Departments of Pediatrics and Environmental Medicine, Strong Children's Research Center, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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Acute lung
inflammation is complicated by altered pulmonary surfactant
phospholipid and protein composition. The proinflammatory cytokine
tumor necrosis factor- (TNF-
) and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate
(TPA) inhibit expression of surfactant-associated proteins A and B
(SP-A and SP-B), both important for normal surfactant function. The
transcription factor nuclear factor-
B (NF-
B) frequently mediates
regulation of gene expression by TPA and TNF-
. In the present study,
electrophoretic mobility shift assays (EMSAs) and pyrrolidine
dithiocarbamate (PDTC), an inhibitor of NF-
B activation, were
utilized to determine the role of NF-
B activation in TPA and TNF-
inhibition of the surfactant proteins in NCI-H441 cells. Pentoxifylline
(PTX), which inhibits TNF-
cellular effects without preventing
NF-
B activation, was also tested. By EMSA, TPA and TNF-
increased
nuclear NF-
B binding activity in temporally distinct patterns. PDTC
decreased TPA- and TNF-
-induced NF-
B binding activity but did not
limit their inhibition of SP-A and SP-B mRNAs. PDTC independently
decreased both SP-A and SP-B mRNAs. PTX partially reversed TNF-
- but
not TPA-mediated inhibition of SP-A and SP-B mRNAs without altering NF-
B binding. The effects of PDTC and PTX on NF-
B and the
surfactant proteins suggest that NF-
B activation does not mediate
TPA or TNF-
inhibition of SP-A and SP-B mRNA accumulation.
tumor necrosis factor-; nuclear factor-
B; pyrrolidine
dithiocarbamate; pentoxifylline; dexamethasone; adenocarcinoma
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INTRODUCTION |
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PULMONARY SURFACTANT is a complex mixture of
phospholipids and associated proteins. Qualitative and quantitative
abnormalities in pulmonary surfactant contribute to lung dysfunction in
patients with acute respiratory distress syndrome (ARDS)
and pneumonia (16). Three surfactant-associated proteins, designated
surfactant protein (SP) A, SP-B, and SP-C, contribute to the formation,
stabilization, and metabolism of pulmonary surfactant. SP-A and SP-B
augment alveolar stability and, particularly in bronchiolar epithelium, host immune defense and distal airway patency (7, 11). Decreased expression or function of these specific surfactant-associated proteins
is associated with surfactant dysfunction and respiratory failure in
mice and humans (8, 12, 17, 21). Heterozygous transgenic SP-B
(+/) mice, expressing ~50% of normal SP-B mRNA and protein
levels, demonstrate abnormal pulmonary function (7). Acquired
reductions in SP-A and SP-B are reported in the bronchoalveolar lavage
fluid of patients with ARDS and pneumonia, suggesting that decreased SP
levels contribute to the pathophysiology of inflammatory pulmonary
diseases (5, 10).
It is likely that the effects of tumor necrosis factor (TNF)- on
alveolar type II cells, the pulmonary epithelial cells that produce
surfactant, contribute to human lung diseases including ARDS. The
cytokine TNF-
is a principal mediator of ARDS associated with
invasive bacterial infection and reperfusion injury (for a review, see
Ref. 9). TNF-
is increased in the bronchoalveolar lavage fluid and
serum of patients with ARDS or sepsis syndrome (27). Clinically or
experimentally elevated pulmonary TNF-
is associated with abnormal
surfactant phospholipid and surfactant-associated protein content in
vivo (29). In the human pulmonary adenocarcinoma cell line NCI-H441
(H441), SP-A and SP-B mRNA levels decreased within 2 h and reached
~20% of the control level 24 h after treatment with recombinant
human (rh) TNF-
or the phorbol ester
12-O-tetradecanoyl phorbol-13-acetate
(TPA) (22, 30). TNF-
also decreased surfactant phospholipid
synthesis by human alveolar type II cells in vitro (4). Intratracheal
instillation of TNF-
resulted in decreased SP mRNA in mice (19).
Understanding the mechanisms by which TNF-
and TPA alter surfactant
synthesis may suggest novel therapies for inflammatory lung diseases.
The cellular effects of TNF- and TPA are frequently mediated by
activation of the transcription factor nuclear factor-
B (NF-
B)
(13). The present study examines the relationship of NF-
B activation
to the inhibition of SP mRNA by TPA and TNF-
. Before treatment with
TNF-
or TPA, H441 cells were pretreated with pyrrolidine
dithiocarbamate (PDTC), previously utilized as a potent, specific
inhibitor of NF-
B, or with 1-(5-oxohexyl)-3,7-dimethylxanthine [pentoxifylline (PTX)], an inhibitor of TNF-
synthesis
and cellular activity (4, 24). Nuclear NF-
B binding activity was
demonstrated by electrophoretic mobility shift assay (EMSA). Changes in
the expression of SP-A, measured in the absence of dexamethasone (Dex), and SP-B, measured in the presence of Dex, were assessed by Northern blot.
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METHODS |
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Cell culture and reagents. The H441
cells express SP-A and SP-B and have morphological features most
closely related to bronchiolar epithelial cells (18). H441 cells were
maintained in RPMI 1640 medium (GIBCO BRL, Grand Island, NY) with 10%
heat-inactivated fetal bovine serum (Intergen, Purchase, NY) and 1%
antibiotic solution (100 U/ml of penicillin, 100 µg/ml of
amphotericin B, and 100 µg/ml of streptomycin; GIBCO BRL) as
previously described (18). SP-B mRNA was studied in cells
pretreated with Dex (Sigma, St. Louis, MO) prepared as a 10 mM stock
solution in 95% ethanol, diluted further in medium to 50 nM, and
applied 48 h before the addition of TPA (10 nM; Sigma) or rhTNF- (25 ng/ml; R&D Systems, Minneapolis, MN). rhTNF-
was resuspended in
endotoxin-free saline. TPA was prepared as a 2 mM stock solution in
dimethyl sulfoxide before further dilution in medium. Where indicated,
PDTC (0.1-100 µM) or PTX (100 µg/ml), resuspended in RPMI
medium, was added 2 h before TNF-
or TPA treatment.
Northern blot analysis of SP-A, SP-B, and
-actin mRNAs. Cellular RNA was isolated from
cells lysed in situ in 4 M guanidinium isothiocyanate (Kodak Chemical,
Rochester, NY), 0.5%
N-lauroylsarcosine (Sigma), and 25 mM
sodium citrate and was stored at
80°C. Total cell RNA was
extracted by the method of Chomczynski and Sacchi (6) modified for
phase lock gel II columns (5 Prime
3 Prime, Boulder, CO). The
RNA was quantitated by absorbance at 260 nm. Samples (15 µg) of RNA
were fractionated on a 1.2% agarose-formaldehyde gel and transferred
to Nytran membranes (Schleicher & Schuell, Keene, NH). After
ultraviolet cross-linking, the Nytran was stained with 1% methylene
blue in 0.3 M sodium acetate to assess the integrity of the RNA and to
verify the uniformity of loading. The Nytran was then prehybridized for
3-4 h in a 65°C hybridization oven (Robbins Scientific,
Sunnyvale, CA) in 500 mM Na phosphate, 1 mM EDTA, 1% bovine serum
albumin, and 7% sodium dodecyl sulfate. Hybridization was performed at
65°C overnight in the same solution with the addition of
random-primed
[
-32P]dCTP-radiolabeled
human SP-A or SP-B cDNA as previously described (20). After
hybridization, the blots were washed once at room temperature and again
at 65°C, both for 20 min, in 40 mM Na phosphate, 1 mM EDTA, and 1%
sodium dodecyl sulfate. All filters were exposed to a phosphorimaging
storage screen for 1-24 h. The hybridization signal was
quantitated with ImageQuant software (Molecular Dynamics, Sunnyvale,
CA). After imaging and quantitation, the radiolabel was removed in Na
pyrophosphate at 65°C for 2 h. The filters were rehybridized with
-32P-labeled
3'-untranslated region of human
-actin cDNA, washed, and
quantitated as described above.
Quantitative data from Northern blot assays were analyzed by
single-factor analysis of variance and Fisher's protected least significant difference statistics utilizing Statview 4.0 statistical-analysis software (Abacus Concepts, Berkeley, CA) on a
Macintosh computer system. The experimental data are expressed as a
percentage of mRNA detected in the control cells normalized to
-actin mRNA content.
EMSAs. Nuclear protein extracts were
prepared from H441 cells, as indicated, 0-24 h after treatment.
Cells were harvested by gentle scraping. Cytoplasmic membranes were
lysed in 0.1% Igepal CA-630 nonionic detergent (Sigma), 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10 mM KCl, and 1.5 mM
MgCl2, pH 7.9, for 10 min on ice.
The cytoplasmic protein fraction was removed by centrifugation at
12,000 g. Nuclear proteins were
extracted in high-salt buffer (420 mM NaCl, 20 mM HEPES, 1.5 mM
MgCl2, 0.2 mM EDTA, and 25% glycerol, pH 7.9) at 4°C. The supernatant was diluted in 50 mM KCl,
20 mM HEPES, 0.2 mM EDTA, and 20% glycerol, pH 7.9, and stored at
80°C until utilized as nuclear protein extracts for EMSA. Lysis, extraction, and diluting buffers each contained dithiothreitol (0.5 mM), Pefabloc (0.5 mM; Boehringer Mannheim, Indianapolis, IN), and
leupeptin (10 µg/ml). Nuclear protein (10 µg), quantified by
bicinchoninic acid assay (26), was incubated with 0.01 pmol [
-32P]ATP-labeled,
double-stranded consensus NF-
B DNA binding sequence in 4% glycerol,
1 mM MgCl2, 0.5 mM EDTA, 0.5 mM
dithiothreitol, 50 mM NaCl, 10 mM
tris(hydroxymethyl)aminomethane · HCl, pH 7.5, and
0.05 mg/ml of polydeoxyinosinic-deoxycytidylic acid for 20 min at room
temperature. The sequence of the NF-
B oligonucleotide utilized was
5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Promega, Madison, WI)
(14). Nuclear protein extract derived from TPA-stimulated HeLa cells
was utilized as an EMSA positive control (Promega). Supershift assays
were performed by subsequent incubation of the DNA-nuclear protein
solution with 1 µg of anti-p65, anti-p52, or anti-p50 antibody (Santa
Cruz Biotechnology, Santa Cruz, CA). The radiolabeled DNA-protein
extract mixture was fractionated by nondenaturing, 4% polyacrylamide
gel electrophoresis (40:1 acryl-bis). The polyacrylamide gel was dried
and visualized by phosphorimaging utilizing ImageQuant software.
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RESULTS |
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The concentrations of TPA, TNF-, PDTC, and PTX utilized did not
cause cytotoxicity as indicated by trypan blue exclusion and
light-microscopic evaluation. Under the experimental conditions chosen,
the quality and quantity of total RNA recovered and of
-actin mRNA
levels detected were not influenced by these agents. As previously
demonstrated in the H441 cell line, Dex enhanced SP-B mRNA content,
whereas TNF-
and TPA decreased SP-A and SP-B mRNA levels (18, 30).
Induction of NF-B binding activity by TNF-
and TPA. EMSAs utilizing a radiolabeled NF-
B
consensus binding site are represented in Fig.
1. Increased NF-
B binding was detected
in nuclear protein extracts harvested from H441 cells 24 h after
treatment with TNF-
or TPA (Fig.
1A). Specificity of two of the
shifted bands (solid arrowheads) was confirmed by further retardation
of the protein-DNA complexes by antibodies specific for the NF-
B p65
(Fig. 1B) and NF-
B p50 (Fig.
1C) proteins and by competition with
100-fold excess of unlabeled NF-
B oligonucleotides (Fig.
1D). Anti-p65 antibody resulted in
retarded migration of the upper, more slowly migrating band, whereas
anti-p50 antibody retarded the upper band partially and the lower band
completely. Anti-NF-
Bp52 antibody did not supershift the observed
bands (data not shown). The lower set of bands in Fig. 1 (brackets)
were consistently observed in the mobility assays and were partially
competed with 100-fold excess of unlabeled NF-
B oligonucleotides but
were not competed or shifted by the anti-NF-
B antibodies tested,
anti-p65, -p52 and -p50. The intensity and pattern of these lower bands
varied and were not consistently altered by TPA, TNF-
, PDTC, or PTX treatment (Fig. 1; also see Fig. 3).
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To study SP-B mRNA expression, H441 cells were pretreated with Dex.
SP-A was studied in cells not treated with the steroid because Dex
decreases SP-A mRNA to nearly undetectable levels in H441 cells. EMSAs
were performed with nuclear extracts taken from cells that were and
were not pretreated with Dex to determine the impact of the steroid on
NF-B activity. Consistent with a previous report (3) in
other cell types, Dex decreased both basal TNF-
- and TPA-induced
NF-
B binding activity (Fig. 2). Changes
in NF-
B binding induced by TPA, TNF-
, PDTC, or PTX relative to
the appropriate RPMI- or Dex-treated control cells were not appreciably
altered by Dex pretreatment (Fig. 2; also see Fig. 6).
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The activation of NF-B binding by TNF-
or TPA in H441 cells
followed specific temporal patterns (Fig.
3). NF-
B binding was markedly increased
within 2 h of TNF-
treatment and was decreased at 4 h but
progressively increased at 8 and 24 h to levels at least as high as at
3 h (Fig. 3). The pattern of TPA-induced NF-
B binding differed from
the TNF-
time course. TPA induction of NF-
B binding was maximal
2-4 h after treatment and subsequently decreased, although
remaining greater than the control level at 24 h (Fig. 3).
The resolution of the current EMSAs was inadequate to determine
differences in the temporal patterns of induction of the upper p65-p50
band versus the lower p50-p50 band.
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Effect of PDTC on NF-B activation by TPA and
TNF-
. PDTC was demonstrated in other cell types
to specifically inhibit NF-
B binding and block TNF-
activity
(24). As shown in Fig. 3, PDTC blunted or prevented the increase in
NF-
B binding observed 2-24 h after TNF-
and TPA treatment.
The inhibition of TNF-
-induced NF-
B complex formation by PDTC was
greatest at 2 and 24 h, the times of peak NF-
B activation.
TPA-induced NF-
B was also maximally decreased by PDTC at 2 and 24 h
after TPA treatment. Both upper p65-p50 and lower p50 complexes were
inhibited by PDTC.
Effect of PDTC on SP-A and SP-B mRNAs.
Despite the ability to limit NF-B activation, PDTC did not block
TNF-
- or TPA-mediated inhibition of SP mRNA levels (Fig.
4). In the presence of Dex, PDTC decreased
SP-B mRNA levels (Fig. 4B). PDTC may
also decrease basal non-Dex-induced levels of SP-B mRNA, but the basal
levels were too low to accurately quantify. PDTC also decreased SP-A mRNA levels in non-Dex-treated cells (Fig.
4A). Inhibition of SP-A and SP-B
mRNAs by PDTC was dose dependent (Fig. 5).
The 50% inhibitory concentration for both SP-A and SP-B was between 10 and 50 µM.
-Actin mRNA levels were not altered by PDTC.
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Effect of PTX on SP-A and SP-B mRNAs.
H441 cells were also treated with PTX 2 h before the addition of
TNF- or TPA. Representative Northern blots shown in Fig.
6, A and
B, demonstrate that PTX enhanced the
level of SP-B mRNA in the presence of and SP-A mRNA in the absence of
Dex. PTX induction was greater for SP-B than for SP-A. PTX also
reversed the inhibitory effect of TNF-
on SP-B and SP-A mRNAs by
~80 and 40%, respectively. In contrast, PTX did not alter the
inhibitory effect of TPA on the SP mRNAs. There was no detectable
effect of PTX on non-Dex-induced expression of SP-B mRNA.
-Actin
mRNA levels were not altered by PTX. PTX did not alter NF-
B EMSA
binding activity when utilized alone or when added before TNF-
treatment in either the presence or absence of Dex (Fig.
6C).
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DISCUSSION |
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The present study demonstrates specific patterns of activation of
NF-B binding in the H441 cell line in response to TPA and TNF-
.
Incubation with antibodies to the NF-
B p65 and p50 proteins resulted
in further retardation of two of the DNA-protein complexes, suggesting
that TPA and TNF-
induce binding of a p65-p50 heterodimer, a p50
homodimer, and either a p65 homodimer or a non-p50-containing p65
heterodimer. The temporal patterns of NF-
B induction by TPA and
TNF-
differ. Early activation of NF-
B, within 2 h, was observed after both TPA and TNF-
treatment. However, NF-
B activation after
TNF-
treatment was biphasic and prolonged, whereas TPA induced a
rapid but transient response. Temporal characteristics of TNF-
- and
TPA-mediated NF-
B activation are likely due to cell- and
stimulus-specific transduction pathways (1, 28).
The NF-B p65-p50 heterodimers and p50 homodimers were consistently
induced by TPA and TNF-
in association with the inhibition of SP-A
and SP-B mRNAs. The present study, however, dissociates changes in SP-A
and SP-B mRNA levels from patterns of NF-
B p65 and p50 DNA binding.
For example, PDTC inhibited, at least partially, activation of NF-
B
binding by TPA and TNF-
. However, PDTC did not block the inhibitory
effects of TPA or TNF-
on SP-A or SP-B mRNA. Instead, in a
dose-dependent manner, PDTC alone decreased both SP-A and SP-B mRNA
levels. PTX did inhibit the effect of TNF-
on SP-B and, to a lesser
extent, SP-A mRNAs, but it did not decrease TNF-
-mediated NF-
B
activation. PTX did not alter TPA inhibition of SP-A or SP-B mRNA
level. This study demonstrates differential time-dependent induction of
NF-
B by TPA and TNF-
and suggests that the signal transduction
pathways mediating the effects of TPA and TNF-
inhibition of SP-B
are divergent such that PTX blocks TNF-
but not TPA. This study does
not support a role for NF-
B activation in the regulation of SP-A and
SP-B mRNAs.
The EMSAs reported were performed with a consensus binding sequence for
NF-B present, for example, in immunoglobulin-
light chain genes
and the human immunodeficiency virus LTR promoter (15). SP-specific
NF-
B binding sequences have not been identified and therefore cannot
be used in EMSA. Sequences in the human SP-B gene share 75-80%
homology with the NF-
B consensus sequence; however, there is no
evidence that they are active NF-
B binding sites. Because minor
differences in DNA sequence can influence the binding and activity of
transcription factors, NF-
B species that bind nonconsensus binding
sites and regulate SPs in cis or in
trans may have been overlooked in the
present study. Rapidly migrating NF-
B oligonucleotide-protein
complexes were partially competed by nonlabeled NF-
B
oligonucleotides but were not competed or further shifted by the
anti-NF-
B p65, p50, and p52 antibodies tested (Fig. 1, brackets).
Similar bands have been reported and may represent less common NF-
B
complexes, NF-
B-like proteins, or nonspecific DNA-protein
associations (24, 25). However, these unidentified bands were not
consistently altered by TPA, TNF-
, PDTC, or PTX, and, therefore, the
DNA-protein interactions they represent are not likely to regulate SP
mRNA expression by these agents.
As reported, H441 cells studied for SP-B mRNA were stimulated with Dex,
whereas cells analyzed for SP-A were not treated with the steroid (19,
22, 31). A low constitutive level of NF-B binding activity in H441
cells was noted. Dex reduced the basal nuclear NF-
B binding activity
by roughly 50%, yet the steroid also reduced SP-A mRNA levels (Fig. 2)
(18, 23). Dex also induced SP-B mRNA by 10- to 20-fold, a degree of
induction that is not likely to be explained by such a relatively small
decrease in NF-
B activity (Fig. 4) (18). In addition, SP-B mRNA
levels were greater in Dex-pretreated, TNF-
-treated cells than in
non-Dex-pretreated, non-TNF-
-treated cells, even though
NF-
B binding activity was greater in the former than in the latter.
Thus alterations in SP-A and SP-B mRNA expression did not correlate
with the changes in NF-
B activity induced by Dex with or without
TNF-
, supporting the concept that NF-
B does not regulate SP mRNA
levels.
The response of SP-A mRNA to TPA, TNF-, and PDTC was similar to the
SP-B response despite the lack of steroid induction, supporting the
hypothesis that the effects of PDTC, TPA, and TNF-
on SP mRNA are
independent of mechanisms that mediate Dex induction of SP-B. However,
PTX resulted in a greater induction of SP-B mRNA than of SP-A mRNA,
suggesting that PTX may augment steroid induction of SP-B expression.
Interactions between Dex, TPA, TNF-
, PDTC, and PTX on SP mRNA
expression will become clear as the signaling pathway of each is
defined.
Mechanisms by which PTX increased SP-A and SP-B mRNAs remain to be
elucidated. PTX is a methylxanthine derivative and nonspecific phosphodiesterase inhibitor and may increase intracellular levels of
adenosine 3',5'-cyclic monophosphate (cAMP). SP-A and SP-B mRNAs are enhanced by cAMP (for a review, see Ref. 30). We speculate that PTX increases SP-A and SP-B mRNAs by causing the accumulation of
intracellular cAMP. Recently, beneficial effects of PTX in human septic
shock and ARDS were reported (2, 32). The effects of TNF- and PTX on
SP-B mRNA expression in the present study are similar to the effects of
these agents on the synthesis of surfactant lipids (4). The ability of
PTX to reverse TNF-
-associated inhibition of surfactant phospholipid
synthesis and SP expression, as shown in the present study, may
contribute to its therapeutic efficacy.
The present study demonstrates specific temporal patterns of induction
of nuclear NF-B binding activity by TNF-
and TPA in a pulmonary
epithelial cell line. The expression of SP-A and SP-B mRNAs was,
however, independent of changes in NF-
B binding activity. Further
studies will be needed to determine the consequences of TNF-
-induced
NF-
B binding activity in pulmonary epithelial cells. Improved
understanding of the intracellular responses of pulmonary epithelial
cells to inflammatory mediators such as TNF-
will contribute to the
development of novel clinical interventions for pulmonary disease.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Clinical Investigator Award 5-K08-HL-03318-02 and the Strong Children's Research Center.
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FOOTNOTES |
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Address for reprint requests: G. S. Pryhuber, Strong Children's Research Center, Division of Neonatology, 601 Elmwood Ave., Box 651, Rochester, NY 14642.
Received 20 November 1996; accepted in final form 12 November 1997.
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