Nitric oxide-induced reduction of lung cell and whole lung thioredoxin expression is regulated by NF-kappa B

Jianliang Zhang1, Leonard W. Velsor2, Jawaharlal M. Patel1,3, Edward M. Postlethwait2, and Edward R. Block1,3

1 Department of Medicine, University of Florida, and 3 Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608; and 2 Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined whether nitric oxide (NO)-induced inhibition of thioredoxin (Thx) expression is regulated by a mechanism mediated by a transcription factor, i.e., nuclear factor-kappa B (NF-kappa B), in cultured porcine pulmonary artery endothelial cells (PAEC) and in mouse lungs. Western blot analysis revealed that Ikappa B-alpha content was reduced by 20 and 60% in PAEC exposed to 8.5 ppm NO for 2 and 24 h, respectively. NO exposure also caused significant reductions of cytosol fraction p65 and p52 content in PAEC. The nuclear fraction p65 and p52 contents were significantly reduced only in PAEC exposed to NO for 24 h. Exposure to NO resulted in a 50% reduction of p52 mRNA but not of the Ikappa B-alpha subunit. DNA binding activity of the oligonucleotide encoding the NF-kappa B sequence in the Thx gene was significantly reduced in PAEC exposed to NO for 24 h. Exposure of mice to 10 ppm NO for 24 h resulted in a significant reduction of lung Thx and Ikappa B-alpha mRNA and protein expression and in the oligonucleotide encoding Thx and NF-kappa B/DNA binding. These results 1) demonstrate that the effects of NO exposure on Thx expression in PAEC are comparable to those observed in intact lung and 2) suggest that reduced expression of the NF-kappa B subunit, leading to reduced NF-kappa B/DNA binding, is associated with the loss of Thx expression in PAEC and in intact mouse lungs.

nuclear factor-kappa B; transcription factor; gene regulation; mouse lung; lung endothelium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO), a free radical gas produced by vascular endothelial and other mammalian cells, plays a critical role in lung physiology and pathophysiology (5, 14). Despite the important role of NO in the regulation of vascular tone in the pulmonary circulation, in vivo and in vitro studies indicate that NO is cytotoxic to a number of cells, including vascular endothelial cells (24, 27, 29, 31). For example, NO has been reported to exert a toxic role during such diverse pathological events as chemically induced pulmonary edema, ischemia-reperfusion injury, allograft transplant rejection, and inflammatory lung disease in which endothelial cell dysfunction is common (6, 18, 39). We recently reported that 1) exposure of porcine pulmonary artery endothelial cells (PAEC) to NO significantly reduced the catalytic activity of the constitutive (endothelial cell) isoform of NO synthase (ecNOS) (31), 2) disulfide reducing chemicals, e.g., dithiothreitol or 2-mercaptoethanol or enzymatic reduction of disulfide by thioredoxin/thioredoxin reductase (Thx/Thx-R), but not by glutaredoxin, restored NO-induced loss of ecNOS catalytic activity (30, 31), and 3) exposure to NO significantly reduced the expression of the redox regulatory proteins Thx and Thx-R in PAEC (42). Although the effect of NO on lung Thx expression is not known, the level of lung Thx content is critical for maintaining redox regulation of proteins under oxidative stress.

Thx plays a critical role in the regulation of catalytic activity and the function of redox-sensitive proteins, enzymes, receptors, and transcription factors, including nuclear factor-kappa B (NF-kappa B) (1, 17, 35). Rel family transcription factors NF-kappa B1 and NF-kappa B2 are involved in the expression of a wide variety of genes, including ecNOS and Thx genes (20, 25, 26). NF-kappa B exists primarily as a heterodimer composed of 65- and 50-kDa (p65 and p50, respectively, NF-kappa B1) or 52-kDa (p52, NF-kappa B2) subunits that is sequestered in the cytoplasm with the inhibitory subunit Ikappa B-alpha , Ikappa B-beta , Ikappa B-gamma , or Bcl-3 (11, 38, 40). Homodimers of p50 and p65 have also been reported (4). Activation of the NF-kappa B complex involves dissociation of the heterodimer complex from the Ikappa B subunit in the cytosol and translocation to the nucleus, which results in DNA binding and activation of gene expression (40). NO has been shown to inhibit transcription factor/DNA binding in mammalian cells (23, 28, 33). In the present study we report 1) the in vitro effects of NO exposure on NF-kappa B subunit dissociation, translocation, and expression and on NF-kappa B/DNA binding activity of the Thx promoter in cultured PAEC and 2) the in vivo effects of NO exposure on Thx and Ikappa B-alpha expression and NF-kappa B/DNA binding to the Thx promoter in mouse lungs.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue culture. Endothelial cells were isolated from the main pulmonary artery of 6- to 7-mo-old pigs and propagated in monolayer cultures, as reported by Patel and Block (30). Fifth- to seventh-passage PAEC in postconfluent monolayers maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) and antibiotics were used for all experiments.

Exposure of PAEC and mice to NO. Confluent PAEC monolayers were exposed to a continuous flow of 8.5 ppm NO gas premixed with 5% CO2 and air for 2 and 24 h, as previously described (32, 42). Exposure to NO for 24 h under these conditions did not alter the pH (7.4) of the culture medium. NO2 was not detectable in the premixed gas or in the exposure chamber.

For the mouse exposure study, male C57BL/6J mice (26-28 g; Jackson Laboratories) were continuously exposed to 10 ppm NO for 24 h. Animal procedures met standards of the University of Texas Medical Branch (Galveston) Animal Care and Use Committee. Mice had free access to food and water throughout the exposure period. With use of mass flow controllers, a high concentration of NO (1,000 ppm NO in nitrogen; Liquid Carbonic, Pasadena, TX) was bled into a stream of compressed air to achieve the desired concentration and an exposure chamber turnover rate of 15 volume changes per hour. Chamber concentrations of NO and NO2 were continuously monitored using a environmental chemiluminescence NOx analyzer (model 42, Thermo Environmental, Franklin, MA) and were maintained at 10 ppm NO. During exposure, NO2 concentrations averaged 0.17 ± 0.04 ppm. Immediately on removal from the chamber, animals were anesthetized with pentobarbital sodium (60 mg/kg ip), with the depth of anesthesia verified via foot pinch. A midline thoracotomy was performed to expose the lungs, and after transection of the abdominal aorta, the lungs were removed en bloc and immediately frozen in liquid nitrogen for later analysis. NO-exposed PAEC or lungs were used for the isolation of cytosol and nuclear fractions, Western blot analysis, RNA extraction, and protein determination. Lungs were also used for isolation of total membrane fractions and for assays of NO synthase (NOS) activity.

Isolation of cytosol, total membrane, and nuclear fractions. Lung cytosol and total membrane fractions were isolated to compare the in vivo effect of NO on NOS activity with the previously reported in vitro effect of NO on ecNOS activity in PAEC (32). PAEC cytosol and nuclear fractions and whole lung nuclear fractions were isolated to examine expression of NF-kappa B subunits and NF-kappa B/DNA binding activity. For isolation of cytosol fractions from PAEC and cytosol and total membrane fractions from intact lung, control and NO-exposed cell monolayers and lungs were washed twice and homogenized using a motor-driven Potter-Elvehjem Teflon-glass homogenizer with 10 strokes at 4°C in buffer A (50 mM Tris · HCl, pH 7.4, containing EDTA and EGTA at 0.1 mM each, 1 mM phenylmethylsulfonyl fluoride, and leupeptin at 1.0 mg/l). The homogenates were centrifuged at 100,000 g for 60 min at 5°C in an ultracentrifuge (model L3-50, Beckman, Irvine, CA), the total membrane pellets were resuspended in buffer A, and the supernatants (cytosol) were collected separately as described previously (43).

The nuclear fraction was isolated as previously described (3). Cell monolayers and lungs were washed twice with ice-cold PBS and suspended in lysing buffer [10 mM HEPES buffer, pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM dithiothreitol, 0.2% (vol/vol) Igepal CA-630 (Sigma Chemical, St. Louis, MO), 74 µM antipain, 0.1 µM aprotinin, 130 µM bestatin, 50 µM chymostatin, 1 µM E-64, 1 µM leupeptin, 2 mM Pefabloc SC, 1 µM pepstatin, and 300 nM phosphoramidon (Boehringer Mannheim, Indianapolis, IN)] for 5 min and homogenized as described above. After centrifugation of homogenates at 500 g for 5 min, the supernatant was removed. The pelleted nuclear fraction was gently rinsed twice with 1 ml of lysing buffer and then resuspended in 300 µl of the same buffer. The nuclear fraction was layered on top of 300 µl of Igepal CA-630-free lysing buffer containing 30% saccharose and centrifuged at 2,900 g for 10 min. The supernatant was gently removed, and pelleted nuclei were resuspended in 200 µl of nuclear resuspension buffer (250 mM Tris · HCl, pH 7.8, 1 mM EDTA, 60 mM KCl, 1 mM dithiothreitol, 74 µM antipain, 0.1 µM aprotinin, 130 µM bestatin, 50 µM chymostatin, 1 µM E-64, 1 µM leupeptin, 2 mM Pefabloc SC, 1 µM pepstatin, and 300 nM phosphoramidon). The nuclear suspension was subjected to freezing and thawing three times and then cleared by ultracentrifugation at 135,000 g for 15 min. Cytosol, total membrane, and nuclear fraction proteins were quantified by the Bradford assay (8).

Measurement of lung NOS activity. Exposure to NO is known to reduce ecNOS activity in PAEC (30). We examined whether exposure to NO has similar effects on calcium-dependent NOS activity in the total membrane fraction of mouse lungs. Total membrane and cytosol fraction NOS activity or cytosol NOS activity alone was measured by monitoring the formation of L-[3H]citrulline from L-[3H]arginine (8). For total NOS activity, membranes (100-120 µg of protein) were incubated (total volume 0.4 ml) in buffer A containing 1 mM NADPH, 100 nM calmodulin, 10 µM tetrahydrobiopterin, 2.5 mM CaCl2, and 5 µM combined L-arginine and purified L-[3H]arginine (0.6 µCi, specific activity 69 Ci/mmol; NEN, Boston, MA) for 30 min at 37°C. Calcium-independent NOS, i.e., inducible NOS (iNOS), activity was measured under identical conditions in a similar incubation mixture containing 1 mM EDTA and no CaCl2. Blanks were incubated under identical conditions but in the absence of total membrane protein. Purification of commercially available L-[3H]arginine was carried out as described previously (31). Total NOS activity was determined after subtraction of the activity of the blank. Calcium-dependent NOS activity was determined after subtraction of iNOS activity from total NOS activity. The specific activity of NOS is expressed as picomoles of L-citrulline per minute per milligram of protein.

Western blot analysis of Ikappa B-alpha , p65, and p52 proteins. The effects of NO exposure on expression of PAEC cytosol and nuclear fraction Ikappa B-alpha , p65, and p52, as well as on lung expression of Thx, were examined. PAEC cytosol and nuclear fractions as well as mouse lung homogenate proteins (40 µg each) from control and NO-exposed PAEC and lungs were separated by 12% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Bio-Rad, Richmond, CA), as described previously (42). The blots were incubated in blocking solution [0.2% nonfat milk in TBS (20 mM Tris · HCl, pH 7.5, and 500 mM NaCl)] and then hybridized with anti-Ikappa B-alpha , anti-p65, or anti-p52 (Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal antibodies (1:2,000 dilution) or human anti-Thx monoclonal antibody (American Diagnostica, Greenwich, CT) (1:1,000 dilution) in antibody buffer (0.2% nonfat milk and 0.1% Tween 20 in TBS) at room temperature for 1 h. After they were washed with buffer (0.1% Tween 20 in TBS), the membranes were incubated in 1:3,000-diluted anti-rabbit IgG, alkaline phosphatase-linked whole antibody (Immunstar chemiluminescent protein detection system, Bio-Rad) for 1 h. The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL) with Kodak XAR-5 film for 5-15 min. The blots were scanned using the Fluor-S MultImager system (Bio-Rad) to quantify these protein contents.

RNA isolation and RNase protection assay. Total RNA was extracted directly from control and NO-exposed PAEC and mouse lung by use of the RNAgents Total RNA Isolation System (Promega, Madison, WI). RNA contents of the extracts were determined by measuring levels of 18S rRNA as an internal standard, since the gene expression of 18S RNA was not altered by NO exposure (unpublished data). mRNA was extracted from the total RNA preparations by use of the PolyATtract mRNA isolation system (Promega) according to the manufacturer's instructions. Digoxigenin-UTP (DIG)-labeled RNA probes for Ikappa B-alpha (600 bp), Thx (330 bp), and p52 (120 bp) were transcribed in vitro from Pvu II-digested pBS-Ikappa B (kindly provided by Dr. Stephen Haskill, University of North Carolina, Chapel Hill, NC), linearized pThx-A, as described by Zhang et al. (42), and linearized pTRI-p52 (obtained from reagent donor Dr. Gary Nabel through the AIDS Research and Reference Reagent Program, Division of Acquired Immune Deficiency Syndrome, National Institute of Allergy and Infectious Diseases), respectively. The RNase protection assay was carried out to determine the mRNA levels with use of an RNase protection kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) (19, 44). mRNA extracts (5 µl, 200 ng/µl) from control and NO-exposed PAEC or whole mouse lungs and 1.5 µl of DIG-labeled Ikappa B, Thx, or p52 RNA probe (40 ng/µl each) were mixed, denatured, and incubated at 45°C overnight. After RNase treatment, the partial RNA:RNA hybrids were subjected to electrophoresis in a polyacrylamide (4%)-7 M urea gel at ~30 W for 2.5 h in Tris-borate buffer. After being blotted onto a nylon membrane (Zeta-Probe GT, Bio-Rad), the digestion products were immunodetected with anti-digoxigenin-AP and the chemiluminescence substrate CSPD (Genius Labeling and Detection kits, Boehringer Mannheim), as described by the manufacturer, and then exposed to Kodak BioMax film for 5-15 min. Quantitation of mRNA for the Ikappa B, Thx, and p52 was performed using the Fluor-S MultiImager system (Bio-Rad). The levels of specific mRNA content were standardized to the beta -actin mRNA (Ambion, Austin, TX) levels, which are not affected by NO exposure.

Electrophoretic mobility shift assay. The DNA band-shift analysis of PAEC and mouse lung nuclear fractions was performed as reported by Baldassarre et al. (3). Briefly, the Thx NF-kappa B double-stranded oligonucleotide (5'-AGA CCT GGG ACT CTC CCT CCC AGC-3') was synthesized by the Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL). The nuclear fraction (10 µg protein) was incubated with 1 µg of poly(dI-dC) and 0.1 µg of poly-L-lysine in 20 mM HEPES buffer, pH 7.6, containing 1 mM EDTA, 10 mM (NH4)2SO4, 0.2% (wt/vol) Tween 20, and 30 mM KCl for 15 min at room temperature. At the end of incubation, 5× loading buffer was added, and the samples were electrophoresed in a native 7% polyacrylamide gel in buffer (pH 8.5) containing 90 mM Tris, 90 mM boric acid, and 2 mM EDTA. The DIG nucleotide/NF-kappa B and free probes were electroblotted onto a nylon membrane and immunodetected with anti-digoxigenin-AP and the chemiluminescence substrate CSPD (DIG gel shift kit, Boehringer Mannheim), as described by the manufacturer, and then exposed to Kodak XAR-5 film for 5-15 min. Specificity of Thx oligonucleotide/NF-kappa B binding was determined by using a 10-fold excess of unlabeled oligonucleotide as well as by supershift assay with p65 antibody. Densitometric analysis for the NF-kappa B/DNA band was performed using the Fluor-S MultiImager system.

Statistical analysis. Statistical significance for the effects of NO exposure on the Ikappa B-alpha , p65, and p52 protein contents, Ikappa B-alpha , p52, and Thx mRNA levels, Thx oligonucleotide-NF-kappa B/DNA binding activity, and NOS activity was determined using Student's paired t-test (41). Values are means ± SE for n experiments. P < 0.05 was taken as significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure of PAEC monolayers to 8.5 ppm NO for 2 or 24 h had no significant effect on endothelial cell morphology assessed by phase-contrast microscopy. Similarly, protein contents of cytosol and membrane fractions from PAEC exposed to NO for 2 and 24 h were comparable to controls (data not shown). In addition, exposure of mice to 10.1 ± 0.2 (SD) ppm NO for 24 h produced no alterations in lung weight or overt signs of gross pathology (e.g., pulmonary edema).

Effect of NO exposure on mouse lung NOS activity and Thx mRNA and protein expression. Because NO exposure results in reduction of ecNOS activity and Thx expression in cultured PAEC (32, 42), we examined the in vivo response to NO exposure by measuring total membrane fraction calcium-dependent NOS activity and Thx mRNA and protein expression in the lungs of mice exposed to NO gas. Total membrane fraction calcium-dependent NOS activities in control and NO-exposed lungs were 3.38 ± 1.0 and 1.21 ± 0.6 nmol L-citrulline · min-1 · mg protein-1 (P < 0.001, n = 4), respectively. Calcium-dependent NOS activity in cytosolic fractions from control and NO-exposed lungs was not detectable under our experimental conditions. Exposure of mice to 10 ppm NO gas for 24 h resulted in decreased expression of lung Thx mRNA (Fig. 1, A and B) and protein (Fig. 1C) contents (P < 0.05 for both).


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Fig. 1.   Northern and Western blot analysis of thioredoxin (Thx) in lungs of mice exposed to NO gas. For Northern analysis of Thx mRNA (A and B), mRNA was isolated from total RNA extracts from lungs of control (C) and NO-exposed (10 ppm for 24 h) mice. Glyoxal-denatured total mRNA (1 µg/lane) was fractionated and blotted onto nylon membranes. Membranes were hybridized with digoxigenin-UTP (DIG)-labeled human Thx cDNA, and mRNA was detected using immunochemiluminescence method. Thx mRNA content was quantified after standardization with beta -actin mRNA. For Western blot analysis of Thx protein (C and D), lung homogenate proteins (4 µg) from control and NO-exposed (10 ppm for 24 h) mice were separated by SDS-PAGE, electrophoretically transferred to polyvinylidine difluoride membranes, and immunoanalyzed. Thx expression was quantified by densitometric analysis of 4 separate blots. * P < 0.05.

Effect of NO exposure on NF-kappa B subunits Ikappa B-alpha , p65, and p52 in PAEC. To examine whether NO exposure alters NF-kappa B subunit dissociation and translocation, cytosol and nuclear fraction Ikappa B-alpha , p65, and p52 protein levels were measured. As shown in Fig. 2, exposures to NO for 2 and 24 h reduced the Ikappa B-alpha protein levels in the cytosol by 20% (P < 0.05) and 60% (P < 0.01), respectively, compared with controls. As shown in Fig. 3, exposure of PAEC to NO for 2 and 24 h significantly decreased the protein levels of p65 in the cytosol fractions (P < 0.01 for both). In addition, p65 protein content in the nuclear fraction of PAEC exposed to NO for 24 h, but not for 2 h, was significantly (P < 0.01) reduced compared with controls. The p52 protein contents in the cytosol and nuclear fractions of cells exposed to NO for 24 h, but not for 2 h, were significantly reduced (P < 0.01 for both; Fig. 4). These results suggest that exposure to NO reduces the translocation and/or expression of the NF-kappa B subunits in PAEC.


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Fig. 2.   Western blot analysis of nuclear factor-kappa B (NF-kappa B) inhibitory protein Ikappa B-alpha in pulmonary artery endothelial cells (PAEC). A: cytosolic proteins (40 µg) from control (C) and NO-exposed (8.5 ppm for 2 and 24 h) PAEC were fractionated, blotted, and immunodetected. B: Ikappa B-alpha protein quantified by densitometric analysis of 3 separate blots. * P < 0.05, ** P < 0.01 vs. control.



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Fig. 3.   Western blot analysis of NF-kappa B subunit p65 in cytosolic (Cyto) and nuclear fractions (Nuc) of PAEC. A: cytosolic and nuclear fraction proteins (40 µg each) from control (C) and NO-exposed (8.5 ppm for 2 and 24 h) PAEC were fractionated, blotted, and immunodetected. B: p65 protein quantified by densitometric analysis of 3 separate blots. * P < 0.01 vs. respective controls.



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Fig. 4.   Western blot analysis of NF-kappa B subunit p52 in cytosolic and nuclear fractions of PAEC. A: cytosolic and nuclear proteins (40 µg each) from control (C) and NO-exposed (8.5 ppm for 2 and 24 h) PAEC were fractionated, blotted, and immunodetected. B: p52 protein quantified by densitometric analysis of 3 separate samples. * P < 0.01 vs. respective controls.

Effect of NO on Ikappa B-alpha and p52 mRNA expression. To determine whether the NO-induced reductions of NF-kappa B subunit proteins in the cytosol and/or the nuclear fraction are associated with reduced mRNA expression for these proteins, we monitored Ikappa B-alpha and p52 mRNA contents in PAEC exposed to NO for 24 h, in control PAEC, and in the lungs of mice. As shown in Fig. 5, Ikappa B-alpha mRNA content in NO-exposed cells was slightly, but not significantly, reduced, whereas the lung Ikappa B-alpha mRNA contents of mice exposed to NO were significantly (P < 0.01) reduced compared with controls. The p52 mRNA content in NO-exposed cells was significantly (P < 0.01) reduced compared with controls, whereas the p52 mRNA content of mice exposed to NO was comparable to controls (Fig. 6).


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Fig. 5.   Effect of NO exposure on Ikappa B-alpha mRNA in PAEC and mouse lungs. Total mRNAs were extracted from control (C) and NO-exposed (8.5 ppm for 24 h) PAEC and from lungs of control and NO-exposed (10 ppm for 24 h) mice. Total mRNA (1 µg) and 1.5 µl of DIG-labeled Ikappa B-alpha RNA probe (40 ng/µl) were mixed, denatured, and incubated at 45°C overnight. After RNase treatment, partial RNA:RNA hybrids were subjected to electrophoresis. A: representative blot for Ikappa B-alpha in mouse lungs and PAEC, respectively. B: respective densitometric analysis of 3 separate blots in A. * P < 0.01 vs. control.



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Fig. 6.   Effect of NO exposure on p52 mRNA in PAEC and mouse lungs. Total mRNAs were extracted from control (C) and NO-exposed (8.5 ppm for 24 h) PAEC and from lungs of control and NO-exposed (10 ppm for 24 h) mice. Total mRNA (1 µg) and 1.5 µl DIG-labeled p52 RNA probe (40 ng/µl) were mixed, denatured, and incubated at 45°C overnight. After RNase treatment, partial RNA:RNA hybrids were subjected to electrophoresis. A: representative blot for p52 in PAEC and mouse lungs, respectively. B: respective densitometric analysis of 3 separate blots in A. * P < 0.01 vs. control.

Effect of NO exposure on Thx-oligonucleotide/NF-kappa B binding activity. Alterations in the levels of the NF-kappa B subunits p65/p52 and/or the extent of their nuclear translocation have been shown to influence NF-kappa B/DNA binding. Consequently, we examined the effects of transcription factor NF-kappa B/DNA binding on Thx gene expression. The oligonucleotide-NF-kappa B/DNA binding activity was standardized by gel mobility shift assay with use of a 10-fold excess of unlabeled oligonucleotide, as well as by supershift assay with use of p65 antibody (Fig. 7A). As shown in Fig. 7, B and C, exposure to NO gas for 24 h significantly reduced Thx-oligonucleotide-NF-kappa B/DNA binding activity in PAEC and in the lungs of mice compared with controls (P < 0.01 for both). However, Thx-oligonucleotide-NFkappa B/DNA binding activity was comparable to controls in PAEC exposed to NO gas for 2 h (data not shown). This NO-induced reduction of NF-kappa B/DNA binding activity may account for the NO-induced reductions of Thx mRNA and protein expression in PAEC and mouse lungs.


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Fig. 7.   Effect of NO exposure on Thx-oligonucleotide (Oligo)/NF-kappa B binding activity in PAEC and mouse lungs. Electrophoretic mobility shift assay was performed using nuclear fraction protein (10 µg) from control (C) and NO-exposed (8.5 ppm for 24 h) PAEC and lungs of control and NO-exposed (10 ppm for 24 h) mice, and Thx-oligonucleotide/NF-kappa B binding was assayed. A: specificity determined by excess unlabeled NF-kappa B oligonucleotide and supershift assay with use of p65 antibody. B: representative assay blot for PAEC and lung. C: densitometric analysis of Thx-oligonucleotide/NF-kappa B complex from 3 separate experiments of corresponding blots in B. In A, lanes 1 and 3 represent nuclear extract (NF-kappa B) + labeled oligonucleotide, lane 2 represents NF-kappa B + labeled oligonucleotide + excess unlabeled oligonucleotide, and lane 4 represents NF-kappa B + labeled oligonucleotide + p65 antibody. * P < 0.01 vs. respective control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We recently reported that exposure to NO resulted in the loss of catalytic activity of NOS as well as reduced Thx and Thx-R mRNA and protein expression in PAEC (32, 42). The results of the present study demonstrate for the first time that exposure to NO also results in loss of NOS catalytic activity and Thx mRNA and protein expression in the lungs of mice exposed to NO gas and suggest that NO-induced reduction of Thx gene expression is mediated through NF-kappa B modulation in PAEC and mouse lung. NO-induced reduction in Thx oligonucleotide-NF-kappa B binding activity was associated with decreased expression and/or impaired translocation of NF-kappa B subunits from cytosol to nuclear fractions in PAEC and intact lungs. This indicates that NO-induced diminished expression of Thx in PAEC and intact lung is regulated through an NF-kappa B-mediated mechanism. Alternatively, this mechanism may also be Thx dependent with respect to the level of Thx expression and/or its redox status.

NF-kappa B-activated gene expression requires dissociation of the inhibitory protein Ikappa B-alpha in the cytosol followed by translocation of the NF-kappa B dimer (p65/p52) to the nucleus. A number of extracellular stimuli, including oxidative stress, viral and bacterial infection, drugs, and environmental chemical agents, physical stress, and biological mediators such as inflammatory cytokines have been implicated in alterations of NF-kappa B/DNA binding activity and gene expression (11, 33, 38, 40). Although a number of studies have reported that exposure to NO or NO donors modulates NF-kappa B/DNA binding activity in various mammalian cells (12, 23, 28, 33, 37), the results of the present study establish a specific association between NO-induced NF-kappa B modulation and reduced binding to the DNA sequences for Thx gene transcription in PAEC and mouse lung. These observations are consistent with and support our previous report in which cultured PAEC were used (42), and the present results in mouse lungs demonstrate that NO-induced diminished expression of Thx mRNA and protein occurs in vivo and is mediated by a transcriptional mechanism.

The precise mechanisms responsible for the NO-induced reduced expression of Ikappa B-alpha , p65, and p52 subunit proteins and of Ikappa B-alpha and p52 mRNA contents as well as for the NO-induced reduction in the nuclear translocation of the p65/p52 dimer in the present study are not known. However, it is possible that NO-induced inhibition of total protein synthesis or synthesis of specific proteins such as Ikappa B-alpha , p65, and p52 or the 100-kDa precursor protein (p100) of p52 may account for the reduced expression of these subunits in PAEC and intact lung, inasmuch as NO and NO-generating agents are known to inhibit total as well as specific protein synthesis in a variety of mammalian cells (13, 21, 22). Precise concentrations of NO generated from donors (100-400 µM) were not characterized in these studies.

NO is also known to interact with protein sulfhydryls to form S-nitrosothiols, resulting in inactivation of a number of redox-sensitive proteins, enzymes, receptors, and transcription factors, including NF-kappa B (1, 2, 17, 29, 32). Thus direct oxidation of NF-kappa B subunit proteins by NO may result in reduced dissociation, translocation, and/or DNA binding activity. Several other mechanisms critical for dissociation of the inhibitory protein Ikappa B-alpha from NF-kappa B may also be partially responsible for reduced translocation of the p65/p52 dimer to the nucleus. For example, a number of factors, including phosphorylation, proteolytic degradation, and activation of Ikappa B-specific kinase, are known to play a role in NF-kappa B activation (10, 15, 23), and NO may affect any one of these.

The implications of our observations are significant for NO-induced modulation of the redox-sensitive transcription factor NF-kappa B and its component proteins, which play a critical role in the regulation of a variety of genes. Similarly, tissue level of Thx may be critical in maintaining redox status of a variety of enzymes in the signal transduction cascade. The tissue level of Thx is especially important, since Thx plays a critical role in the cellular defense against oxidative stress, including protection against NO-induced loss of ecNOS activity in PAEC (32). This is particularly important in pathological conditions where NO plays a role, including septic shock, organ transplant rejection, inflammation, radiation damage, chemical- or drug-induced injury, and atherosclerosis. In addition, exogenous administration of inhaled NO gas for extended periods is commonly used to restore lung hemodynamics and gas exchange in newborn or adult patients with primary pulmonary hypertension, respectively (34, 36), and adult respiratory distress syndrome (7, 16). Therapeutic use of NO gas has the potential to alter the function of a number of redox-sensitive proteins in lung cells. Thus maintaining Thx levels is critical for regulation of lung cell redox regulatory processes and pulmonary function.


    ACKNOWLEDGEMENTS

We thank Bert Herrera for tissue culture assistance, Di-hua He for technical assistance, Addie Heimer for manuscript preparation, and Janet Wootten for excellent editorial help.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-58679 (to J. M. Patel) and HL-54679 (to E. M. Postlethwait) and by the Department of Veterans Affairs Medical Research Service.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. M. Patel, Research Service (151), Dept. of Veterans Affairs Medical Center, 1601 SW Archer Rd., Gainesville, FL 32608-1197 (E-mail: Pateljm{at}medicine.ufl.edu).

Received 18 December 1998; accepted in final form 28 May 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abate, C., L. Patel, F. J. Rauscher, and T. Curran. Redox regulation of fos and jun DNA-binding activity in vitro. Science 249: 1157-1161, 1990[Medline].

2.   Arnelle, D. R., and J. S. Stamler. NO+, NO, and NO- donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Arch. Biochem. Biophys. 318: 279-285, 1995[Medline].

3.   Baldassarre, F., M. Mallardo, E. Mezza, G. Scala, and I. Quinto. Regulation of NF-kappa B through the nuclear processing of p105 (NF-kappa B1) in Epstein-Barr virus-immortalized B cell lines. J. Biol. Chem. 270: 31244-31248, 1995[Abstract/Free Full Text].

4.   Baldwin, A. S. The NFkappa B and Ikappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 14: 649-681, 1996[Medline].

5.   Barnes, P. J., and M. G. Belvisi. Nitric oxide and lung disease. Thorax 48: 1034-1043, 1993[Medline].

6.   Berisha, H. I., H. Pakbaz, A. Absood, and S. I. Said. Nitric oxide as a mediator of oxidant lung injury due to paraquat. Proc. Natl. Acad. Sci. USA 91: 7445-7449, 1994[Abstract].

7.   Bigatello, L. M., W. E. Hurford, R. M. Kacmarek, J. D. Roberts, and W. M. Zapol. Prolonged inhalation of low concentrations of nitric oxide in patients with severe adult respiratory distress syndrome. Anesthesiology 80: 761-770, 1994[Medline].

8.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein ultilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

9.   Bredt, D. S., and S. H. Snyder. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA 87: 682-690, 1990[Abstract].

10.   Brown, K., S. Park, T. Kanno, G. Franzoso, and U. Siebenlist. Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, Ikappa B-alpha . Proc. Natl. Acad. Sci. USA 90: 2532-2536, 1995[Abstract].

11.   Chiao, P. J., S. Miyamoto, and I. M. Verma. Autoregulation of Ikappa B-alpha activity. Proc. Natl. Acad. Sci. USA 91: 28-32, 1994[Abstract].

12.   Colasanti, M., T. Persichini, M. Menegazzi, S. Mariotto, E. Giordano, C. M. Caldarera, V. Sogos, G. M. Lauro, and H. Suzuki. Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide. J. Biol. Chem. 270: 26731-26733, 1995[Abstract/Free Full Text].

13.   Curran, R. D., F. K. Ferrari, P. H. Kispert, J. Stadler, D. J. Stuehr, R. J. Simmons, and T. R. Billiar. Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis. FASEB J. 5: 2085-2092, 1991[Abstract/Free Full Text].

14.   Davies, M. G., G. J. Fulton, and P. O. Hagen. Clinical biology of nitric oxide. Br. J. Surg. 82: 1598-1610, 1995[Medline].

15.   Didonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, and M. Karin. A cytokine-responsive Ikappa B kinase that activates the transcription factor NF-kappa B. Nature 388: 458-554, 1997.

16.   Gerlach, H., D. Pappert, K. Lewandowski, R. Rossaint, and K. J. Falke. Long-term inhalation with elevated low doses of nitric oxide for selective improvement of oxygenation in patients with adult respiratory distress syndrome. Intensive Care Med. 19: 443-449, 1993[Medline].

17.   Hayashi, T., Y. Ueno, and T. Okamoto. Oxidoreductive regulation of nuclear factor-kappa B. Involvement of a cellular reducing catalyst thioredoxin. J. Biol. Chem. 268: 11380-11388, 1993[Abstract/Free Full Text].

18.   Isaacson, T. C., V. Hampl, E. K. Weir, D. P. Nelson, and S. L. Archer. Increased endothelium-derived NO in hypertensive pulmonary circulation of chronically hypoxic rats. J. Appl. Physiol. 76: 933-940, 1994[Abstract/Free Full Text].

19.   Lee, J. J., and N. A. Costlow. A molecular titration assay to measure transcript prevalence levels. Methods Enzymol. 152: 633-648, 1987[Medline].

20.   Kaghad, M., F. Dessarps, H. Jacquemin-Sablon, D. Caput, D. Fradelizi, and E. E. Wollman. Genomic cloning of human thioredoxin-encoding gene: mapping of the transcription start point and analysis of the promoter. Gene 140: 273-278, 1994[Medline].

21.   Kim, Y. M., S. Kyonghee, S. J. Hong, A. Green, J. J. Chen, E. Tzeng, C. Hierholzer, and T. R. Billiar. Inhibition of protein synthesis with cytostatic activity: nitric oxide induces phosphorylation of initiation factor eIF-2alpha . Mol. Med. 4: 179-190, 1998[Medline].

22.   Kolpakov, V., D. Gordon, and T. Kulik. Nitric oxide-generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ. Res. 76: 305-309, 1995[Abstract/Free Full Text].

23.   Lin, Y. C., K. Brown, and U. Siebenlist. Activation of NF-kappa B requires proteolysis of the inhibitor Ikappa B-alpha : signal-induced phosphorylation of Ikappa B-alpha alone does not release active NF-kappa B. Proc. Natl. Acad. Sci. USA 92: 552-556, 1995[Abstract].

24.   Ma, X. L., B. L. Lopez, T. A. Christopher, D. S. Birenbaum, and J. Vinten-Johansen. Exogenous NO inhibits basal NO release from vascular endothelium in vitro and in vivo. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H2045-H2051, 1996[Abstract/Free Full Text].

25.   Marsden, P. A., H. H. Heng, S. W. Scherer, R. J. Stewart, A. V. Hall, X. M. Shi, L. C. Tsui, and K. T. Schappert. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J. Biol. Chem. 268: 17478-17488, 1993[Abstract/Free Full Text].

26.   Nadaud, S., A. Bonnardeaux, M. Lathrop, and F. Soubrier. Gene structure, polymorphism and mapping of the human endothelial nitric oxide synthase gene. Biochem. Biophys. Res. Commun. 198: 1027-1033, 1994[Medline].

27.   Oka, M., M. Ohnishi, H. Takahashi, S. Soma, K. Hasunuma, K. Sato, and S. Kira. Altered vasoreactivity in lungs isolated from rats exposed to nitric oxide gas. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L419-L424, 1996[Abstract/Free Full Text].

28.   Park, S. K., H. I. Lin, and S. Murphy. Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-kappa B binding to DNA. Biochem. J. 322: 609-613, 1997[Medline].

29.   Patel, J. M., A. J. Abeles, and E. R. Block. Nitric oxide exposure and sulfhydryl modulation alter L-arginine transport in cultured pulmonary artery endothelial cells. Free Radic. Biol. Med. 20: 629-637, 1996[Medline].

30.   Patel, J. M., and E. R. Block. Acrolein-induced injury to cultured pulmonary artery endothelial cells. Toxicol. Appl. Pharmacol. 122: 46-53, 1993[Medline].

31.   Patel, J. M., and E. R. Block. Sulfhydryl-disulfide modulation and the role of disulfide oxidoreductases in regulation of the catalytic activity of nitric oxide synthase in pulmonary artery endothelial cells. Am. J. Respir. Cell Mol. Biol. 13: 352-359, 1995[Abstract].

32.   Patel, J. M., J. L. Zhang, and E. R. Block. Nitric oxide-induced inhibition of lung endothelial cell nitric oxide synthase via interaction with allosteric thiols: role of thioredoxin in regulation of catalytic activity. Am. J. Respir. Cell Mol. Biol. 15: 410-419, 1996[Abstract].

33.   Peng, H. B., P. Libby, and J. K. Liao. Induction and stabilization of Ikappa B-alpha by nitric oxide mediates inhibition of NF-kappa B. J. Biol. Chem. 270: 14214-14219, 1995[Abstract/Free Full Text].

34.   Pepke-Zaba, J., T. W. Higenbottam, A. T. Dinh-Xuan, D. Stone, and J. Wallwork. Inhaled nitric oxide as a cause of selective pulmonary vasodilation in pulmonary hypertension. Lancet 338: 1173-1174, 1991[Medline].

35.   Powis, G., M. Briehl, and J. Oblong. Redox signalling and the control of cell growth and death. Pharmacol. Ther. 68: 149-173, 1995[Medline].

36.   Roberts, J. D., J. R. Fineman, F. C. Morin III, P. W. Shaul, S. Rimar, M. D. Schreiber, R. A. Polin, M. S. Zwass, M. M. Zayek, I. Gross, M. A. Heymann, and W. M. Zapol. Inhaled nitric oxide and persistent pulmonary hyptertension of the newborn. N. Engl. J. Med. 336: 605-610, 1997[Abstract/Free Full Text].

37.   Sheffler, L. A., D. A. Wink, G. Melillo, and G. W. Cox. Exogenous nitric oxide regulates IFN-gamma plus lipopolysaccharide-induced nitric oxide synthase expression in mouse macrophages. J. Immunol. 155: 886-894, 1995[Abstract].

38.   Siebenlist, U., G. Franzoso, and K. Brown. Structure, regulation and function of NF-kappa B. Annu. Rev. Cell Biol. 10: 405-455, 1994.

39.   Tanaka, H., Y. Chiba, M. Sasaki, S. Matsukawa, and R. Muraoka. Relationship between flushing pressure and nitric oxide production in preserved lungs. Transplantation 65: 460-464, 1998[Medline].

40.   Thanos, D., and T. Maniatis. NF-kappa B: a lesson in family values. Cell 80: 529-532, 1995[Medline].

41.   Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw Hill, 1971, p. 210-219.

42.   Zhang, J. L., Y. D. Li, J. M. Patel, and E. R. Block. Thioredoxin overexpression prevents NO-induced reduction of NO synthase activity in lung endothelial cells. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L288-L293, 1998[Abstract/Free Full Text].

43.   Zhang, J. L., J. M. Patel, Y. D. Li, and E. R. Block. Proinflammatory cytokines downregulate gene expression and activity of constitutive nitric oxide synthase in porcine pulmonary artery endothelial cells. Res. Commun. Mol. Pathol. Pharmacol. 96: 71-78, 1997[Medline].

44.   Zinn, K., D. DiMaio, and T. Maniatis. Identification of two distinct regulatory regions adjacent to the human beta -interferon gene. Cell 34: 865-879, 1993.


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