Regulation of surfactant proteins A and B by TNF-alpha and phorbol ester independent of NF-kappa B

Gloria S. Pryhuber, Rubia Khalak, and Qian Zhao

Departments of Pediatrics and Environmental Medicine, Strong Children's Research Center, University of Rochester Medical Center, Rochester, New York 14642

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Acute lung inflammation is complicated by altered pulmonary surfactant phospholipid and protein composition. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha ) 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-kappa B (NF-kappa B) frequently mediates regulation of gene expression by TPA and TNF-alpha . In the present study, electrophoretic mobility shift assays (EMSAs) and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation, were utilized to determine the role of NF-kappa B activation in TPA and TNF-alpha inhibition of the surfactant proteins in NCI-H441 cells. Pentoxifylline (PTX), which inhibits TNF-alpha cellular effects without preventing NF-kappa B activation, was also tested. By EMSA, TPA and TNF-alpha increased nuclear NF-kappa B binding activity in temporally distinct patterns. PDTC decreased TPA- and TNF-alpha -induced NF-kappa 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-alpha - but not TPA-mediated inhibition of SP-A and SP-B mRNAs without altering NF-kappa B binding. The effects of PDTC and PTX on NF-kappa B and the surfactant proteins suggest that NF-kappa B activation does not mediate TPA or TNF-alpha inhibition of SP-A and SP-B mRNA accumulation.

tumor necrosis factor-alpha ; nuclear factor-kappa B; pyrrolidine dithiocarbamate; pentoxifylline; dexamethasone; adenocarcinoma

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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)-alpha on alveolar type II cells, the pulmonary epithelial cells that produce surfactant, contribute to human lung diseases including ARDS. The cytokine TNF-alpha is a principal mediator of ARDS associated with invasive bacterial infection and reperfusion injury (for a review, see Ref. 9). TNF-alpha is increased in the bronchoalveolar lavage fluid and serum of patients with ARDS or sepsis syndrome (27). Clinically or experimentally elevated pulmonary TNF-alpha 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-alpha or the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) (22, 30). TNF-alpha also decreased surfactant phospholipid synthesis by human alveolar type II cells in vitro (4). Intratracheal instillation of TNF-alpha resulted in decreased SP mRNA in mice (19). Understanding the mechanisms by which TNF-alpha and TPA alter surfactant synthesis may suggest novel therapies for inflammatory lung diseases.

The cellular effects of TNF-alpha and TPA are frequently mediated by activation of the transcription factor nuclear factor-kappa B (NF-kappa B) (13). The present study examines the relationship of NF-kappa B activation to the inhibition of SP mRNA by TPA and TNF-alpha . Before treatment with TNF-alpha or TPA, H441 cells were pretreated with pyrrolidine dithiocarbamate (PDTC), previously utilized as a potent, specific inhibitor of NF-kappa B, or with 1-(5-oxohexyl)-3,7-dimethylxanthine [pentoxifylline (PTX)], an inhibitor of TNF-alpha synthesis and cellular activity (4, 24). Nuclear NF-kappa 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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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-alpha (25 ng/ml; R&D Systems, Minneapolis, MN). rhTNF-alpha 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-alpha or TPA treatment.

Northern blot analysis of SP-A, SP-B, and beta -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 right-arrow 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 [alpha -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 alpha -32P-labeled 3'-untranslated region of human beta -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 beta -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 [gamma -32P]ATP-labeled, double-stranded consensus NF-kappa 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-kappa 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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The concentrations of TPA, TNF-alpha , 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 beta -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-alpha and TPA decreased SP-A and SP-B mRNA levels (18, 30).

Induction of NF-kappa B binding activity by TNF-alpha and TPA. EMSAs utilizing a radiolabeled NF-kappa B consensus binding site are represented in Fig. 1. Increased NF-kappa B binding was detected in nuclear protein extracts harvested from H441 cells 24 h after treatment with TNF-alpha 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-kappa B p65 (Fig. 1B) and NF-kappa B p50 (Fig. 1C) proteins and by competition with 100-fold excess of unlabeled NF-kappa 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-kappa 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-kappa B oligonucleotides but were not competed or shifted by the anti-NF-kappa 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-alpha , PDTC, or PTX treatment (Fig. 1; also see Fig. 3).


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Fig. 1.   Induction of nuclear factor-kappa B (NF-kappa B) binding activity by tumor necrosis factor (TNF)-alpha and 12-O-tetradecanoylphorbol-13-acetate (TPA). Nuclear protein extracts were prepared from NCI-H441 cells 24 h after treatment with control medium (lanes a), TPA (10 nM; lanes b), or TNF-alpha (25 ng/ml; lanes c). A: electrophoretic mobility shift assays (EMSAs) were performed as described in METHODS. Lane H, TPA-stimulated HeLa cell nuclear protein extract utilized as a positive control. Supershift assays were performed in which anti-NF-kappa B p65 (B) or anti-NF-kappa B p50 (C) antibodies were added to binding assay for 30 min of incubation before electrophoresis. D: TPA-treated extract (lane b) was competed with 100-fold excess nonradiolabeled NF-kappa B oligonucleotide (lane b'). Solid arrowheads, identified NF-kappa B-protein complexes; open arrowheads, unbound, radiolabeled NF-kappa B oligonucleotide; brackets, unidentified complexes.

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-kappa B activity. Consistent with a previous report (3) in other cell types, Dex decreased both basal TNF-alpha - and TPA-induced NF-kappa B binding activity (Fig. 2). Changes in NF-kappa B binding induced by TPA, TNF-alpha , 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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Fig. 2.   NF-kappa B binding activity after exposure to pyrrolidine dithiocarbamate (PDTC), TNF-alpha , TPA, and dexamethasone (Dex). Cells were pretreated for 48 h with control medium (-) or with (+) Dex (50 nM) and then incubated for 24 h, as indicated, with PDTC (100 µM), TNF-alpha (25 ng/ml), or TPA (10 nM). When utilized in cotreatments, PDTC was applied 2 h before TPA or TNF-alpha . EMSAs were performed with nuclear protein extracts (10 µg) and 32P-labeled NF-kappa B consensus oligonucleotides. Solid arrowheads, complexes containing p65 and p50 NF-kappa B proteins; brackets, unidentified complexes.

The activation of NF-kappa B binding by TNF-alpha or TPA in H441 cells followed specific temporal patterns (Fig. 3). NF-kappa B binding was markedly increased within 2 h of TNF-alpha 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-kappa B binding differed from the TNF-alpha time course. TPA induction of NF-kappa 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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Fig. 3.   Time course of TNF-alpha and TPA induction of and PDTC inhibition of NF-kappa B binding activity. Cells were pretreated with control medium or PDTC 2 h before treatment at 0 h with medium that contained TNF-alpha (A) or TPA (B). Nuclear protein extracts were prepared at 0, 2, 4, 8, and 24 h after TNF-alpha or TPA treatment. EMSAs and phosphorimaging were performed as described in METHODS, utilizing 32P-labeled NF-kappa B consensus oligonucleotide. Solid arrowheads, complexes containing p65 and p50 NF-kappa B; brackets, unidentified complexes.

Effect of PDTC on NF-kappa B activation by TPA and TNF-alpha . PDTC was demonstrated in other cell types to specifically inhibit NF-kappa B binding and block TNF-alpha activity (24). As shown in Fig. 3, PDTC blunted or prevented the increase in NF-kappa B binding observed 2-24 h after TNF-alpha and TPA treatment. The inhibition of TNF-alpha -induced NF-kappa B complex formation by PDTC was greatest at 2 and 24 h, the times of peak NF-kappa B activation. TPA-induced NF-kappa 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-kappa B activation, PDTC did not block TNF-alpha - 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. beta -Actin mRNA levels were not altered by PDTC.


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Fig. 4.   Inhibition of surfactant protein (SP) A (A) and SP-B (B) mRNAs by PDTC, TNF-alpha , and TPA. Cells were pretreated for 48 h with control medium (A) or Dex (50 nM; B). Cells were then treated with medium or PDTC (100 µM) 2 h before control medium, TNF-alpha (25 ng/ml), or TPA (10 nM) as indicated. Total RNA was isolated 24 h after treatment with TPA or TNF-alpha . Northern blot assay and phosphorimaging were performed as described in METHODS. Nytran filters were hybridized with 32P-labeled human SP-A or SP-B cDNA, imaged, stripped, and rehybridized with 32P-labeled beta -actin 3'-untranslated region.


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Fig. 5.   Dose-dependent inhibition of SP-A and SP-B mRNAs by PDTC. Cells were pretreated for 48 h with control medium for SP-A analysis or with Dex (50 nM) for SP-B analysis. Total RNA was isolated 24 h after treatment with PDTC (0-100 µM). A: Northern blot assay and phosphorimaging were performed as described in METHODS. Nytran filters were hybridized with 32P-labeled human SP-A or SP-B cDNA, imaged, then stripped, and rehybridized with 32P-labeled beta -actin 3'-untranslated region. B: cumulative data are represented as a fraction of SP-A or SP-B mRNA levels present in control cells normalized to beta -actin mRNA levels. Values are means ± SE; n = 4 samples. * P < 0.05 vs. control.

Effect of PTX on SP-A and SP-B mRNAs. H441 cells were also treated with PTX 2 h before the addition of TNF-alpha 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-alpha 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. beta -Actin mRNA levels were not altered by PTX. PTX did not alter NF-kappa B EMSA binding activity when utilized alone or when added before TNF-alpha treatment in either the presence or absence of Dex (Fig. 6C).


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Fig. 6.   Effect of pentoxifylline (PTX) on SP-A (A) and SP-B (B) mRNA levels. Cells were pretreated for 48 h with control medium or with Dex (50 nM) as indicated. Cells were then treated with control medium or PTX (100 µg/µl) 2 h before addition of control medium or medium that contained TNF-alpha (25 ng/ml) or TPA (10 nM) as indicated. Total RNA was isolated 24 h after treatment. A and B: Northern blot assay and phosphorimaging, respectively, as described in METHODS. Nytran filters were hybridized with 32P-radiolabeled SP-A or SP-B cDNA, imaged, then stripped, and rehybridized with 32P-radiolabeled beta -actin 3'-untranslated region. C: nuclear protein extracts were prepared 24 h after treatment with medium or TNF-alpha . NF-kappa B EMSA and imaging are described in METHODS. Solid arrowheads, identified NF-kappa B-protein complexes; brackets, unidentified complexes.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present study demonstrates specific patterns of activation of NF-kappa B binding in the H441 cell line in response to TPA and TNF-alpha . Incubation with antibodies to the NF-kappa B p65 and p50 proteins resulted in further retardation of two of the DNA-protein complexes, suggesting that TPA and TNF-alpha 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-kappa B induction by TPA and TNF-alpha differ. Early activation of NF-kappa B, within 2 h, was observed after both TPA and TNF-alpha treatment. However, NF-kappa B activation after TNF-alpha treatment was biphasic and prolonged, whereas TPA induced a rapid but transient response. Temporal characteristics of TNF-alpha - and TPA-mediated NF-kappa B activation are likely due to cell- and stimulus-specific transduction pathways (1, 28).

The NF-kappa B p65-p50 heterodimers and p50 homodimers were consistently induced by TPA and TNF-alpha 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-kappa B p65 and p50 DNA binding. For example, PDTC inhibited, at least partially, activation of NF-kappa B binding by TPA and TNF-alpha . However, PDTC did not block the inhibitory effects of TPA or TNF-alpha 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-alpha on SP-B and, to a lesser extent, SP-A mRNAs, but it did not decrease TNF-alpha -mediated NF-kappa 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-kappa B by TPA and TNF-alpha and suggests that the signal transduction pathways mediating the effects of TPA and TNF-alpha inhibition of SP-B are divergent such that PTX blocks TNF-alpha but not TPA. This study does not support a role for NF-kappa B activation in the regulation of SP-A and SP-B mRNAs.

The EMSAs reported were performed with a consensus binding sequence for NF-kappa B present, for example, in immunoglobulin-kappa light chain genes and the human immunodeficiency virus LTR promoter (15). SP-specific NF-kappa 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-kappa B consensus sequence; however, there is no evidence that they are active NF-kappa B binding sites. Because minor differences in DNA sequence can influence the binding and activity of transcription factors, NF-kappa 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-kappa B oligonucleotide-protein complexes were partially competed by nonlabeled NF-kappa B oligonucleotides but were not competed or further shifted by the anti-NF-kappa B p65, p50, and p52 antibodies tested (Fig. 1, brackets). Similar bands have been reported and may represent less common NF-kappa B complexes, NF-kappa B-like proteins, or nonspecific DNA-protein associations (24, 25). However, these unidentified bands were not consistently altered by TPA, TNF-alpha , 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-kappa B binding activity in H441 cells was noted. Dex reduced the basal nuclear NF-kappa 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-kappa B activity (Fig. 4) (18). In addition, SP-B mRNA levels were greater in Dex-pretreated, TNF-alpha -treated cells than in non-Dex-pretreated, non-TNF-alpha -treated cells, even though NF-kappa 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-kappa B activity induced by Dex with or without TNF-alpha , supporting the concept that NF-kappa B does not regulate SP mRNA levels.

The response of SP-A mRNA to TPA, TNF-alpha , 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-alpha 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-alpha , 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-alpha 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-alpha -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-kappa B binding activity by TNF-alpha and TPA in a pulmonary epithelial cell line. The expression of SP-A and SP-B mRNAs was, however, independent of changes in NF-kappa B binding activity. Further studies will be needed to determine the consequences of TNF-alpha -induced NF-kappa B binding activity in pulmonary epithelial cells. Improved understanding of the intracellular responses of pulmonary epithelial cells to inflammatory mediators such as TNF-alpha will contribute to the development of novel clinical interventions for pulmonary disease.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 274(2):L289-L295
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