Department of Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas 75708-3154
Submitted 24 March 2003 ; accepted in final form 29 July 2003
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ABSTRACT |
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inflammation; transcription; lung injury; Clara cells; type II cells
The importance of SP-B is highlighted by the incidence of fatal respiratory failure in infants with congenital alveolar proteinosis who lack SP-B as a result of a frame-shift mutation in codon 121 (121ins2) and other mutations within SP-B gene (39). Targeted disruption of SP-B causes abnormalities of surfactant metabolism and respiratory failure in newborn mice, further supporting the important role of SP-B in lung function (16). Partial deficiency of SP-B in heterozygous SP-B knockout mice is associated with reduced lung compliance and increased air trapping, indicating that less than optimal levels of SP-B can lead to pulmonary dysfunction (15). Reduced SP-B levels are also encountered in a variety of respiratory disorders other than newborn respiratory distress syndrome (RDS) and congenital alveolar proteinosis, which include acute respiratory distress syndrome (ARDS) (23), respiratory syncytial virus (RSV) infection in infants (32), familial interstitial lung disease (3), and in animal models of Pneumocystis carinii pneumonia (8) and bleomycin-induced lung injury (45).
Nitric oxide (NO) is an important regulatory molecule that plays a central role in a variety of physiological and pathological processes (47). NO is produced from L-arginine by the actions of three isoforms of nitric oxide synthases, endothelial and neuronal nitric oxide synthases, which are constitutively expressed, and inducible nitric oxide synthase, which is induced by proinflammatory stimuli. The respiratory epithelium expresses constitutive and inducible forms of NO synthases (48, 49) and is thus capable of producing elevated levels of NO for extended periods of time during inflammatory conditions affecting the lung. Additionally, activated alveolar macrophages can produce high levels of NO, which can easily diffuse, owing to its small size and lipophilicity, to affect the neighboring epithelium. Once induced, inducible NO synthase remains active for time periods ranging from hours to days to produce NO at levels 1,000-fold greater than the constitutive NO synthases (40). Inhaled NO is used therapeutically to treat pulmonary hypertension and improve oxygenation in a variety of pulmonary disorders such as neonatal RDS, ARDS, pulmonary fibrosis, chronic obstructive lung disease, and primary pulmonary hypertension (14, 21, 33). Thus the respiratory epithelium can be exposed to elevated levels of endogenous or exogenous NO, which can adversely affect its function.
Although NO is important for diverse physiological processes in the lung, elevated NO levels produced during inflammatory conditions could have deleterious effects on lung function. Elevated NO production that occurs in a number of inflammatory diseases of the lung such as ARDS (50), murine model of RSV infection (31), asthma (26), and others has been attributed to contribute to lung injury. SP-B levels are significantly reduced in ARDS (23), RSV infections (32), and acute pneumonia (8), indicating that elevated NO production in inflammatory diseases affecting the lung is associated with reduced SP-B levels. Because SP-B serves critical roles in surfactant function, NO-mediated dysregulation of SP-B gene expression could lead to impairment of surfactant function contributing to lung injury.
In the present study, we tested the hypothesis that elevated NO levels decrease SP-B gene expression in lung epithelial cells. We found that NO donors decreased SP-B mRNA levels in a concentration- and time-dependent manner in H441 and MLE-12 cells, cell lines with characteristics of Clara and type II cells, respectively. Our studies also show that NO decreased SP-B gene expression by reducing SP-B promoter activity and that NO inhibition occurred independently of the cyclic guanosine 3',5'-monophosphate (cGMP) signaling pathway.
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EXPERIMENTAL PROCEDURES |
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NO donors. By definition, all NO donors release the active mediator NO when applied to biological systems. The following compounds were used as NO donors in experiments outlined in this study: S-nitroso-N-acetylpenicillamine (SNAP; Sigma), sodium nitroprusside (SNP, Sigma), diethylenetriamine/nitric oxide adduct (DETA/NO, Sigma), and spermine NONOate (SP/NO, Calbiochem). With the exception of SNP, all the other NO donors undergo decomposition in aqueous medium at physiological pH to release NO (20). The mechanism of release of NO from SNP remains incompletely understood. In biological systems, both enzymatic and nonenzymatic release of NO from SNP has been implicated to occur. Because SNAP, an NO donor belonging to the S-nitrosothiol group of compounds, can participate in transnitrosation reactions, i.e., transfer of the bound NO to thiol groups of other molecules such as enzymes and proteins, cells were exposed to SNAP in serum-free medium. Cells were exposed to DETA/NO and SP/NO in serum-containing medium.
RNA isolation and Northern blot analysis. Experimental procedures for isolation of RNA and Northern blotting analysis are as described previously (35). Total RNA from cells was isolated by the acid-guanidinium thiocyanate-phenol method with TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Equivalent amounts of total RNA were separated by electrophoresis on agarose gels (1%) containing 20 mM MOPS and 1.1% formaldehyde and transferred to Hybond N+ membranes by capillary action with saline sodium citrate (SSC, 20x) as the transfer solution. The membranes were hybridized to full-length human SP-B (ATCC 65985) and glyceraldehyde-3-phosphate (GAPDH, ATCC 78463) cDNAs and then washed. The final wash was routinely set at 1x SSC containing 0.1% SDS at 65°C. RNA bands were quantified with a PhosphorImager system, and SP-B RNA levels were normalized to 18S rRNA levels to correct for variations in quantification, loading, and transfer of RNA.
Immunohistochemical analysis of SP-B expression. H441 cells grown on plastic tissue-culture slides were fixed in Excell PLUS (American Master Tech Scientific) for 2-3 h, and endogenous peroxidase activity was blocked by incubation with Peroxide Block solution (BioGenex Laboratories, San Ramon, CA). Afterward, cells were serially incubated with polyclonal human SP-B antibodies (1:200 dilution; Chemicon International, Temecula, CA), biotinylated secondary antibody, and horseradish peroxidase-conjugated streptavidin (Innovex Biosciences, Richmond, CA). Cells were then exposed to aminoethylcarbazole and counterstained with Contrast Blue solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
PCR amplification of human SP-B 5'-flanking DNA. DNA fragment containing -911/+41 bp of human SP-B 5'-flanking sequence (43) was amplified by polymerase chain reaction (PCR) with H441 cell genomic DNA as the template and SP-B primers, 5'-CGAGCTCCGAGTATAGAGGCCTGTGAGC-3'(-913/-892) (sense) and 5'-CCCAAGCTTCCACTGCAGCAGGTGTGACTCAGCCAGGGCACCTCT-3' (+9/+41) (antisense) that contained introduced SacI and HindIII sites (underlined). The antisense primer contained a single mutation (AC) at nucleotide +16 to mutate the SP-B translation initiation codon ATG. The SP-B ATG codon was mutated to eliminate potential interference with the translation of the luciferase reporter gene. PCR amplification was performed with Ex Taq polymerase (Takara, Shiga, Japan) in a reaction containing 100 ng of H441 cell genomic DNA and 0.3 µM SP-B primers. The reaction consisted of an initial denaturation step at 95°C for 5 min followed by a 30-cycle amplification that consisted of denaturation at 94°C for 30 s followed by annealing at 55°C for 30 s and extension at 72°C for 30 s. Amplification was followed by a final extension step at 72°C for 10 min. Amplified DNA corresponding to the expected size of the fragment was gel purified, digested with SacI and HindIII, and inserted upstream of the luciferase reporter gene in the plasmid pGL3luc (basic) (Promega, Madison, WI). The sequence of the amplified DNA was determined to establish its identity and ensure that it was free of nucleotide changes.
Plasmid DNA isolation. Plasmid DNAs were amplified in Escherichia coli Top10 strain (Invitrogen, Carlsbad, CA) and purified by anion exchange chromatography with a QIAfilter plasmid purification kit (Qiagen, Valencia, CA). The quality of plasmid DNAs was verified by agarose gel electrophoresis, and at least two independent plasmid preparations were used in transfection assays.
Transient transfection and reporter gene assays. Plasmid DNAs were transiently transfected into cells by liposome-mediated DNA transfer with Lipofectamine 2000 (Invitrogen). Approximately 250,000 H441 cells or 150,000 MLE-12 cells per well were plated on 12-well plastic tissue-culture dishes, and 24 h later they were transfected with the liposome-DNA complexes. For each well, we formed the liposome-DNA complex by incubating 0.7 µg of SP-B promoter plasmid plus 0.07 µg of pcDNA3.1 (Invitrogen), -galactosidase expression plasmid, and 2 µl of Lipofectamine 2000 in a total volume of 200 µl of OptiMEM medium. After incubation for 15-20 min at room temperature, the liposome-DNA complex was diluted to 1 ml with OptiMEM medium (Invitrogen), and the mixture was overlaid onto cells. Cells were incubated for 5 h, after which the medium was replaced with serum containing RPMI 1640 or HITES medium, and incubation continued overnight. After overnight incubation, cells were exposed to NO donors and other agents for the indicated periods of time.
Transfected cells were lysed with 200 µl of reaction lysis buffer (Promega) by freeze-thaw, and the lysate was cleared by centrifugation at 14,000 rpm for 2 min. The supernatant was assayed immediately for reporter gene activities or stored at -70°C for future use. Luciferase activity was measured with a luciferase assay kit (Promega) in a 96-well plate luminometer (Berthold). -Galactosidase activity was measured by chemiluminescent assay (Tropix, Bedford, MA) with GalactoLight Plus as the substrate. Luciferase activities of cell lysates were normalized to cotransfected
-galactosidase activity to correct for variations in transfection efficiencies. In some experiments, luciferase activities were normalized to total protein content of cell extracts. Normalization of luciferase activity to cotransfected
-galactosidase activity or protein content produced similar results (data not shown).
Statistical analysis. Data are shown as means ± SD. In experiments where SP-B mRNA level/promoter activity in control cells was arbitrarily set at 100%, statistical significance was analyzed by one sample t-test. For other samples, unpaired t-test was used to analyze statistical significance. One-tailed P values that are <0.05 were considered significant.
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RESULTS |
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A variety of NO donors decrease SP-B mRNA levels in H441 cells. Although all NO donors release NO when applied to biological systems, the pathways leading to the formation and release of NO differ among individual compounds. We determined the effects of donors other than SNAP on SP-B mRNA levels to confirm the inhibitory effects of NO. H441 cells were exposed to SNP (0.1-0.5 mM), SP/NO (0.1-1.0 mM), and DETA/NO (0.1-1.0 mM) for 24 h, and their effects on SP-B mRNA levels were analyzed by Northern blotting. Data (Fig. 2) show that all the NO donors tested inhibited SP-B mRNA levels in a dose-dependent manner similar to SNAP. At the highest concentration used, SNP (0.5 mM), SP/NO (1 mM), and DETA/NO (1 mM) reduced SP-B mRNA levels to 50% of the levels in control cells. Together, these data indicate that elevated levels of NO inhibit SP-B gene expression. NO donors did not have any significant cytotoxic effects on cell viability as assessed by light microscopic examination. The total RNA yield from cells treated with NO donors was similar to untreated cells, further indicating a lack of cytotoxic effects of NO donors on cells. As RNA is highly susceptible to degradation, cytotoxic effects resulting in diminished cell viability would result in lower RNA yields. The apparent lack of cytotoxic effects of the NO donors is also indicated by their distinct effects on H441 cell gene expression. Whereas NO donors decreased SP-B mRNA levels, they increased IL-8 and tissue factor mRNA levels and had no effect on GAPDH mRNA levels. Due to its long half-life (20 h at pH 7.4 and 37°C) and first-order kinetics of decomposition (20), DETA/NO was preferred as a NO donor in many experiments.
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NO donors antagonize glucocorticoid induction of SP-B mRNA levels. Glucocorticoids are clinically used to enhance fetal lung maturation in women at risk of premature labor. The beneficial effects of glucocorticoids are due to their positive effects on surfactant synthesis and in particular on SP-B synthesis (37). Glucocorticoids increase SP-B gene expression by increasing gene transcription and mRNA stability (11). We investigated whether NO donors have similar inhibitory effects on glucocorticoid induction of SP-B mRNA levels. H441 cells were incubated with dexamethasone (10-7 M) in the absence or presence of SNAP, DETA/NO, and SP/NO at concentrations of 0.1-1.0 mM for 24 h, and SP-B mRNA levels were analyzed by Northern blotting. Data (Fig. 3) show that dexamethasone increased SP-B levels by greater than sixfold, and NO donors inhibited dexamethasone induction in a dose-dependent manner. At 1 mM concentration, the NO donors reduced SP-B levels to 30% of the levels in dexamethasone-treated cells. These data indicate that elevated levels of NO have dominant inhibitory effects on SP-B mRNA levels.
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NO donor inhibition of SP-B mRNA is associated with inhibition of SP-B protein. To determine whether the inhibitory effects of NO donors on SP-B mRNA levels are associated with similar effects on SP-B protein levels, we investigated the effects of DETA/NO on SP-B levels in H441 cells by immunochemical staining. H441 cells were exposed to DETA/NO (1 mM) for 24 h, and SP-B levels were visualized by immunostaining with polyclonal human SP-B antibodies. Results (Fig. 4) show that in control cells SP-B was readily detected as a reddish precipitate, and in cells treated with DETA/NO only trace amounts of the precipitate were detected. These data indicate that DETA/NO inhibition of SP-B mRNA is associated with reduced SP-B protein levels.
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NO donors inhibit SP-B mRNA levels in MLE-12 cells. Our data thus far show that NO donors inhibit SP-B mRNA levels in H441 cells, a cell line of Clara cell lineage. To determine whether NO donors have similar effects on SP-B gene expression in alveolar type II cells, we investigated the effects of NO donors on SP-B mRNA levels in MLE-12 cells, a mouse cell line with characteristics of type II cells. MLE-12 cells were exposed to increasing concentrations of SNAP and SNP for 24 h, and SP-B mRNA levels were analyzed by Northern blotting. Results (Fig. 5) show that SNAP and SNP decreased SP-B mRNA levels in a dose-dependent manner; at 1 mM concentration, SNAP and SNP decreased SP-B mRNA to 30 and 45% of the levels in control cells. In other experiments we found that DETA/NO (1 mM) also decreased SP-B mRNA levels in MLE-12 cells (data not shown). These data indicate that elevated levels of NO inhibit SP-B mRNA levels in type II cells similar to Clara cells.
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NO donor inhibition of SP-B mRNA levels is due to NO. The NO donors used in this study are pharmacological tools that serve as sources of NO with the understanding that their effects are primarily caused by the released NO. However, this may not always be the case, in that the metabolites and the decomposed products of the NO donors may exert similar effects as NO. To determine whether the inhibitory effects of the NO donors are indeed due to NO released and not due to decomposition products, we investigated the effects of N-acetylpenicillamine (NAP), the byproduct of decomposition of SNAP and decomposed DETA/NO on SP-B mRNA levels. H441 cells were exposed to dexamethasone (10-7 M) with or without SNAP (1 mM) or NAP (1 mM) and DETA/NO (1 mM) or decomposed DETA/NO for 24 h, and SP-B mRNA levels were analyzed by Northern blotting. To achieve decomposition, we incubated medium containing 1 mM DETA/NO at 37°C for 7 days. After 7 days of incubation, the amount of DETA/NO was negligible as determined by its absorbance at 252 nm. The effects of the NO donors and their byproducts were analyzed in dexamethasone-treated cells, as dexamethasone strongly induces SP-B mRNA facilitating rapid detection of changes in SP-B mRNA levels. Results (Fig. 6) show that whereas SNAP and DETA/NO reduced SP-B mRNA levels to <50 and 10% of the levels in dexamethasone-treated cells, NAP and decomposed DETA/NO did not significantly alter SP-B mRNA levels. DETA produced similar results as decomposed DETA/NO (data not shown). Similar results were obtained from cells maintained in control medium (data not shown). These data indicate that the inhibitory effects of the NO donors must be due to NO per se and not to the byproducts of decomposition.
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In other experiments we found that the NO scavenger carboxyphenyl-tetramethylimidazoline-oxyl-oxide (C-PTIO) and hemoglobin partially reversed the inhibitory effects of SNAP and DETA/NO on SP-B mRNA levels in control and dexamethasone-treated cells (data not shown), further indicating the role of NO as the causative agent. The inability of C-PTIO to completely block NO inhibition of SP-B mRNA is likely due to its inactivation by reducing agents such as ascorbate and thiols present in cells and culture medium (20). Partial effects of hemoglobin could be due to its inability to scavenge NO released inside the cell owing to its large size, which prevents its entry into the cell.
NO inhibition of SP-B mRNA levels is not mediated via TNF- and IL-8. NO is known to increase the production of TNF-
in monocytic U-937 cells (52) and IL-8 in human endothelial (51) and melanoma (4) cells and neutrophils (18). We investigated whether the inhibitory effects of DETA/NO on SP-B mRNA levels are primary or secondary to the release of TNF-
and IL-8. TNF-
is a potent inhibitor of SP-B gene expression (9, 44, 54), but the effects of IL-8 on SP-B gene expression are not known. To determine whether DETA/NO alters the levels of TNF-
and IL-8, we treated H441 cells with control medium or medium containing DETA/NO (1 mM) for 24 h and determined the levels of TNF-
and IL-8 in cell medium by ELISA. Results show that whereas NO donors increased IL-8 levels by three- to fourfold over levels in control cells (IL-8 levels in control medium were in the range of 2-9 ng/ml), they did not significantly alter TNF-
levels (TNF-
levels in control medium were in the range of 5-20 pg/ml). We sought to determine the involvement of TNF-
and IL-8 in the NO inhibition of SP-B mRNA levels. Although ELISA measurements indicate that DETA/NO did not significantly alter TNF-
levels, we thought that it was important to determine whether the inhibitory effects of NO are mediated via TNF-
, as TNF-
is a potent inhibitor of SP-B gene expression. H441 cells were exposed to dexamethasone (10-7 M) for 24 h with or without DETA/NO in the absence or presence of a TNF-
-neutralizing antibody (1 µg/ml; ND50 to neutralize the bioactivity of 0.25 ng/ml of recombinant human TNF-
is 0.003-0.01 µg/ml;R&D Systems, Minneapolis, MN), and SP-B mRNA levels were analyzed by Northern blotting. Results (Fig. 7) show that DETA/NO decreased SP-B mRNA levels to
30% of the levels in cells treated with dexamethasone alone and that TNF-
-neutralizing antibodies failed to reverse the inhibitory effects of DETA/NO. Similar results were obtained for cells in control medium (data not shown). The effect of the neutralizing antibody on TNF-
inhibition of SP-B promoter activity was analyzed to validate the neutralizing effects of the antibody. Data showed that exposure of H441 cells to TNF-
(5 ng/ml) reduced SP-B promoter activity to 15% of the levels in untreated cells and that, in the presence of neutralizing TNF-
IgG (0.5 µg/ml), the promoter activity was restored to 80% of the level in untreated cells. These data indicate that the inhibitory effects of DETA/NO are not mediated through TNF-
.
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We next determined the effects of IL-8 on SP-B mRNA levels in control and dexamethasone-treated cells. H441 cells in control and dexamethasone (10-7 M)-containing medium were exposed to IL-8 (0.1-10 ng/ml) for 24 h, and SP-B mRNA levels were analyzed. Data (Fig. 8) show that IL-8 did not alter SP-B mRNA levels in control and dexamethasone-treated cells, indicating that the inhibitory effects of DETA/NO are not mediated by IL-8. Together, these data indicate that the inhibitory effects are due to NO per se and are not the result of secondary effects of the NO donor to alter the levels of TNF- and IL-8.
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Continued transcription is necessary for NO inhibition of SP-B mRNA levels. NO inhibition of SP-B mRNA levels could be due to decreases in the transcription rate of the gene or mRNA stability or both mechanisms. We investigated the involvement of transcriptional mechanisms in the NO inhibition of SP-B mRNA levels by studying the effects of the transcriptional inhibitor 5,6-dichloro-1--D-ribofuranozyl-benzimidazole (DRB) on DETA/NO inhibition of SP-B mRNA levels. DRB is a purine analog that inhibits the kinase activity of the positive transcription elongation factor b to prevent productive elongation and thereby maintain RNA polymerase II in a repressed state. H441 cells were first incubated with DRB (20 and 40 µg/ml) for 2 h, and incubation continued with DETA/NO for 24 h. SP-B mRNA levels were analyzed by Northern blotting. Results (Fig. 9) showed that treatment of cells with DRB blocked DETA/NO inhibition of SP-B mRNA, indicating that continued transcription and transcriptional mechanisms are necessary for NO inhibition of SP-B gene expression. DRB alone did not have any significant effects on SP-B mRNA levels.
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NO donors inhibit SP-B promoter activity. The transcriptional inhibitor DRB blocked DETA/NO inhibition of SP-B mRNA, indicating that continued transcription and transcriptional mechanisms are necessary for NO inhibition of SP-B gene expression. We assessed the role of transcription by studying the effects of the NO donors on SP-B promoter activity in H441 cells. In vitro cell culture studies have shown that relatively short promoter regions (-236/+41 bp) are sufficient and necessary for high-level expression of reporter genes in a cell type-specific manner in H441 (13, 36) and MLE-12 (1) cells, suggesting that the minimal promoters contain all the necessary cis-DNA elements to achieve cell-specific expression. The effects of NO donors on SP-B promoter expression were investigated by transient expression analysis of promoter plasmids containing a human SP-B -913/+41-bp fragment linked to the luciferase reporter. H441 and MLE-12 cells were transiently transfected with SP-B-luciferase promoter plasmid and then treated with increasing concentrations of DETA/NO for 24 h. Results (Fig. 10, A and B) show that DETA/NO decreased SP-B promoter activity in a dose-dependent manner in both cell types. At the highest concentration tested (1.0 mM for H441 cells and 0.5 mM for MLE-12 cells), DETA/NO reduced SP-B promoter activity to 40 and 25% of the levels in controls in H441 and MLE-12 cells, respectively. These data show that NO donors inhibited SP-B promoter activity similarly to SP-B mRNA levels, indicating that transcriptional mechanisms mediate NO inhibition of SP-B gene expression. Together, these data indicate that NO donors inhibit SP-B gene expression in Clara and type II cells primarily at the transcriptional level.
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NO donors inhibit SP-B gene expression independently of cGMP signaling pathway. Many of the biological actions of NO are mediated via activation of soluble guanylate cyclase (sGC), which catalyzes the conversion of GTP into cGMP (46). NO binds to the heme moiety of the sGC by forming a nitrosyl complex with iron, causing a conformational change that activates the enzyme 400-fold. The activation of sGC and the subsequent increase in cGMP levels alter the activities of cGMP-regulated ion channels, cGMP-regulated phosphodiesterases, and protein kinases to transmit the NO signal to downstream elements to alter gene expression. Protein kinase G regulates the expression of c-fos (25), junB (27), heme oxygenase-1 (30), and others by transcriptional and posttranscriptional mechanisms. cAMP is a positive regulator of surfactant synthesis and increases SP-B gene expression by increasing gene transcription (37). NO stimulation of cGMP levels could modulate the activities of phosphodiesterases to alter intracellular levels of cAMP. In particular, activation of type II phosphodiesterases by cGMP can increase the hydrolysis of cAMP, resulting in decreased intracellular levels of cAMP that can lead to diminished expression of SP-B gene.
We analyzed the effects of inhibitors that block different components of the cGMP signaling pathway, such as sGC, protein kinase G, and cGMP-activated phosphodiesterases, on the NO inhibition of SP-B mRNA levels to determine the involvement of the cGMP-signaling pathway. H441 cells in serum-free medium were first treated with LY-83583 (1 µM) (6), an sGC inhibitor, or KT-5823 (5 µM) (24), a protein kinase G inhibitor, for 1 h, and then incubation continued with dexamethasone (10-7 M) or dexamethasone (10-7 M) plus DETA/NO (1 mM) for 24 h. SP-B mRNA levels in cells were analyzed by Northern blotting. Results (Fig. 11A) showed that dexamethasone increased SP-B mRNA by severalfold, and LY-83583 and KT-5823 did not significantly alter dexamethasone induction. DETA/NO markedly antagonized dexamethasone induction of SP-B mRNA, and LY-83583 and KT-5823 failed to reverse the inhibitory effects of DETA/NO on SP-B mRNA levels. Studies on cells in control medium produced similar results (data not shown). In other experiments we found that 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor (7), failed to reverse the inhibitory effects of DETA/NO in control and dexamethasone-treated cells, indicating the lack of involvement of phosphodiesterases in the NO inhibition of SP-B gene expression (data not shown). Together, these data rule out the involvement of protein kinase G and cGMP-activated phosphodiesterases in the NO inhibition of SP-B gene expression.
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We confirmed the lack of involvement of the cGMP pathway by investigating the effects of 8-bromo-cGMP, a cell-permeable analog of cGMP, on SP-B mRNA levels. H441 cells in control and dexamethasone (10-7 M)-containing medium were exposed to increasing concentrations of 8-bromo-cGMP for 24 h, and SP-B mRNA levels were analyzed. Data (Fig. 11B) show that the cGMP analog did not alter SP-B mRNA levels in control and dexamethasone-treated cells. Similar results were obtained with another cell-permeable analog of cGMP, dibutyryl cGMP (data not shown). These data, along with the data of the lack of effects of cGMP pathway inhibitors on NO inhibition of SP-B mRNA levels, indicate that NO inhibits SP-B mRNA expression independently of cGMP levels.
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DISCUSSION |
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Published reports have shown that during cell culture of A549 cells in Ham's F-12K medium, 2 mM SNAP produced a peak level of 3 µM NO after 2 h, which was sustained for 6 h before declining to basal levels after 10 h, and 0.5 mM DETA/NO produced a peak level of 1.5 µM after 1-2 h, which was sustained for at least 10 h (38). Because the rate of decomposition of the NO donors is determined primarily by the pH and the temperature of the medium and not affected by biological reactants, it can be predicted that the amounts and duration of NO release from SNAP, SP/NO, and DETA/NO in our studies are not vastly different. On the basis of the rate of release of NO from SNAP and DETA/NO, it can be predicted that the NO donors used in our study produce comparable amounts of NO for a minimum of 5 h. It is possible that exposure of cells to relatively short periods of time (5 h) is sufficient to cause inhibition of SP-B gene expression, presumably via activation of signal transduction mechanisms that are yet to be understood. This may explain the inhibition of SP-B gene expression by NO donors with different decomposition rates.
Information on the NO regulation of surfactant protein gene expression is lacking except for a recent study that reported inhibition of SP-A gene expression by the NO donor SNAP in H441 cells (5). Our results on the inhibitory effects of NO on SP-B gene expression are consistent with recent data demonstrating the negative effects of NO and its reactive metabolite peroxynitrite on surfactant homeostasis. Exposure of freshly isolated rat alveolar type II cells to various NO donors in the presence of superoxide dismutase decreased the rate of synthesis of surfactant by 60%, indicating the inhibitory effects of NO on surfactant synthesis (28). Surfactant isolated from newborn lambs exposed to high levels of NO (200 ppm) displayed abnormal surface properties, and SP-A isolated from such animals displayed a decreased ability to aggregate lipids in vitro (29).
Both NO and superoxide anion produced by macrophages, neutrophils, monocytes, and other cells are important mediators of the inflammatory response. NO and superoxide anion rapidly combine to form the potent oxidant peroxynitrite. The relative contributions of NO, superoxide anion, and peroxynitrite in mediating an inflammatory response are not known. Our studies have shown that NO alone is capable of inhibiting SP-B gene expression. Whether superoxide anion and peroxynitrite can also influence SP-B gene expression is not known. Long-term inhalation of high-dose NO (40 ppm) increased total protein concentration in the bronchoalveolar lavage fluid and activated the intra-alveolar coagulation system, indicators of lung injury, in mice (34). These data suggest that NO alone has the potential to cause lung injury, a notion that is supported by our findings of NO inhibition of SP-B gene expression.
Our studies showed that the transcriptional inhibitor DRB blocked DETA/NO inhibition of SP-B mRNA levels, indicating that continued transcription and transcriptional mechanisms are necessary for NO inhibition. To confirm the inhibitory effects of NO on SP-B gene transcription, we determined the effects of NO donors on SP-B promoter activity. Our data show that NO donors decreased SP-B promoter activity in a dose-dependent manner, indicating that NO inhibition of SP-B gene expression is mediated at the transcriptional level. The SP-B promoter fragment that we used in promoter assays contained -913/+41 bp of SP-B 5'-flanking DNA and included all the necessary cis-DNA elements for lung cell-specific expression of the promoter. This suggests that NO inhibition of SP-B promoter activity accurately reflects effects on SP-B gene transcription. NO inhibited SP-B mRNA levels and SP-B promoter activity to a similar extent, indicating that transcriptional mechanisms control NO inhibition of SP-B gene expression. Lack of involvement of mRNA stability as a contributing factor toward inhibition of SP-B mRNA levels is further suggested by the time-course effects of NO donors on SP-B mRNA levels. The inhibitory effects of NO donors were apparent only after 12 h and maximal after 24 h of incubation. If one considers that the half-life of SP-B mRNA is in the range of 7.5-9 h (11), an effect to destabilize the RNA would result in lowered SP-B mRNA levels at an earlier time of incubation than 12 h.
NO influences gene expression via transcriptional, posttranscriptional, and translational mechanisms. Genes modulated by NO include those encoding protective and proinflammatory mediators, chemokines and cytokines, matrix metabolizing enzymes, adhesion molecules, growth factors, and others (40). NO influences the transcriptional control of gene expression by altering the DNA binding activities of transcription factors. There is accumulating evidence to indicate that NO preferentially modulates transcription factors that are sensitive to the cellular redox state (10). Examples of such factors include NF-B, activator protein-1, c-Myb, p53, Sp1, early growth response, vitamin D3 receptor/retinoid X receptor, and hypoxia-inducible transcription factors (10). NO can alter the DNA binding activities by directly modifying sensitive cysteine residues located at the DNA-binding domains of transcription factors. Alternatively, NO, in its capacity as a signaling molecule, can modulate the activities of transcription factors via activation of MAPK and other signaling cascades (41). Molecular mechanisms underlying NO inhibition of SP-B promoter activity remain to be investigated. SP-B promoter activity is maintained by combinatorial actions of thyroid transcription factor 1, hepatocyte nuclear factor 3, Sp1/Sp3, and activating transcription factor/cAMP response element-binding protein, which bind to cognate sites on the promoter (2). It can be speculated that NO modulates the binding activities of one or more of the transcription factors to inhibit SP-B promoter activity.
As many of the actions of NO are mediated via stimulation of intracellular levels of cGMP, we evaluated the role of cGMP signaling pathway in the NO inhibition of SP-B gene expression. Our data show that pharmacological inhibitors that block different components of the cGMP signaling pathway such as sGC, protein kinase G, and phosphodiesterases failed to reverse NO inhibition of SP-B gene expression. These data indicate that NO inhibited SP-B gene expression independently of cGMP levels. Consistent with these data, a cell-permeable analog of cGMP such as 8-bromo-cGMP did not alter SP-B levels.
In conclusion, our studies show that elevated levels of NO inhibited SP-B gene expression in H441 and MLE-12 lung epithelial cells. NO inhibited SP-B gene expression, independently of cGMP levels, primarily at the transcriptional level. As SP-B serves critical roles in surfactant functions, our findings of NO inhibition of SP-B gene expression are novel and highly significant. Because NO production in the lung is elevated in a number of inflammatory diseases of the lung, NO-mediated dysregulation of SP-B gene expression can contribute to the development of lung injury in such diseases. Our findings may also have important implications on the use of NO as a therapeutic agent. Inhaled NO is used to treat a variety of pulmonary disorders in infants and adults, including neonatal RDS, ARDS, pulmonary hypertension, and others. Because elevated NO levels inhibit SP-B gene expression, prolonged use of high levels of NO as a therapeutic agent can be harmful.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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