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Regular Articles |
From the Departments of Surgery,*
Pathology,
Medicine,
and Physiology and Cellular Biophysics,§
College of Physicians and Surgeons of Columbia University, New York,
New York
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Introduction |
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The increasing frequency with which lung reduction surgery is being performed has resulted in the availability of fresh emphysematous tissue for molecular and other analyses. Although this tissue represents only a snapshot of the pathophysiological picture of evolving emphysema, necessarily slanted toward end-stage disease, it provides a useful resource. By hybridization studies with a cDNA expression array, we have identified Egr-1 as a gene product whose expression is selectively enhanced in emphysematous lung, compared with controls. Levels of Egr-1 antigen appear to be increased in a variety of cells, including bronchial epithelium, vascular and bronchial smooth muscle cells, endothelium, and alveolar macrophages. In the apparent absence of an intercurrent infection in the patients with lung disease in our study, the most likely inference is that sustained Egr-1 induction occurs in late stage emphysema. We propose that emphysema may provide a setting to analyze functions of Egr-1 relevant to tissue remodeling and chronic inflammation, new facets of the biology of this transcription factor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Emphysematous lung from 28 patients was obtained from the block of grossly diseased tissue excised at the time of lung reduction surgery from the sample directed to the Division of Surgical Pathology. The patients ranged in age from 45 to 80 years, all were smokers, and all had severe emphysema (FEV1 < 40% of predicted). Six control samples were obtained from patients undergoing excisional procedures for lung nodules, either primary (malignant or benign) or metastatic (ages 45–70 years). The tissue designated as control was distant from the nodule and appeared grossly normal. Two control samples were obtained from brain-dead individuals from whom consent had earlier been obtained for organ harvesting and research. These patients, despite absence of an identifiable cause of death, had suboptimal arterial pO2 for lung donation likely related to prolonged brain death and associated systemic decompensation.
RNA Purification
Total RNA was purified from freshly excised lung tissue using
TRIzol reagent according to the manufacturer’s protocol (GIBCO/BRL).
The tissue was cut into small pieces, placed in TRIzol, homogenized,
and total RNA was purified. Poly A+ mRNA was
isolated using an oligo-dT column (FastTrack 2.0 Kit from Invitrogen,
Carlsbad, CA). In brief, 0.5–1 mg of total RNA resuspended in
10 mmol/L Tris/HCl (pH 7.5) buffer was added to FastTrack lysis buffer
(10 ml) containing RNase/protein degrader. The solution was heated to
65°C for 15 minutes, oligo(dT) cellulose was added, and the mixture
was incubated for 2 hours at room temperature. The resin was then
washed and mRNA eluted. RNA was monitored by absorbance at 260/280 nm
(the ratio was generally 
1.8) and agarose gel electrophoresis.
cDNA Expression Array Study
32P-labeled cDNA probes were prepared by
transcribing 1 µg of each RNA population (poly
A+ RNA derived from emphysematous or control
lung) using reagents purchased from Atlas cDNA Expression Arrays Kit
(Clontech, Palo Alto, CA) and
[
32P]dATP (Amersham, Piscataway, NJ,
no. PB 10204; 10 µCi/µl; 3,000 Ci/mmol). Labeled cDNA probes were
separated from unincorporated 32P-labeled
nucleotides and small (<0.1 kb) cDNA fragments, using Chroma Spin-200
DEPC-H2O columns. Then, equal amounts of
32P-labeled cDNA probes were separately
hybridized for 4 hours at 68°C to duplicate Atlas Array membranes
with immobilized cDNA arrays (Clontech, 588 genes). Membranes were
subjected to high stringency washing conditions (0.1 x SSC with
0.5% sodium dodecyl sulfate) for 2 hours at 68°C, and
autoradiography was performed at -70°C with Kodak XAR film.
Hybridization signals for different poly A+ RNA
samples were compared using NIH Image software. Four
preparations from normal controls and four from emphysema patients were
studied using the arrays. Levels of Egr-1, based on hybridization
signals, were normalized according to the average levels of four
housekeeping genes, ubiquitin, GAPDH, ß-actin, and 23-kd highly basic
protein, as suggested by the manufacturer.
Northern Blotting
Total cellular RNA (10–20 µg/sample) was subjected to
denaturing gel electrophoresis in 0.8% agarose-formaldehyde gels and
transferred to Duralon UV membranes (Stratagene, La Jolla, CA).
Membranes were hybridized for 1 hour at 68°C with
32P-labeled partial cDNAs for human Egr-1
(271–1902CDS; GenBank NM 001964) and Sp1 (human cDNA, 2.1-kb
fragment),6
and autoradiography was performed as above.
Gel-purified DNA fragments were labeled using
-32P-dCTP with a random primer labeling kit
(Stratagene).
Immunohistochemistry
Lung tissue was collected, immediately cut into small pieces, and placed in buffered formalin (10%). Following fixation, tissue was paraffin-embedded and stained for routine histochemistry (H&E) or processed for immunohistochemistry to detect Egr-1 antigen. Lung tissue was washed with phosphate-buffered saline (pH 7.0), fixed in formalin, and embedded in paraffin. Sections were first stained with rabbit anti-Egr-1 IgG (8 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), and then were incubated with secondary antibody, an affinity-purified peroxidase-conjugated anti-rabbit IgG (Sigma, St. Louis, MO). Where indicated, an excess of Egr-1 peptide (Santa Cruz Biotechnology) used as immunogen to generate the anti-Egr-1 antibody was preincubated with tissue sections, and then the primary antibody was added for a further incubation period.
Western Blotting
Small pieces of frozen lung were thawed in RIPA buffer (sodium dodecyl sulfate, 0.1%; sodium deoxycholate, 0.5%; Nonidet P-40, 1%; Tris-HCl, 20 mmol/L, pH 7.5, and NaCl, 150 mmol/L) with freshly added inhibitors (phenylmethylsulfonyl fluoride, 200 µmol/L; leupeptin, 1 µg/ml; pepstatin, 1 µg/ml; and aprotonin, 1 µg/ml) using 1 ml of ice-cold buffer per gram of tissue. Tissue was further disrupted by dounce homogenization; the temperature was maintained at 4°C throughout the procedure. The mixture was centrifuged at 10,000 x g for 10 minutes at 4°C, and the supernatant (total tissue lysate) was harvested for further study. Protein concentration was determined by the method of Bradford, lysate (50 µg) was mixed with 2x electrophoresis sample buffer, and electrophoresis was performed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% reduced). Proteins in the gel were transferred to nitrocellulose membranes, and the membranes were blocked by incubation with nonfat milk (10%) at 4°C overnight.18 Membranes were then exposed to anti-Egr-1-specific antibody (1:2000 dilution; no. 588, Santa Cruz Biotechnology) for 90 minutes at room temperature and washed 6x for 10 minutes each using Tris-buffered saline containing Tween-20 (0.05%). Horseradish peroxidase-conjugated secondary antibody was used to detect sites of primary antibody binding, and the enhanced chemiluminescence method was used. Blots were also incubated with antibody to ß-actin purchased from Sigma.
Gel Shift Analysis
Nuclear extracts were prepared from lung tissues stored at
-80°C by the procedure of Dignam et al19
as modified by
Schrieber et al.20
The following double-stranded
oligonucleotide probes, purchased from Santa Cruz, were used (only the
5'-3' sequence is shown): Egr consensus sequence
(5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3'), mutationally inactivated Egr
consensus sequence (5'-GGATCCAGCTAGGGCGAGCTAGGGGGA-3'), and Sp1
consensus sequence (5'-ATTCGATCGGGGCGGGGCGAG-3'). Oligonucleotides
were radiolabeled using [
32P]ATP (Amersham)
and T4 polynucleotide kinase (Promega, Madison, WI). For
electrophoretic mobility shift assay (EMSA), reaction mixtures (20
µl) containing nuclear extract (5 µg) and
32P-labeled oligonucleotide probe (50,000 cpm) in
gel shift reaction buffer (Promega) were incubated for 20 minutes at
room temperature. Supershift experiments were performed by adding
anti-Egr-1 antibody (no. 588, Santa Cruz Biotechnology) or the same
concentration of nonimmune IgG to reaction mixtures (1 µl antibody
per 20 µl of reaction mixture) for 1 hour at 4°C. DNA-protein
complexes were resolved by nondenaturing gel electrophoresis on 4%
polyacrylamide gels in 0.5x TBE buffer at 120 V at 4°C. Gels were
dried and subjected to autoradiography.
Cell Culture
Human lung fibroblasts (HLF; strain CCL-202, American Type Culture
Collection, Manassas, VA) were grown in complete medium (minimal
essential medium with nonessential amino acids, L-glutamine, and
penicillin/streptomycin supplemented with fetal calf serum [10%;
GIBCO/BRL]). Human bronchial smooth muscle cells (BSMC) and human
pulmonary artery smooth muscle cells (PASMC) were obtained from
Clonetics (San Diego, CA). BSMC and PASMC were grown in complete medium
(Smooth Muscle Cell Basal Medium [Clonetics], human epidermal growth
factor [0.5 ng/ml], insulin [10 µg/ml], human fibroblast growth
factor [2 ng/ml], gentamicin [500 µg/ml] and amphotericin B [0.1
µg/ml]) supplemented with 5% fetal calf serum. Confluent cells were
washed with phosphate-buffered saline and stimulated in medium
containing cytokines, IL-1 (R&D Systems, Minneapolis, MN), TNF-
(R&D), IFN- (Sigma), TGF-ß1 (R&D) or phorbol myristate acetate
(PMA; Sigma) alone. Total cellular RNA was extracted from adherent
cells and used for Northern blot analysis.
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Results |
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cDNA made from poly A+ RNA derived from four
emphysema patients and four controls was analyzed using the cDNA
expression array system. Figure 1
is a
representative autoradiogram of one emphysema (Figure 1B)
and one
control patient (Figure 1A)
. Visual inspection suggested a strong
increase in a cDNA corresponding to Etr103 (note arrow in Figure 1A
), a
gene encoding a zinc finger protein induced during phorbol
ester-stimulated differentiation of HL60 cells identical to
Egr-1.21
This impression was confirmed by image analysis,
based on data from all eight individuals (Figure 1C)
. The relative
expression of Egr-1 cDNA (the Egr-1 hybridization signal was normalized
based on levels of housekeeping genes; see Methods section) in
emphysema patients appeared to be increased 
3.3-fold compared with
controls (P = 0.036). In contrast, multiple
housekeeping genes, including ubiquitin, phospholipase A2,
glyceraldehyde-3-phosphate dehydrogenase (G3PDH), ß-actin, and 23-kd
highly basic protein, displayed similar levels in the cDNA expression
array assay comparing control and emphysema tissue (not shown).
Expression of several other potentially relevant genes was also
assessed: transforming growth factor (TGF)-ß1 because of its role in
fibrotic disorders,22
two apoptosis-related genes, Mch4
(apoptotic cysteine proteinase) and TRAF-2 (TNF receptor-associated
factor-2),23,24
and interleukin (IL)-13, a cytokine
implicated in allergic disorders such as asthma.25,26
Although there appeared to be small and variable increases in the
hybridization signal for TGF-ß1, Mch4, TRAF-2, and IL-13 in cDNA
prepared from poly A+ RNA from some samples of
emphysematous lung, they did not reach statistical significance (Figure 1C)
. Note that levels of Sp1 transcripts assessed by this method also
did not appear different in emphysema and control patients. Taken
together, these results focused our attention on analyzing Egr-1
expression in lung tissue from patients with emphysema.
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Total RNA from 28 patients with emphysema and 8 non-emphysematous
controls was analyzed by Northern blotting using a labeled cDNA for
Egr-1. Figure 2
shows the results with 15
samples (10 emphysema and 5 controls); a strong increase in Egr-1
transcripts in the patients with emphysema (lanes 6–15) is observed
compared with the controls (lanes 1–5). When these data were pooled
with those obtained from Northern blot analysis of the other samples
(18 emphysema and 3 controls), 21/28 samples from emphysema patients
showed a strong increase in Egr-1 (Figure 3
, lanes 6–15), whereas the others
(7/28) showed a lesser or no increase in Egr-1 transcripts. The control
samples displayed low levels of Egr-1 in 7/8 samples, though one showed
higher levels of Egr-1.
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Increased levels of Egr-1 transcripts in emphysematous lung did not reflect a general enhancement in expression of transcription factors. For example, levels of Sp1 were virtually the same in total RNA harvested from emphysema and control groups study by Northern blotting (not shown). This is especially relevant since GC-rich DNA binding motifs for Sp1 and Egr-1 can be distributed in an overlapping topology, and it has been shown that increased expression of Egr-1 can displace Sp1 from such sites, thereby modulating gene expression.3,27
Expression of Egr-1 Antigen in Emphysematous Lung
Levels of Egr-1 antigen in lungs of patients with emphysema
appeared to be directly proportional to the amount of Egr-1 mRNA.
Figure 3, A and B
, displays results of Western (Figure 3A)
and Northern
(Figure 3B)
blot analysis in representative emphysema patients with low
(lanes 1 and 2) and high (lanes 3–6) levels of Egr-1 expression. It is
evident that patients with low levels of Egr-1 transcripts display low
levels of Egr-1 antigen (lanes 1 and 2). The latter does not reflect
RNA or protein degradation in the samples, as ß-actin transcripts and
antigen were represented by discrete bands and ß-actin was highly
expressed in these two individuals (lanes 1 and 2). Emphysema patients
whose lung samples displayed high levels of Egr-1 transcripts showed,
in parallel, elevated Egr-1 antigen (lanes 3–6).
Sites of Egr-1 expression in emphysematous lung were visualized
immunohistologically in freshly procured lung samples fixed in
formalin. Six samples from emphysema patients and four controls were
analyzed in this fashion, and results in one representative emphysema
patient and one control are shown in Figure 4
. All patients had severe emphysema; evidence of greatly dilated alveolar
airspaces and typical clubbing is observed by H&E staining (Figure 4, A and B)
. Egr-1 antigen was observed in bronchial epithelium and smooth
muscle, and in many peribronchial cells, presumed to be
monocyte-macrophages (Figure 4, C and D)
. Immunoreactive Egr-1 was also
seen in alveolar macrophages (Figure 4E)
. In each case, Egr-1
immunoreactivity appears to be principally cytoplasmic. Similar strong
staining for Egr-1 was seen in the other five emphysema patients whose
lungs were subjected to immunohistochemical analysis (not shown). When
samples were preincubated with the Egr-1 peptide used as immunogen, and
then anti-Egr-1 IgG was added, the specific staining pattern was
prevented (Figure 4, F
-H). In addition, replacement of anti-Egr-1 IgG
with nonimmune IgG, also resulted in no staining (Figure 4, I
-K). In
contrast to these results in lung from emphysema patients, lungs from
controls (Figure 4M)
displayed virtually no detectable Egr-1 under the
same conditions in a sample from a control individual (Figure 4L)
.
Similar results were obtained when tissue from three other control
individuals was analyzed (not shown).
|
. Nuclear extracts from a patient with
emphysema and a high level of Egr-1 transcripts demonstrated a strong
gel shift band (lane 1) with consensus
32P-labeled Egr oligonucleotide probe, which was
not observed with mutationally inactivated Egr probe (lane 2).
Supershift experiments were performed to determine whether the gel
shift band observed with consensus Egr oligonucleotide probe and
nuclear extracts from the patient with emphysema represented
interaction with Egr-1: addition of anti-Egr-1 IgG to the reaction
mixture resulted in a supershift (lane 3), whereas the same
concentration of nonimmune IgG was without effect (lane 4). Further
evidence for the specificity of protein-DNA interaction was shown by
competition experiments in which 100-fold excess of unlabeled Egr probe
blocked appearance of the gel shift band (lane 5) whereas similar
experiments with unlabeled Sp1 were without effect (lane 6). Although
these results provide strong support for the concept that Egr-1 might
direct transcription in emphysematous lung, when nuclear extracts from
five patients were studied, only two samples provided strong gel shift
bands (as those shown in Figure 5
), whereas the other three produced
small to no increases in the gel shift band (though high levels of
Egr-1 antigen were present). This apparent discrepancy between the
presence of Egr-1 antigen and its activity in EMSA might be explained
by the tissue from which the nuclear extracts were prepared. The
latter extracts were prepared from frozen lung, and we have
subsequently found that storage of lung at -80°C for intervals of
weeks to months (in this case, up to 2 months) often results in loss of
activity in the gel shift assay, although antigenicity by
immunoblotting is maintained.
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In view of the expression of Egr-1 in a range of cells in
emphysematous lung, experiments were performed to determine the
potential of some of these cells to produce this transcription factor
under basal conditions and in response to stimulation in cell culture.
Human lung fibroblasts (HLF), human bronchial smooth muscle cells
(BSMC), and human pulmonary artery smooth muscle cells (PASMC)
displayed low levels of Egr-1 transcripts under quiescent conditions in
cell culture. After exposure of these cultures to a generic stimulus,
PMA (50 ng/ml), there was a striking, time-dependent increase in the
level of Egr-1 transcripts in each of these cell types (Figure 6A)
. Egr-1 mRNA was strongly up-regulated
in HLF, PASMC, and BSMC after 60 minutes of incubation with phorbol
ester. In HLF and PASMC, the level of transcripts was reduced by 120
minutes, whereas Egr-1 transcripts were more sustained in BSMC.
Cultured cells were also stimulated with cytokines likely to be present
in the inflammatory environment of emphysematous lung at various stages
of the disease, including IL-1, tumor necrosis factor (TNF)-,
interferon (IFN)-, and transforming growth factor (TGF)-ß1.
Addition of IL-1, TNF-, and TGFß1, individually and together
(combinations of IL-1 + TNF, IL-1 + TGFß1, and TNF +
TGFß1) to BSMC (Figure 6C)
resulted in robust induction of Egr-1
transcripts that was clearly sustained at the 2-hour time point,
especially compared with results on PASMC and HLF (see Figure 6, D and E
). Cultured PASMC (Figure 6D)
and HLF (Figure 6E)
exposed to the same
combinations of cytokines displayed prominent up-regulation of Egr-1
transcripts, though the level was clearly reduced by the 2-hour time
point. Experiments with IFN- demonstrated that this cytokine also
induced Egr-1 transcripts, with maximal levels of Egr-1 mRNA by 60
minutes for each of the three cell types (Figure 6B)
. Although levels
of steady-state Egr-1 mRNA had clearly declined by 2 hours, the most
persistent elevation was again seen in BSMC. These data indicate the
responsiveness of cultured cells derived from the lung for expression
of Egr-1 after exposure to cytokines. In the case of BSMC,
up-regulation of Egr-1 appeared to have a somewhat longer time course,
in that message levels were still high after 120 minutes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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75%) for
induction of Egr-1 in a broad range of lung cells. Compared with the
rapid induction and decay of Egr-1 in response to acute injuries or
environmental perturbations,1,3-6,8-12
it seems that
Egr-1 expression is much more prolonged in emphysema. Consistent with
these in vivo data, treatment of cultured lung-derived human
fibroblasts and bronchial and pulmonary artery smooth muscle cells with
a cocktail of cytokines resulted in enhanced Egr-1 transcripts. These
data suggest the relevance of examining the contribution of Egr-1 to
the biology of lung remodeling, including the active airway destruction
and fibrosis characteristic of the earlier course of emphysema.
Because our studies involved analysis of only 28 patients with late
stage emphysema, the relevance of these data to earlier stages of
disease pathogenesis remains uncertain. This may be why it was
difficult to identify increased expression of genes thought to be
regulated by Egr-1 in previous in vitro
studies.4
Using cDNA expression arrays and the patient
samples, enhanced expression of several genes thought (based on
in vitro evidence) to be regulated by Egr-1 was not found.
For example, evaluation of cDNA derived from lung RNA did not reveal
increases in transcripts for TGF-ß1,28
CD44,29
TNF-,30
macrophage-colony
stimulating factor-1 (M-CSF),31
PDGF-A or
-B,32,33
ICAM-1,34
and NF-
B
p10535
in emphysematous lung. There are several possible
explanations for these results: 1) more sensitive methods are required
to accurately detect possibly subtle changes in low abundance
transcripts, such as those indicated above; 2) Egr-1-regulated genes
are not expressed at this late stage of emphysema, despite high levels
of the transcription factor; 3) other, yet to be identified, genes
under control of Egr-1 are involved; 4) corepressors of Egr-1,
especially NAB2, are also induced at high levels in emphysematous
lung;36
5) Egr-1 has principally a repressive effect on
gene expression under these conditions; and 6) Egr-1 in emphysematous
lung is not transcriptionally active.
For genes traditionally associated with Egr-1, based on studies in vitro,4 these data must be confirmed in vivo. To the best of our knowledge, only three genes have been shown to be regulated by Egr-1 in vivo by study of the Egr-1 knockouts: luteinizing hormone-ß in females,17 apo A-1,37 and tissue factor.6 Thus, although the Egr-1 response element is present in a multitude of genes and its contribution to gene expression can be dissected by manipulation of environmental conditions in culture, it is possible that other factors predominate in vivo. In this context, it is likely that genes may prove to be regulated by Egr-1 in vivo that have been overlooked by in vitro analyses. Egr-1-null animals subject to a range of stresses clearly provide a rational experimental system in which to perform such studies. Finally, it is important to note that assessment of Egr-1-regulated genes by analysis of total RNA extracted from lung, or cDNA produced from total lung mRNA, principally detects striking changes in the expression of transcripts of relatively greater abundance. In view of the likelihood that focal changes in gene expression will occur in emphysema, especially earlier in the disease, careful in situ hybridization and immunohistochemical studies will be necessary for sensitive identification of such induced transcripts and the transcribed proteins. However, at this point, candidate Egr-1-regulated genes for this more in-depth analysis remain to be identified.
Two important issues should be clarified with respect to our data pertaining to the potential transcriptional competency of Egr-1 extracted from the lung tissue and concerning the control group for our study. In other work performed after the current study was completed, we have found that to reproducibly maintain sequence-specific DNA binding activity of Egr-1 extracted from emphysematous lung tissue, it is necessary to prepare the nuclear extracts immediately and to minimize storage time at -80°C. We believe this is the reason that the DNA binding activity detected in our samples with increased amounts of Egr-1 antigen was variable. However, it remains possible that the Egr-1 produced in emphysematous lung is not fully competent in terms of its transcriptional activity, possibly reflecting events at the level of protein phosphorylation.38 This would also explain the principally cytoplasmic, rather than nuclear, distribution of Egr-1, and the lack of expression of Egr-1-regulated genes. Our next approach to this question will be to perform studies on optimally prepared nuclear extracts from lung samples. This cannot be decided with the current samples because of their extended time in storage and the limited amounts of remaining material. In terms of our control group, apparently uninvolved lung at the margins of resected nodules or samples from donors which proved unacceptable for clinical lung transplantation represent nonideal normal tissue to serve as the basis for comparison. However, these samples are remarkably similar to those harvested from emphysema patients in that they are freshly obtained from a living patient. Clearly, procuring the best normal control tissue for this type of study will always have to be somewhat of a compromise, as it is not possible to obtain tissue from healthy, normal volunteers.
Despite these reservations, our study is the first to report increased
expression of Egr-1 in patients with late stage emphysema. Our
observations should serve as a stimulus for additional studies using a
range of lung samples to determine whether Egr-1 is expressed earlier
in the disease, if genes presumably regulated by Egr-1 are responsive,
and, ultimately, to analyze whether Egr-1 contributes to the
pathogenesis or progression of this destructive pulmonary disease.
Induction of Egr-1 in lung-derived cultured lung fibroblasts and smooth
muscle cells by a range of cytokines, including IL-1, TNF-, IFN-,
and TGF-ß1, which could potentially contribute to airway damage and
fibrosis at different stages of chronic lung disease, suggests that
induction of Egr-1 may be relevant to the pathology of this disorder.
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Footnotes |
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Supported by funds from the Surgical Research Fund and grants from the U. S. Public Health Service (HL63967, AG16233).
Accepted for publication June 30, 2000.
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 E. Harja, L. G. Bucciarelli, Y. Lu, D. M. Stern, Y. S. Zou, A. M. Schmidt, and S.-F. Yan Early Growth Response-1 Promotes Atherogenesis: Mice Deficient in Early Growth Response-1 and Apolipoprotein E Display Decreased Atherosclerosis and Vascular Inflammation Circ. Res., February 20, 2004; 94(3): 333 - 339. [Abstract] [Full Text] [PDF]
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