emission ratio was calculated for each time point as an indicator of Fura 2-bound Ca2+.

Statistical analysis

Grouped results from at least four different mice or samples were expressed as mean ± SEM, and differences between groups were assessed for significance by Student’s t test when data were available in only two groups. When data were available in more than two groups, ANOVA was used to perform pairwise comparison. A value of p < 0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Brief IFN-{gamma} treatment results in STAT1 activation and cell death

The effect of IFN-{gamma} concentrations on reducing human AEC viability was determined by treating AALEB cells daily with concentrations ranging from 0 to 500 ng/ml and measuring viability using the MTT assay 6 days later. The lowest concentration resulting in the lowest viability was 50 ng/ml with the higher IFN-{gamma} doses showing no difference in viability from the 50 ng/ml dose (Fig. 1A). Thus, 50 ng/ml IFN-{gamma} was used for all subsequent experiments. The kinetics of IFN-{gamma}-induced loss of viability was characterized by MTT assay of IFN-{gamma}-treated AALEB cells over 0, 24, 48, or 72 h of continual IFN-{gamma} treatment and expressed as a percentage of viability of untreated AALEB cells. A major drop in viability occurs between 48 and 72 h of continual IFN-{gamma} treatment (Fig. 1B). IFN-{gamma} exerts much of its effect on cells through the transcription factor STAT1 (22). The ability to detect phosphorylated tyrosine 701 STAT1 (pY701STAT1) is a marker of STAT1 activation (23). A dramatic increase in pY701STAT1 levels was detected even at 15 min of IFN-{gamma} treatment and this activation was reduced to barely detectable levels after 24, 48, and 72 h of continual IFN-{gamma} treatment (Fig. 1C). Total STAT1 protein was also increased at 24, 48, and 72 h of treatment with IFN-{gamma}.


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 1. Brief IFN-{gamma} results in STAT1 phosphorylation and AEC death. AALEB AECs were treated with 0, 0.1, 0.5, 1, 10, 50, 100, and 500 ng/ml IFN-{gamma} (A) for 6 days (n = 6/group) or with 0 or 50 ng/ml IFN-{gamma} (B) for 24, 48, or 72 h followed by viability assessment using MTT assay. Viability is expressed as a percentage of treated cells normalized to untreated (n = 6/group). C, AALEB cells were treated with 50 ng/ml IFN-{gamma} for 0, 0.25, 24, 48, or 72 h, harvested, and processed for electrophoresis and immunoblotting. Immunoblot for phosphorylated tyrosine 701 STAT1 (STAT 1-P). The membrane was stripped and reprobed with anti-STAT1, which detects all forms of STAT1. D, AALEB cells were treated with 50 ng/ml IFN-{gamma} or vehicle (C) for 1, 5, and 24 h and maintained in IFN-{gamma}-free medium for 96 h or continuously treated for 96 h. At 96 h, viability was assessed using the MTT assay and expressed as a percentage of vehicle-treated controls (n = 6/group). Error bars represent group mean ± SEM. *, p < 0.05 vs other groups.

 
We further tested whether this brief activation of STAT1 is sufficient to signal cell death in AECs. Therefore, AALEB cells were treated with IFN-{gamma} for 1, 5, or 24 h and medium was replaced to maintain cells in IFN-{gamma}-free medium for 96 h. Viability was measured 96 h after initiation of IFN-{gamma} treatment and was compared with cultures treated continually for 96 h with IFN-{gamma}. Cultures treated for only 1, 5, or 24 h with IFN-{gamma} showed significantly reduced viability 4 days later compared with untreated cells, similar to cells continuously treated for 4 days with IFN-{gamma} (Fig. 1D).

STAT1 is required for IFN-{gamma}-induced cell death

To define the role of STAT1 activation in IFN-{gamma}-induced cell death, AECs were transfected with STAT1 mutants, including a Y701F mutation that interferes with STAT1 dimerization (Y701F STAT1), and a form truncated at aa 713 that can dimerize and bind DNA but cannot activate transcription (histidine 713 STAT1). Detection of anti-FLAG in cell extracts demonstrated successful transfection and expression of the mutant STAT1 in four different experiments (Fig. 2A). In each of the four experiments, minimal cell death was detected in both histidine 713 STAT1 and vector-only transfectants when not treated with IFN-{gamma} (data not shown). However, IFN-{gamma}-induced cell death was significantly reduced in histidine 713 STAT1 compared with vector-transfected cells in all four experiments (Fig. 2B). The Y701F STAT1 mutant was marginally effective in reducing cell death (data not shown), suggesting that activation of various sites within the full length STAT1 is needed for IFN-{gamma}-mediated cell death.


Figure 2
View larger version (53K):
[in this window]
[in a new window]

  		FIGURE 2. STAT1 is crucial for IFN-{gamma}-induced cell death. A and B, AALEB cells were transfected with STAT1 dominant negative mutant truncated at residue 713 (His 713 STAT1) or vector only or were left untransfected. After 24 to 48 h of transfection, cells were treated with 50 ng/ml IFN-{gamma} for 24 h and percentage of cell death was assessed by counting detached cells in the medium and trypan blue exclusion of cells still attached to the plate 72 h after initiation of IFN-{gamma} treatment. A, Anti-FLAG immunoblot of transfected cells showing protein expression of transfected mutant STAT1 truncated at aa 713 (Trunc. 713). B, Ratio of dead cells to viable cells as representative of four independent experiments. *, p < 0.05 significant difference between histone 713 STAT1- and vector-transfected cells. C, IFN-{gamma} signaling through STAT1 is crucial for resolution of MCH during prolonged exposure to allergen. Sensitized STAT1–/– and STAT1+/+ mice were exposed daily to OVA for 15, 30, or 45 days, then sacrificed. Lungs were processed for Alcian blue/periodic acid-Schiff staining and image analysis. Mucous cell numbers per millimeter basal lamina are shown for epithelia in the third, fourth, and fifth generations of STAT1+/+ and STAT1–/– mice (n = 5 mice/group). D, Photomicrograph represents immunohistochemistry staining for Bax in STAT1+/+ and STAT1–/– mice. Arrows show diffuse Bax staining in mucous cells (blue) of STAT1+/+ mice, whereas Bax immunostaining is concentrated to specific areas in mucous cells of STAT1–/– mice. Scale bar, 20 µm. Error bars for group mean ± SEM.

 
Previously, we reported that MCH is sustained in STAT1–/– mice exposed to allergen for 15 days but is significantly reduced in STAT1+/+ mice (4). In the present study, we sought to investigate whether MCH would be reduced in STAT1–/– mice if mice are exposed for longer periods than 15 days. Exposure of sensitized STAT1+/+ and STAT1–/– mice to allergen for 15, 30, or 45 days showed that MCH remained unchanged over the time course in STAT1–/– mice and was 10-fold more elevated compared with STAT1+/+ mice even after 45 days of allergen exposure (Fig. 2C).

Our previous studies demonstrated that Bax is crucial for the resolution of MCH (11); therefore, we hypothesized that sustained MCH in STAT1–/– mice may have been due to the lack of Bax expression. However, the immunostaining of the tissues from allergen-exposed STAT1–/– and STAT1+/+ mice showed that Bax protein is detected in metaplastic mucous cells, but the pattern of Bax expression appeared more concentrated in specific areas within mucous cells in STAT1–/– mice and more diffuse in STAT1+/+ mice (Fig. 2D).

IFN-{gamma}-induced cell death does not involve induced expression of Bax or release of cytochrome c

We further examined whether Bax mRNA and protein levels are affected by treating human AECs with IFN-{gamma}. Bax mRNA levels were reduced in IFN-{gamma}-treated NHBECs both at 24 or 48 h (Fig. 3A). Immunoblotting revealed that Bax protein levels remained unchanged over 24–72 h of IFN-{gamma} treatment (Fig. 3B). The protein levels were also not changed at 6 and 12 h of treatment (data not shown). These findings supported the observations from Bax immunostaining in STAT1+/+ and STAT1–/– mice and suggested that the role of Bax may be affected by its localization. Translocation of Bax from cytosolic pools to mitochondria followed by release of cytochrome c or other factors from the mitochondria are accepted to be critical steps in the apoptotic pathway in many cell types (24, 25, 26). To assess whether this occurs in IFN-{gamma}-stimulated human AEC death, AALEB cells were treated with IFN-{gamma} for 24, 48, or 72 h followed by fractionation into mitochondria-enriched and cytosolic compartments. During the 24–72 h of cell culture, cell death was readily apparent in the IFN-{gamma}-treated cultures, but not in untreated cultures (data not shown). Surprisingly, no differences were observed in Bax levels in the mitochondria-enriched or cytosolic fractions from IFN-{gamma}-treated compared with untreated cells at 24–72 h (Fig. 3C, upper panels), suggesting that Bax does not translocate to the mitochondria in response to IFN-{gamma} in these cells. Bax protein levels in the cellular compartments were normalized using mitochondria heat shock protein 70 for mitochondria and actin for the cytosol. Approximately equivalent signals between the untreated and IFN-{gamma}-treated samples were observed for these markers. Cytochrome c appeared only in the mitochondrial-rich, but not in the cytosolic fraction in the IFN-{gamma}-treated samples (Fig. 3C, middle panels). These results show that Bax levels did not increase in the mitochondrial compartment and that cytochrome c was not released from the mitochondrial compartment.


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 3. Bax levels are not increased and cytochrome c is not released from mitochondria in cells treated with IFN-{gamma}. A, NHBEs were treated with 0, 25, or 50 ng/ml IFN-{gamma} for 24 or 48 h, followed by assessment of Bax mRNA. B, AALEB cells were treated with 50 ng/ml IFN-{gamma}, harvested, and processed for protein electrophoresis at 24, 40, and 48 h after initiation of IFN-{gamma} treatment. Results are representative of five experiments. C, No movement of Bax or cytochrome c to or from mitochondria in IFN-{gamma}-treated cells. AALEB cells were treated with 50 ng/ml IFN-{gamma} for 72 h with daily changes of medium and IFN-{gamma}. The cells were harvested and processed for a mitochondrial-enriched fraction (Mito) and cytosol, as described in Materials and Methods. Immunoblot of Bax (upper), cytochrome c (Cyt c) (middle), and the markers for the respective fractions of mitochondria heat shock protein 70 (mtHsp70) and actin (lower). Results are representative of six experiments.

 
IFN-{gamma} triggers Bax translocation to the ER

Because overall levels of Bax protein were apparently not the key to IFN-{gamma}-triggered cell death, we hypothesized that IFN-{gamma} may cause translocation of Bax to initiate cell death. Immunofluorescent staining and confocal fluorescent microscopy were used to study the possible translocation of Bax in individual cells. After treatment of human AECs with IFN-{gamma} for 24, 48, or 72 h, no change in colocalization of Bax with mitochondria at any of the time points was observed (Fig. 4, A–C). However, increased punctate Bax staining that was often perinuclear was observed in IFN-{gamma}-treated cells (Fig. 4E) compared with untreated controls (Fig. 4H).


Figure 4
View larger version (78K):
[in this window]
[in a new window]

  		FIGURE 4. Bax is translocated to the ER and not to mitochondria. A–F, AALEBs were grown on coverslips, treated with 50 ng/ml IFN-{gamma} for 24, 48, and 72 h followed by processing for immunofluorescence imaging for mitochondria (A, B, and C), Bax (B, E, and H), and ER marker calnexin (A, D, and G). Overlays of mitochondria (Mit) or calnexin (Cal) with Bax are shown in C, F, and I, respectively. Colocalization of Bax and calnexin is seen as the yellow color in F. AALEB cells not treated with IFN-{gamma} showed no colocalization of Bax to the ER (G, H, and I). Images shown are from the 48 h time point and are representative of seven (mitochondria) and four (calnexin) independent experiments performed for Bax colocalization. Scale bar, 20 µm. IFN-{gamma} causes structural changes in ER and mitochondria. J and K, Images represent transmission electron microscopy of AALEB cells cultured with (K) or without (J) 50 ng/ml IFN-{gamma} for 72 h. Arrows point to ER and arrowheads to mitochondria. Scale bar, 0.2 µm. Thapsigargin-induced increases in intracellular Ca2+ are slower in IFN-{gamma}-treated cells. L, AALEB cells were treated with 50 ng/ml IFN-{gamma} for 3, 17, 29, or 50 h and analyzed for increases in intracellular Ca2+ induced by thapsigargin (thap) and measured by Fura 2-AM, as described in Materials and Methods. Ca2+ released from the ER is expressed as a ratio of fluorescent emission from two excitation wavelengths of Fura 2, 340 and 390 nm. The experiment is representative of three independent time course experiments.

 
Because the perinuclear pattern of Bax staining is suggestive of the ER and because ER-stress-induced cell death has been reported for epithelial cells (27), we used calnexin to determine whether Bax protein was localized at the ER (Fig. 4, D–F). Colocalization of Bax with the ER marker was observed at 24, 48, and 72 h following IFN-{gamma} treatment in a pattern that was often perinuclear (Fig. 4F). At each of these time points colocalization of Bax with the ER was observed in 20% of IFN-{gamma}-treated but in <1% of untreated cells. From 60 cells analyzed in each experiment, ~60% of the cells with ER Bax showed condensed nuclei, and essentially all cells with condensed nuclei showed colocalization of Bax with the ER. In nontreated AALEB controls, only 6% of cells showed colocalization of Bax with the ER (Fig. 4, G–I).

The colocalization of Bax with the ER was absent in nontreated NHBECs (Fig. 5, A–C) but was observed in NHBECs treated with IFN-{gamma} for 48 h (Fig. 5, D–F). From 60 cells each, the percentage of NHBECs that showed ER Bax was increased from 9% in nontreated controls to 19% or 30% in cells treated with IFN-{gamma} for 24 or 48 h, respectively. Therefore, IFN-{gamma}-induced localization of Bax to the ER applies for primary AECs that undergo cell death similar to that observed in AALEB cells (4). Similarly, we also observed 61% of cells showing colocalization of Bax with the ER following IFN-{gamma} treatment of the breast cancer cell line MCF-7, whereas only 30% of cells in nontreated controls.


Figure 5
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 5. Bax colocalization with the ER in IFN-{gamma}-treated NHBECs at 48 h compared with nontreated controls. NHBECs were cultured on coverslips and treated with 50 ng/ml IFN-{gamma} for 48 h (D, E, and F) or with nothing (A, B, and C) as control. Detection for calnexin (A and D) and Bax (B and E), and overlays for calnexin with Bax (C and F) are shown. The yellow color denotes colocalization of Bax and calnexin.

 
To determine whether IFN-{gamma} treatment causes visible structural changes in the ER, electron microscopy was performed on AALEB cells were incubated with or without IFN-{gamma} for 72 h, fixed with glutaraldehyde and embedded in epon in preparation for ultra-thin sectioning. Samples were then examined by transmission electron microscopy. These experiments reveal striking structural differences in IFN-{gamma}-treated cells (Fig. 4K) compared with untreated cells (Fig. 4J). These changes include swollen ER, shrunken mitochondria with abnormal cristae, and a paucity of cytoplasm, which are all features characteristic of dying cells.

IFN-{gamma} reduces ER Ca2+ stores

Because of the unexpected finding that Bax was localized to the ER, but not to the mitochondria, and the gross changes in ER structure revealed by electron microscopy in response to IFN-{gamma} treatment, we investigated whether IFN-{gamma} affects the ER calcium stores that have been shown to play a role in the induction of apoptosis (28, 29, 30). To this end, changes in thapsigargin-induced increases in intracellular calcium were measured in human AECs treated with IFN-{gamma} for 3, 17, 29, and 50 h compared with untreated controls. A time-dependent reduction in the thapsigargin-induced rate of increase in intracellular calcium was observed in IFN-{gamma}-treated compared with untreated cultures (Fig. 4L), providing further evidence that IFN-{gamma} causes cell death by inducing changes in the ER.

IFN-{gamma} causes Bax to localize to the ER in STAT1+/+ cells, but not in STAT1–/– cells.

To determine the importance of STAT1 in the IFN-{gamma}-triggered localization of Bax to the ER, AECs were isolated from STAT1+/+ and STAT1–/– mice and treated with murine IFN-{gamma} for 24 and 48 h. Phase contrast microscopy showed that the IFN-{gamma}-treated STAT1+/+ cultures have fewer cells (Fig. 6), and more floating (dead) cells (data not shown) than do IFN-{gamma}-treated STAT1–/– cultures, confirming the importance of STAT1 for IFN-{gamma}-induced cell death. From 60 cells each that were viewed using immunofluorescent staining and confocal fluorescent microscopy, we observed that Bax colocalized with the ER marker calnexin in 40% of the STAT1+/+, but only in 4% of the STAT1–/–, cells treated with IFN-{gamma} (Fig. 6).


Figure 6
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 6. Bax colocalizes with the ER in IFN-{gamma}-treated cultured AECs from STAT1+/+ but not from STAT1–/– mice. Phase-contrast microscopy of IFN-{gamma}-treated AECs isolated from STAT1+/+ and STAT1–/– cells shows that IFN-{gamma} reduced the number of attached epithelial cells in STAT1+/+ but not in STAT1–/– cultures. STAT1+/+ (A, B, and C) and STAT1–/– (D, E, and F) mice were treated with 50 ng/ml murine IFN-{gamma} for 48 h followed by processing for immunofluorescence imaging for ER marker calnexin (green) (A and D) and Bax (red) (B and E). Overlays of calnexin with Bax (C and F) show colocalization of Bax and calnexin (yellow color in C). Arrows point to representative areas of colocalization. Scale bar, 20 µm.

 
Bax localizes to the ER in STAT1+/+ mice recovering from MCH, but not in STAT1–/– mice

To determine the relevancy of the IFN-{gamma}-induced localization of Bax to the ER observed in cell culture in an in vivo model of MCH resolution, we used confocal immunofluorescence to detect Bax localization within airways from nonexposed naive mice or from STAT1+/+, and STAT1–/– mice exposed to allergen for 15 days. This time point was chosen because at day 15 of allergen exposure, STAT1+/+ mice are recovering from MCH, whereas STAT1–/– mice still have substantial MCH. Tissue sections adjacent to those where Bax immunostaining was detected with diaminobenzidine were immunostained with calnexin and Bax to investigate colocalization. These same slides were then stained with Alcian blue/periodic acid-Schiff to identify the mucus-producing cells. Despite extensive immunostaining for Bax and calnexin, Bax colocalization to the ER was not observed in epithelial cells of naive (Fig. 7, A–D) mice and STAT1–/– airways (Fig. 7, I–L). However, epithelial cells from STAT1+/+ airways displayed colocalization of Bax with the ER marker calnexin (Fig. 7, E–G). The colocalization of Bax with calnexin was found in mucus-producing cells of STAT1+/+ mice exposed to allergen for prolonged periods (Fig. 7H). These results support our findings in cell culture and confirm that STAT1 activation promotes the translocation of Bax to the ER in mucous AECs of mice during the resolution of MCH.


Figure 7
View larger version (89K):
[in this window]
[in a new window]

  		FIGURE 7. Bax colocalizes with the ER in STAT1+/+ but not in STAT1–/– mice exposed to allergen for 15 days. Airway tissues from naive, STAT1+/+ and STAT1–/– mice were prepared for confocal immunofluorescent analysis then stained with Alcian blue/periodic acid-Schiff (AB/PAS), as described in Materials and Methods. Calnexin (green) (A, E, and I) and Bax (red) (B, F, and J) in naive, and STAT1+/+ and STAT1–/– mice exposed to allergen for 15 days. Overlay of calnexin and Bax (C, G, and K) shows no colocalization for naive and STAT1–/– mice. Overlay of calnexin and Bax shows colocalization (yellow) in STAT1+/+ mice (G). Brightfield micrographs of epithelia in C, G, and K are shown after staining with Alcian blue/periodic acid-Schiff (D, H, and L). Arrows point to representative areas where Bax is colocalizing to calnexin and the cells after Alcian blue/periodic acid-Schiff staining. Scale bar, 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
ER stress and the apoptotic program coupled to it have been implicated in a range of diseases including ischemia/reperfusion injury, neurodegeneration, and diabetes (31). However, the regulation and execution steps of ER stress-induced apoptosis in mammalian cells remain poorly understood in these diseases, and much less is known in this regard for chronic lung diseases. The present study shows that IFN-{gamma} causes translocation of Bax to the ER in primary human and mouse AECs and in airway and breast cancer cell lines. This STAT1-dependent translocation of Bax to the ER and calcium release was observed in AECs and may provide the stimulus for the resolution of MCH during prolonged exposure to allergen, an under-appreciated but crucial feature in maintaining AEC homeostasis. Our previous study showed that sensitized STAT1–/– mice had 4-fold more MCH than STAT1+/+ after 15 days of allergen exposure (4). We extended those findings in this study by demonstrating that MCH of STAT1+/+ mice had decreased to presensitization levels after 45 days of allergen exposure, whereas MCH in STAT1–/– mice remained elevated and was 10-fold higher than in STAT1+/+ mice. Therefore, obstruction of this resolution pathway, as observed in the STAT1-deficient cells leads to sustained MCH as observed in chronic diseases associated with chronic mucous hypersecretion.

In response to ER stress, cells react rapidly through a set of adaptive mechanisms to reestablish normal ER function. To this end, several pathways are activated that collectively are termed as ER stress response and when the stress is too strong or persistent so that ER dysfunction cannot be corrected, the apoptotic pathway is initiated (32). One of the more commonly reported triggers for ER stress is a change in cellular redox regulation. IFN-{gamma} is a crucial transactivator of the NADPH oxidase 1, Nox1, stimulates expression of Nox1 mRNA and protein in intestinal epithelial cells, leading to 4-fold up-regulation of superoxide anion generation (33). IFN-{gamma} induces ROS by up-regulating enzymes involved in the production or regulation of ROS (33, 34), thus it is possible that this is a mechanism by which IFN-{gamma} exerts its effect on the ER. Also, STAT1 signaling modulates intracellular oxidative stress through a positive-feedback mechanism involving the p38 MAPK/STAT1/ROS pathway (35). The presence of shrunken and altered mitochondria in IFN-{gamma}-treated AECs supports the hypothesis that IFN-{gamma} causes ER stress by generating ROS. The present findings extend the role of IFN-{gamma} and indicate that IFN-{gamma} causes ER stress and calcium release in the setting of resolution of allergen-induced MCH.

It is well established that STAT1 activation by IFN-{gamma} occurs rapidly, and our results show that this brief activation with IFN-{gamma} was sufficient to cause cell death. STAT1 protein levels were increased over 24 to 72 h of IFN-{gamma} treatment, consistent with previous reports (36, 37), suggesting a possible positive feedback mechanism of STAT1 function. Although IFN-{gamma} causes STAT1 phosphorylation at the tyrosine residue 701, inhibition of endogenous STAT1 function by expression of a dominant negative STAT1 form with a mutation at this residue resulted in no significant relief from IFN-{gamma}-triggered cell death. However, the truncation mutant at histidine 713 was effective in significantly reducing cell death compared with controls transfected with vector only. It is possible that phosphorylation on serine 727, a residue absent in histidine 713 STAT1 mutant but present in Y701F mutant that increases STAT1-dependent transcription (38, 39), is critical to facilitate the Bax translocation and cell death.

Despite the observation that mitochondria appeared altered, treatment with IFN-{gamma} did not reduce cytochrome c levels in mitochondria nor was it detected in the cytosol of IFN-{gamma}-treated cells, suggesting that IFN-{gamma}-induced cell death does not involve signaling through cytochrome c, which is associated with cell death channeled through the mitochondrial pathway (26, 40). Many studies have reported that Bax translocates from a monomeric form in the cytosol to the mitochondria, where it forms oligomers that are associated with the release of cytochrome c and other factors from the mitochondria. This process leads to the formation of the apopotosome and the effector caspase activation cascade (25, 41, 42, 43). Our findings with fractionated cells were confirmed by the lack of colocalization of Bax with mitochondria as shown by confocal immunofluorescent microscopy. Because we could not identify involvement of the mitochondrial pathway to cell death, the relationship between the ER and mitochondria in IFN-{gamma}-triggered death needs to be further elucidated.

Our previous findings showed that Bax is important for the resolution of MCH during prolonged exposure to allergen (11). However, Bax mRNA and protein levels were not increased in IFN-{gamma}-treated human and murine AECs and our cell fractionation studies with human AECs did not detect any noticeable movement of Bax from the cytosol to the mitochondria in response to IFN-{gamma}. Moreover, sustained MCH in STAT1–/– mice was not due to lack of Bax expression as shown by the immunostaining for Bax in the airway epithelia from these mice. However, the pattern of Bax immunostaining in metaplastic mucous cells of STAT1–/– mice was different from that in STAT1+/+, supporting the findings in cell cultures that STAT1 plays a role in the translocation of Bax in IFN-{gamma}-induced cell death. This finding was confirmed by Bax showing increased colocalization with calnexin, an integral membrane protein located in the ER membrane (44), in human and murine STAT1+/+ cells treated with IFN-{gamma} and in the AECs from STAT1+/+ mice recovering from MCH. The possibility that an altered immune response due to the lack of STAT1 in immune cells indirectly mediated Bax translocation in vivo was ruled out because Bax localization to the ER in response to IFN-{gamma} treatment was observed in STAT1+/+ but not in STAT1–/– AECs. Bax has been reported to be found in membrane-bound cellular compartments including the ER, in addition to the well-studied mitochondrial location (45, 46, 47). Zong et al. (48) found the presence of Bax and the formation of Bax oligomers in both the mitochondria and the ER following treatment of murine embryonic fibroblasts with endoplasmic stressors; however, they did not report a specific translocation step of Bax from the cytosol to the ER. To our knowledge, this report is the first on Bax localizing to the ER, but not to the mitochondria, following not only IFN-{gamma} but any physiologically relevant death stimulus.

In cultured cells, IFN-{gamma}-induced cell death is not restricted to mucous cells. Rather, all proliferating AECs are susceptible to IFN-{gamma} (4). In wild-type mice exposed to allergen for 15 days, ER Bax was detected in mucous cells, suggesting that during resolution hyperplastic mucous cells are targeted for removal. Several studies have shown that following injury to the airway epithelium, a large proportion of the hyperplastic and cycling cells are secretory cells (49, 50, 51) and mucous cells arise mainly by self-replication and differentiation of pre-existing cells (52, 53). Because mucous cells are the hyperplastic cells, they may be by default susceptible to cell death and elimination in vivo during resolution. The difference in susceptibility of proliferating and resting AECs to IFN-{gamma} may ensure that only hyperplastic AECs are removed during resolution without damaging the resting AECs and thereby maintaining the barrier function of the airway epithelium.

A substantially higher percentage of IFN-{gamma}-treated cells showed ER Bax compared with untreated controls and essentially all cells with condensed nuclei showed colocalization of Bax with the ER, suggesting that ER Bax causes cell death. In addition, electron microscopy shows swollen ER in cells treated with IFN-{gamma}. Further evidence that IFN-{gamma}-mediated cell death proceeds through an ER stress pathway is provided by the reduction of thapsigargin-induced calcium increases in IFN-{gamma}-treated human AEC cultures compared with untreated cultures. Calcium mobilization from the ER is an important aspect of certain types of apoptosis (28, 29) including those induced by glucose deprivation and ROS, among others (54, 55). These observations suggest that STAT1-dependent translocation of Bax to the ER is crucial for IFN-{gamma} to resolve allergen-induced goblet cell hyperplasia during prolonged exposure of mice to allergen. Future studies will elucidate whether ER Bax is responsible for IFN-{gamma}-induced calcium release and cell death.

In summary, our studies show that Bax is obligatory for IFN-{gamma}-induced cell death of AECs and that a brief activation of STAT1 translocates Bax to the ER causing calcium release. ER stress-associated Bax and cell death were essentially absent in STAT1–/– cells, suggesting that activation of STAT1 is upstream of the ER stress. Many of the current cell-based models of ER stress rely on chemical agents, such as inhibitors of protein glycosylation and folding (tunicamycin), vesicle trafficking (brefeldin A), or ER calcium flux (thapsigargin) (56). These chemicals elicit an acute and severe form of ER stress and the relevance of these studies is not clear because such acute response may not reflect what the cell experiences under physiological settings. Our studies, which show that IFN-{gamma} leads to apoptosis involving the ER, provide a physiologically relevant system and form a basis to further explore signals emanating from the ER in its native context and to identify drug candidates to reduce MCH and chronic mucous hypersecretion.


   Acknowledgments
 
We thank Yoneko Knighton for preparing tissue samples for light microscopic analyses and Janet Pfeiffer, Isaac Estrada, Mark Fischer, Karen Cardon, Kurt Schwalm, and Laura Boies for technical assistance in selected experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants HL68111 and ES09237 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Yohannes Tesfaigzi, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. E-mail address: ytesfaig{at}lrri.org Back

3 Abbreviations used in this paper: MCH, mucous cell hyperplasia; ER, endoplasmic reticulum; NHBEC, normal human bronchial epithelial cell; ARC, airway epithelial cell. Back

Received for publication July 3, 2006. Accepted for publication March 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Harris, J. F., M. J. Fischer, J. A. Hotchkiss, B. P. Monia, S. H. Randell, J. R. Harkema, Y. Tesfaigzi. 2005. Bcl-2 sustains increased mucous and epithelial cell numbers in metaplastic airway epithelium. Am. J. Respir. Crit. Care Med. 171: 764-772. [Abstract/Free Full Text]
  2. Bartner, L. R., N. E. Robinson, M. Kiupel, Y. Tesfaigzi. 2006. Persistent mucus accumulation: a consequence of delayed bronchial mucous cell apoptosis in RAO-affected horses?. Am. J. Physiol. 291: L862-L870.
  3. Blyth, D. I., M. S. Pedrick, T. J. Savage, H. Bright, J. E. Beesley, S. Sanjar. 1998. Induction, duration, and resolution of airway goblet cell hyperplasia in a murine model of atopic asthma: effect of concurrent infection with respiratory syncytial virus and response to dexamethasone. Am. J. Respir. Cell Mol. Biol. 19: 38-54. [Abstract/Free Full Text]
  4. Shi, Z. Q., M. J. Fischer, G. T. De Sanctis, M. Schuyler, Y. Tesfaigzi. 2002. IFN{gamma} but not Fas mediates reduction of allergen-induced mucous cell metaplasia by inducing apoptosis. J. Immunol. 168: 4764-4771. [Abstract/Free Full Text]
  5. Tesfaigzi, J., J. A. Hotchkiss, J. R. Harkema. 1998. Expression of Bcl-2 during mucous cell metaplasia and remodeling in F344/N rats. Am. J. Resp. Cell Mol. Biol. 18: 794-799. [Abstract/Free Full Text]
  6. Tesfaigzi, Y., M. J. Fischer, A. J. Martin, J. Seagrave. 2000. Bcl-2 in LPS- and allergen-induced hyperplastic mucous cells in airway epithelia of Brown Norway rats. Am. J. Physiol. 279: L1210-L1217.
  7. Adams, J. M., S. Cory. 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281: 1322-1326. [Abstract/Free Full Text]
  8. Thornberry, N. A., Y. Lazebnik. 1998. Caspases: enemies within. Science 281: 1312-1316. [Abstract/Free Full Text]
  9. Scorrano, L., S. A. Oakes, J. T. Opferman, E. H. Cheng, M. D. Sorcinelli, T. Pozzan, S. J. Korsmeyer. 2003. BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300: 135-139. [Abstract/Free Full Text]
  10. Oakes, S. A., J. T. Opferman, T. Pozzan, S. J. Korsmeyer, L. Scorrano. 2003. Regulation of endoplasmic reticulum Ca2+ dynamics by proapoptotic BCL-2 family members. Biochem. Pharmacol. 66: 1335-1340. [Medline]
  11. Tesfaigzi, Y., M. J. Fischer, F. H. Y. Green, G. T. De Sanctis, J. A. Wilder. 2002. Bax is crucial for IFN{gamma}-induced resolution of allergen-induced mucous cell metaplasia. J. Immunol. 169: 5919-5925. [Abstract/Free Full Text]
  12. Kumar, A., M. Commane, T. W. Flickinger, C. M. Horvath, G. R. Stark. 1997. Defective TNF-{alpha}-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases. Science 278: 1630-1632. [Abstract/Free Full Text]
  13. Chin, Y. E., M. Kitagawa, K. Kuida, R. A. Flavell, X. Y. Fu. 1997. Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol. Cell. Biol. 17: 5328-5337. [Abstract]
  14. Lee, C. K., E. Smith, R. Gimeno, R. Gertner, D. E. Levy. 2000. STAT1 affects lymphocyte survival and proliferation partially independent of its role downstream of IFN-{gamma}. J. Immunol. 164: 1286-1292. [Abstract/Free Full Text]
  15. Lundberg, A. S., S. H. Randell, S. A. Stewart, B. Elenbaas, K. A. Hartwell, M. W. Brooks, M. D. Fleming, J. C. Olsen, S. W. Miller, R. A. Weinberg, W. C. Hahn. 2002. Immortalization and transformation of primary human airway epithelial cells by gene transfer. Oncogene 21: 4577-4586. [Medline]
  16. You, Y., E. J. Richer, T. Huang, S. L. Brody. 2002. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am. J. Physiol. 283: L1315-L1321.
  17. Tesfaigzi, J., M. B. Wood, N. F. Johnson, K. J. Nikula. 1998. Apoptosis is a major pathway responsible for the resolution of endotoxin-induced type II cell hyperplasia in the rat. Int. J. Exp. Pathol. 79: 303-312. [Medline]
  18. Harkema, J. R., J. A. Hotchkiss. 1992. In vivo effects of endotoxin on intraepithelial mucosubstances in rat pulmonary airways: quantitative histochemistry. Am. J. Pathol. 141: 307-317. [Abstract]
  19. Stout, B. A., M. E. Bates, L. Y. Liu, N. N. Farrington, P. J. Bertics. 2004. IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils. J. Immunol. 173: 6409-6417. [Abstract/Free Full Text]
  20. Griffiths, G., G. Warren, P. Quinn, O. Mathieu-Costello, H. Hoppeler. 1984. Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies. J. Cell Biol. 98: 2133-2141. [Abstract/Free Full Text]
  21. Pfeiffer, J. R., J. C. Seagrave, B. H. Davis, G. G. Deanin, J. M. Oliver. 1985. Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells. J. Cell Biol. 101: 2145-2155. [Abstract/Free Full Text]
  22. Schroder, K., P. J. Hertzog, T. Ravasi, D. A. Hume. 2004. Interferon-{gamma}: an overview of signals, mechanisms and functions. J. Leukocyte Biol. 75: 163-189. [Abstract/Free Full Text]
  23. Shuai, K., G. R. Stark, I. M. Kerr, J. E. Darnell, Jr. 1993. A single phosphotyrosine residue of Stat91 required for gene activation by interferon-{gamma}. Science 261: 1744-1746. [Abstract/Free Full Text]
  24. Liu, X., C. N. Kim, J. Yang, R. Jemmerson, X. Wang. 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86: 147-157. [Medline]
  25. Wolter, K. G., Y. T. Hsu, C. L. Smith, A. Nechushtan, X. G. Xi, R. J. Youle. 1997. Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139: 1281-1292. [Abstract/Free Full Text]
  26. Green, D. R., J. C. Reed. 1998. Mitochondria and apoptosis. Science 281: 1309-1312. [Abstract/Free Full Text]
  27. Breckenridge, D. G., M. Germain, J. P. Mathai, M. Nguyen, G. C. Shore. 2003. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 22: 8608-8618. [Medline]
  28. Baffy, G., T. Miyashita, J. R. Williamson, J. C. Reed. 1993. Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J. Biol. Chem. 268: 6511-6519. [Abstract/Free Full Text]
  29. Lam, M., G. Dubyak, L. Chen, G. Nunez, R. L. Miesfeld, C. W. Distelhorst. 1994. Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc. Natl. Acad. Sci. USA 91: 6569-6573. [Abstract/Free Full Text]
  30. He, H., M. Lam, T. S. McCormick, C. W. Distelhorst. 1997. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J. Cell Biol. 138: 1219-1228. [Abstract/Free Full Text]
  31. Xu, C., B. Bailly-Maitre, J. C. Reed. 2005. Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 115: 2656-2664. [Medline]
  32. Boyce, M., J. Yuan. 2006. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ. 13: 363-373. [Medline]
  33. Kuwano, Y., T. Kawahara, H. Yamamoto, S. Teshima-Kondo, K. Tominaga, K. Masuda, K. Kishi, K. Morita, K. Rokutan. 2006. Interferon-{gamma} activates transcription of NADPH oxidase 1 gene and up-regulates production of superoxide anion by human large intestinal epithelial cells. Am. J. Physiol. 290: C433-C443.
  34. Ito, M., R. Karmali, M. Krim. 1985. Effect of interferon on chemiluminescence and hydroxyl radical production in murine macrophages stimulated by PMA. Immunology 56: 533-541. [Medline]
  35. Kim, H. S., M. S. Lee. 2005. Essential role of STAT1 in caspase-independent cell death of activated macrophages through the p38 mitogen-activated protein kinase/STAT1/reactive oxygen species pathway. Mol. Cell. Biol. 25: 6821-6833. [Abstract/Free Full Text]
  36. Wong, L. H., I. Hatzinisiriou, R. J. Devenish, S. J. Ralph. 1998. IFN-{gamma} priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFNs. J. Immunol. 160: 5475-5484. [Abstract/Free Full Text]
  37. Wong, L. H., H. Sim, M. Chatterjee-Kishore, I. Hatzinisiriou, R. J. Devenish, G. Stark, S. J. Ralph. 2002. Isolation and characterization of a human STAT1 gene regulatory element: inducibility by interferon (IFN) types I and II and role of IFN regulatory factor-1. J. Biol. Chem. 277: 19408-19417. [Abstract/Free Full Text]
  38. Wen, Z., Z. Zhong, J. E. Darnell, Jr. 1995. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82: 241-250. [Medline]
  39. Zhang, X., J. Blenis, H. C. Li, C. Schindler, S. Chen-Kiang. 1995. Requirement of serine phosphorylation for formation of STAT-promoter complexes. Science 267: 1990-1994. [Abstract/Free Full Text]
  40. Gross, A., J. Jockel, M. C. Wei, S. J. Korsmeyer. 1998. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17: 3878-3885. [Medline]
  41. Zou, H., W. J. Henzel, X. Liu, A. Lutschg, X. Wang. 1997. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90: 405-413. [Medline]
  42. Li, P., D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479-489. [Medline]
  43. Antonsson, B., S. Montessuit, S. Lauper, R. Eskes, J. C. Martinou. 2000. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem. J. 345: (Pt. 2):271-278. [Medline]
  44. Tjoelker, L. W., C. E. Seyfried, R. L. Eddy, Jr, M. G. Byers, T. B. Shows, J. Calderon, R. B. Schreiber, P. W. Gray. 1994. Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5. Biochemistry 33: 3229-3236. [Medline]
  45. Hoetelmans, R., H. J. van Slooten, R. Keijzer, S. Erkeland, C. J. van de Velde, J. H. Dierendonck. 2000. Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells. Cell Death Differ. 7: 384-392. [Medline]
  46. Gajkowska, B., T. Motyl, H. Olszewska-Badarczuk, M. M. Godlewski. 2001. Expression of BAX in cell nucleus after experimentally induced apoptosis revealed by immunogold and embedment-free electron microscopy. Cell Biol. Int. 25: 725-733. [Medline]
  47. Hetz, C., P. Bernasconi, J. Fisher, A. H. Lee, M. C. Bassik, B. Antonsson, G. S. Brandt, N. N. Iwakoshi, A. Schinzel, L. H. Glimcher, S. J. Korsmeyer. 2006. Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1{alpha}. Science 312: 572-576. [Abstract/Free Full Text]
  48. Zong, W. X., C. Li, G. Hatzivassiliou, T. Lindsten, Q. C. Yu, J. Yuan, C. B. Thompson. 2003. Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J. Cell Biol. 162: 59-69. [Abstract/Free Full Text]
  49. Keenan, K. P., J. W. Combs, E. M. McDowell. 1982. Regeneration of hamster tracheal epithelium after mechanical injury. I. Focal lesions: quantitative morphologic study of cell proliferation. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 41: 193-214. [Medline]
  50. Keenan, K. P., J. W. Combs, E. M. McDowell. 1982. Regeneration of hamster tracheal epithelium after mechanical injury. II. Multifocal lesions: stathmokinetic and autoradiographic studies of cell proliferation. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 41: 215-229. [Medline]
  51. Keenan, K. P., J. W. Combs, E. M. McDowell. 1982. Regeneration of hamster tracheal epithelium after mechanical injury. III. Large and small lesions: comparative stathmokinetic and single pulse and continuous thymidine labeling autoradiographic studies. Virchows Arch. B Cell Pathol. Incl. Mol Pathol. 41: 231-252. [Medline]
  52. Jeffery, P. K., L. Reid. 1975. New observations of rat airway epithelium: a quantitative and electron microscopic study. J. Anat. 120: 295-320. [Medline]
  53. Tesfaigzi, Y., J. F. Harris, J. A. Hotchkiss, J. R. Harkema. 2004. DNA synthesis and Bcl-2 expression during the development of mucous cell metaplasia in airway epithelium of rats exposed to LPS. Am. J. Physiol. 286: L268-L274.
  54. Hockenbery, D. M., Z. N. Oltvai, X. M. Yin, C. L. Milliman, S. J. Korsmeyer. 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241-251. [Medline]
  55. Kaufman, R. J.. 1999. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13: 1211-1233. [Free Full Text]
  56. Li, J., B. Lee, A. S. Lee. 2006. Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J. Biol. Chem. 281: 7260-7270. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page