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The Journal of Immunology, 2004, 173: 4147-4153.
Copyright © 2004 by The American Association of Immunologists

Reduction of Nitric Oxide Synthase 2 Expression by Distamycin A Improves Survival from Endotoxemia1

Rebecca M. Baron, Irvith M. Carvajal, Xiaoli Liu, Rachel O. Okabe, Laura E. Fredenburgh, Alvaro A. Macias, Yen-Hsu Chen, Kuniaki Ejima, Matthew D. Layne and Mark A. Perrella2

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 References
 
NO synthase 2 (NOS2) plays an important role in endotoxemia through overproduction of NO. Distamycin A (Dist A) belongs to a class of drugs termed minor-groove DNA binders, which can inhibit transcription factor binding to AT-rich regions of DNA. We and others have previously shown that AT-rich regions of DNA surrounding transcription factor binding sites in the NOS2 promoter are critical for NOS2 induction by inflammatory stimuli in vitro. Therefore, we hypothesized that Dist A would attenuate NOS2 up-regulation in vivo during endotoxemia and improve animal survival. C57BL/6 wild-type (WT) mice treated with Dist A and LPS (endotoxin) showed significantly improved survival compared with animals treated with LPS alone. In contrast, LPS-treated C57BL/6 NOS2-deficient (NOS2–/–) mice did not benefit from the protective effect of Dist A on mortality from endotoxemia. Treatment with Dist A resulted in protection from hypotension in LPS-treated WT mice, but not in NOS2–/– mice. Furthermore, LPS-induced NOS2 expression was attenuated in vivo (WT murine tissues) and in vitro (primary peritoneal and RAW 264.7 murine macrophages) with addition of Dist A. Dist A selectively decreased IFN regulatory factor-1 DNA binding in the enhancer region of the NOS2 promoter, and this IFN regulatory factor-1 site is critical for the effect of Dist A in attenuating LPS induction of NOS2. Our data point to a novel approach in modulating NOS2 expression in vivo during endotoxemia and suggest the potential for alternative treatment approaches for critical illness.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 References
 
During endotoxemia, toxins such as LPS (endotoxin) are released by Gram-negative bacteria and activate host immune cells, including macrophages, to produce proinflammatory cytokines. When endotoxic shock develops, exaggerated amounts of these proinflammatory cytokines are released, resulting in refractory hypotension, tissue hypoperfusion, and organ dysfunction. Unfortunately, few direct therapeutic options exist once shock has developed, and the mainstays of therapy remain primarily antibiotics and supportive care (1, 2), which often includes use of vasoconstrictor agents in an attempt to maintain adequate systemic blood pressure. However, these agents also have the potential to decrease tissue perfusion and contribute to end-organ ischemia and damage. Thus, additional strategies are needed to modulate this delicate balance between vasodilation and vasoconstriction during endotoxic shock.

The inducible form of NO synthase (NOS2)3 catalyzes the production of NO, a labile and free-radical gas that acts as a potent vasodilator (3). Induction of NOS2 expression by LPS and other inflammatory stimuli during endotoxemia results in overproduction of NO, which plays an important role in the development of shock. NOS2 is induced in a variety of cell types involved inthe inflammatory cascade, including macrophages and vascular smooth muscle cells, as well as in end-organ tissues critical in mediating the pathophysiologic effects of endotoxemia (e.g., lung and kidney) (4). The effects of NOS2 deficiency (using NOS2–/– mice) on mortality in animals subjected to endotoxemia have yielded varying results, leading to hypotheses that NOS2 has a protective effect (5), a detrimental effect (6, 7), or no significant effect (8, 9) on animal survival. The differences in these outcomes have been attributed to the varied experimental conditions, including genetic background and gender of the animals, subtype and dose of LPS administered, and fluid resuscitation of the animals. However, these variable results also most likely reflect the balance of beneficial and harmful effects to the organism as a result of NOS2 expression (10). Hence, the ability to decrease NOS2 expression during endotoxemia, rather than completely eliminating its expression, might be important for improving outcomes from this disease process.

The antibiotic Distamycin (Dist) A belongs to a class of drugs termed minor-groove binders, which specifically bind AT-rich sequences in the minor groove of DNA (11). These drugs can therefore interfere with sequence-specific transcription factor binding (12, 13). We and others have previously shown that AT-rich regions of DNA surrounding transcription factor binding sites in the NOS2 promoter are critical for NOS2 induction by inflammatory stimuli in vitro. We therefore hypothesized that Dist A, through interfering with transcription factor binding to AT-rich regions of DNA, would decrease LPS-induced NOS2 expression in vivo and improve survival from endotoxemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 References
 
Mouse model of endotoxemia

Male C57BL/6 wild-type (WT) mice (Charles River Laboratories, Wilmington, MA; 6–8 wk of age) and male C57BL/6 mice with targeted deletion of NOS2 (NOS2–/–; The Jackson Laboratory, Bar Harbor, ME; 6–8 wk of age) were injected i.p. with 40 mg/kg LPS (Escherichia coli serotype O26:B6; Sigma-Aldrich, St. Louis, MO) or vehicle (saline). Mice also received Dist A (25 mg/kg) injected i.p. 30 min before LPS administration (m.w. 518; Sigma-Aldrich) or vehicle (25% DMSO; Sigma-Aldrich). The total volume of injected solution was ~0.7 ml per mouse. Survival was assessed at 10, 20, 30, 50, and 70 h following injection. In separate experiments, WT mice treated with the NOS2-specific inhibitor 1400W (14, 15) (Cayman Chemical, Ann Arbor, MI; 20 mg/kg s.c. 30 min before Dist A, then administered again at the same dose 8 h following LPS injection) were subjected to survival studies in a similar fashion.

Blood pressure (mean arterial pressure (MAP)) was also determined in LPS/vehicle or LPS/Dist A-treated WT and NOS2–/– animals following surgical implantation of carotid artery catheters, as described (16). Once the mice recovered from surgery/anesthesia (tribromoethanol, 250 mg/kg i.p.), MAP was measured at baseline and 1, 2, and 4 h following injection. In separate experiments, tissue (lung and/or kidney) was harvested for RNA extraction from WT animals 1, 2, and 4 h following injection. All animal experiments were performed in accordance with National Institutes of Health Guidelines and were approved by the Harvard Medical Area Standing Committee on Animals.

Cell culture and reagents

Murine macrophages (RAW 264.7; American Type Culture Collection, Manassas, VA) were cultured, as described (17). Peritoneal macrophages were elicited from WT mice using thioglycolate medium, as previously described (4). LPS and Dist A are described above, and murine rIFN-{gamma} was purchased from PeproTech (Rocky Hill, NJ).

Plasmid constructs and site-directed mutagenesis

The mouse WT NOS2 luciferase reporter plasmid iNOS (–1485/+31) in the pGL2-basic vector (Promega, Madison, WI) was described previously (referred to in this work as WT plasmid) (18). Site-directed mutagenesis of the WT plasmid was performed as described previously (19) to generate constructs containing mutated (m) consensus binding sites in the NOS2 enhancer region for NF-{kappa}B (termed mNF-{kappa}B, –972 to –962, GGGGATTTTCC to TCTAGATTTAA), Stat-1{alpha} (termed mGAS, –944 to –940, TTTTC to AGCTG, and IFN regulatory factor-1 (IRF-1) (termed mISRE, –920 to –915, TCACTT to CCGCGG.

Transient transfections of RAW 264.7 cells, reporter assays, and nitrite measurements

The WT NOS2, mNF-{kappa}B, mGAS, and mISRE plasmids (0.5 µg) were transiently transfected in RAW 264.7 cells using FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN), as described previously (19). Cells were cultured in 0.5% FBS for 48 h before transfection. Twelve hours following transfection of the reporter construct and a {beta}-galactosidase expression vector (to normalize for luciferase activity), cells were pretreated with Dist A (75 µM) or vehicle (ethanol), then with LPS (50 ng/ml) and IFN-{gamma} (100 U/ml) 30 min later. Twenty-four hours following treatment, cells were harvested and assessed for luciferase activity (Promega luciferase assay system) and {beta}-galactosidase, as described previously (19). RAW 264.7 cells were also plated, treated as described, then assayed for nitrite levels in the medium at 48 h following treatment using Greiss reagent, as previously described (20). Nitrite levels were normalized for cellular total protein, which was measured by the Bradford dye-binding method using Bio-Rad protein assay reagent. In separate experiments, RAW 264.7 cells and primary peritoneal macrophages were similarly plated and treated, then harvested for total RNA at 12 h following treatment (described below), or for nuclear protein (from RAW 264.7 cells) following 2 h of treatment (18).

RNA isolation and Northern blot analysis

RNeasy Mini RNA isolation kit (Qiagen, Valencia, CA) was used to extract total RNA from cultured cells and mouse tissues, according to the manufacturer’s instructions. Northern blot analysis using a radiolabeled NOS2 probe was performed, as previously described (20). A radiolabeled rRNA 18S probe (20) was used to confirm equal loading.

EMSAs

EMSAs were performed, as described previously (17), with double-stranded oligonucleotide probes encoding the IRF-1 consensus binding site in the murine NOS2 enhancer region (–933 to –908) (termed IFN-stimulated response element (ISRE): 5'-CACTGTCAATATTTCACTTTCATAAT-3'). Nuclear extract was obtained from RAW 264.7 cells, as described above, and nuclear protein was quantified by the Bradford dye-binding method (Bio-Rad, Hercules, CA). rIRF-1 protein was generated from human IRF-1 cDNA cloned into pCDNA3 plasmid (Invitrogen Life Technologies, Carlsbad, CA) using the TNT T7 Quick Coupled Transcription/Translation system (Promega). For EMSA using rIRF-1, the radiolabeled ISRE probe was incubated along with 100x cold mutant competitor to decrease nonspecific binding from the TNT product (termed mut-ISRE; underlined bases are altered from the ISRE probe: 5'-CCTAACACTGTCAATATTCCGCGGTCATAATGGAAAATTCC-3').

Statistics

Comparisons between groups were made by factorial ANOVA, followed by Fisher’s least significant difference test. Error bars represent SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
References
 
Dist A improves survival from endotoxemia in WT mice, but not in NOS2–/– mice

WT mice were injected with LPS/Dist A or LPS/vehicle i.p. and assessed for survival following injection (Fig. 1A). Dose-response experiments were performed to determine a dose of LPS that would result in mortality comparable to that reported in literature (5, 6, 7, 8, 21), and an effective dose of Dist A whose vehicle (DMSO) would not have an independent effect on the animals (data not shown). WT mice exposed to LPS/vehicle exhibited 100% mortality (0% survival) within 30 h. WT mice injected with LPS/Dist A exhibited significantly higher survival at 30 h (100% survival), 50 h (55% survival), and 70 h (48% survival) following injection when compared with the LPS/vehicle-treated WT animals (p < 0.05 at each of these time points).



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  		FIGURE 1. Dist A improves survival from endotoxemia in WT mice, but not in NOS2–/– mice. A, WT mice were injected with 40 mg/kg LPS + 25 mg/kg Dist A i.p. ({blacksquare}, WT LPS + Dist A, n = 10) or 40 mg/kg LPS + vehicle i.p. ({square}, WT LPS, n = 10). Mice were evaluated at 10, 20, 30, 50, and 70 h following injection to determine percentage of survival (*, p < 0.05 compared with WT LPS). B, NOS2–/– mice were subjected to the same experimental protocol as outlined for the WT animals (n = 6 in each treatment group; p = NS between groups of NOS2–/– animals at each time point). C, WT mice treated with the NOS2-specific inhibitor 1400W were subjected to the same experimental protocol as outlined for the WT animals (n = 7 in LPS/vehicle group and n = 8 in LPS/Dist A group; p = NS between groups of 1400W-treated WT animals at each time point).

 
NOS2–/– mice (Fig. 1B) and WT mice treated with the NOS2-specific inhibitor 1400W (WT-1400W; Fig. 1C) were similarly injected with LPS/Dist A or LPS/vehicle i.p. and assessed for survival following injection (Fig. 1B). NOS2–/– and WT-1400W mice exposed to LPS/vehicle exhibited 100% mortality (0% survival) within 30 h, a rate similar to LPS/vehicle-treated WT mice. However, in contrast to the WT mice, NOS2–/– and WT-1400W mice receiving LPS/Dist A did not exhibit improved survival when compared with the LPS/vehicle-treated NOS2–/– and WT-1400W animals, respectively (p = NS at all time points). These results demonstrate that NOS2 plays a role in the protective effect of Dist A during endotoxemia.

Dist A protects against the development of severe hypotension during endotoxemia in WT mice, but not in NOS2–/– mice

As others have reported that induction of NOS2 expression is important in mediating LPS-induced vasodilation and hypotension (6, 10, 22, 23), we assessed NOS2 expression in WT tissue (lung) during the 4-h period following LPS administration (Fig. 2A). NOS2 was induced as early as 1 h following LPS stimulation, with increasing levels of expression seen over a 4-h period. We next assessed MAP over this same time course following injection of LPS/Dist A or LPS/vehicle in WT animals (Fig. 2B) and NOS2–/– animals (Fig. 2C). Following surgical implantation of carotid artery catheters, the mice were allowed to recover for 2 h before baseline blood pressure measurements were obtained.



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  		FIGURE 2. Dist A protects against the development of severe hypotension during endotoxemia in WT mice, but not in NOS2–/– mice. A, Total RNA was extracted from lung tissue harvested from WT mice at 1, 2, and 4 h following treatment with LPS. A representative Northern blot is shown (10 µg of RNA per lane) using a radiolabeled NOS2 probe and an 18S probe (to confirm equal loading). This experiment was performed two separate times. B, Carotid artery catheters were implanted in WT mice for MAP measurement. Two hours following recovery from surgery/anesthesia, baseline MAP was obtained. Mice were then injected with either 10 mg/kg LPS + 25 mg/kg Dist A i.p. ({blacksquare}, WT LPS + Dist A, n = 12) or 10 mg/kg LPS + vehicle IP ({square}, WT LPS, n = 9). MAP was measured at 1, 2, and 4 h after injection for surviving animals. Data points represent mean MAP in each group of animals ± SE. (*, p < 0.05 compared with WT LPS). C, MAP measurement was determined in NOS2–/– mice in a manner similar to that described for WT mice (NOS2–/– LPS + Dist A, n = 5, and NOS2–/– LPS, n = 5). Percentage of reduction in MAP (compared with baseline) at 4 h following injection is shown for all groups of WT and NOS2–/– mice ± SE ({blacksquare}, LPS + Dist A; {square}, LPS; *, p < 0.05 for WT LPS + Dist A vs WT LPS; p = NS for NOS2–/– LPS + Dist A vs NOS2–/– LPS).

 
At baseline, all WT animals had similar MAP (93 ± 2.5 mm Hg for LPS/Dist A; 95 ± 2.1 mm Hg for LPS/vehicle; p = NS). Following injection, WT animals receiving LPS/vehicle developed significant hypotension (MAP, 57 ± 8.7 mm Hg, 52 ± 5 mm Hg, and 47 ± 6.7 mm Hg at 1, 2, and 4 h postinjection, respectively). However, WT animals receiving LPS/Dist A were protected from the development of significant hypotension (mean MAP, 80 ± 5.6 mm Hg, 72 ± 6.7 mm Hg, and 71 ± 6 mm Hg at 1, 2, and 4 h postinjection, respectively; p < 0.05 when compared with MAP in LPS/vehicle-treated WT animals at 1, 2, and 4 h postinjection). In contrast, both LPS/vehicle- and LPS/Dist A-treated NOS2–/– mice developed equivalent reductions in MAP following treatment (LPS-treated NOS2, 56.8 ± 5.9%, and LPS/DistA-treated NOS2, 53.8 ± 7.1% reduction in MAP at 4 h; p = NS between NOS2–/– treatment groups). Thus, the protective effect of Dist A on survival of WT mice subjected to endotoxemia is associated with protection from significant hypotension in these animals.

Dist A attenuates NOS2 induction in vivo in WT animals subjected to endotoxemia

We hypothesized that the protective effect of Dist A on mortality and hypotension during endotoxemia in WT mice would correlate with reduced NOS2 induction in organs mediating the adverse pathophysiologic consequences of endotoxemia. Therefore, we next determined the effect of Dist A on NOS2 expression in lungs and kidneys of LPS-treated WT mice.

Lungs and kidneys were harvested from WT mice 2 h following treatment with vehicle, LPS/vehicle, or LPS/Dist A (Fig. 3). Basal expression of NOS2 was undetectable (first lane in each panel), and significant induction of NOS2 expression occurred in both organs following LPS/vehicle treatment (second lane in each panel). Addition of Dist A attenuated NOS2 induction in lung and kidney (third lane in each panel).



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  		FIGURE 3. Dist A attenuates NOS2 induction in vivo in WT mice subjected to endotoxemia. Total RNA was extracted from lung and kidney tissue harvested from WT animals 2 h following treatment with vehicle, LPS/vehicle, or LPS/Dist A. A representative Northern blot (10 µg of RNA per lane) is shown using a radiolabeled NOS2 probe and an 18S probe (to confirm equal loading). This experiment was performed three separate times.

 
Dist A attenuates NOS2 induction in vitro in primary peritoneal and RAW 264.7 mouse macrophages under conditions of endotoxemia

To elucidate the mechanisms responsible for the attenuated induction of NOS2 by Dist A during endotoxemia in vivo, we examined the effect of Dist A on murine macrophages in vitro. The induction of NOS2 in this important cell type during endotoxemia has been well characterized in response to stimulation with LPS/IFN-{gamma}, which replicates the downstream effects of LPS stimulation in vivo (24, 25). We thus determined the effect of Dist A on the induction of NOS2 RNA and NOS activity by LPS/IFN-{gamma} in murine macrophages.

Total RNA was extracted from primary peritoneal (Fig. 4A) and RAW 264.7 murine macrophages (Fig. 4B) following exposure to vehicle, LPS + IFN-{gamma}/vehicle, or LPS + IFN-{gamma}/Dist A for 12 h. Similar to the pattern observed in vivo (described above), basal expression of NOS2 RNA was undetectable (primary peritoneal macrophages) or very low (RAW 264.7 cells), and a significant induction of NOS2 RNA was observed following LPS + IFN-{gamma}/vehicle. However, attenuation of this NOS2 induction was observed with the addition of Dist A to LPS + IFN-{gamma}. Nitrite levels as a marker of NO production were measured in RAW 264.7 cells following 48 h of exposure to vehicle, LPS + IFN-{gamma}/vehicle, or LPS + IFN-{gamma}/Dist A (Fig. 4C). Basal nitrite levels were low (vehicle-treated cells), and a 65-fold induction in nitrite levels was observed following treatment with LPS + IFN-{gamma}/vehicle (p < 0.05 compared with vehicle-treated cells). Addition of Dist A to LPS + IFN-{gamma} resulted in significant attenuation of this induction (50% reduction, p < 0.05 compared with LPS + IFN-{gamma}/vehicle-treated cells). Thus, Dist A attenuates the induction of NOS2 RNA and NOS2 activity during endotoxemia in murine macrophages.



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  		FIGURE 4. Dist A attenuates NOS2 induction in vitro in primary peritoneal and RAW 264.7 mouse macrophages under conditions of endotoxemia. A, Primary peritoneal mouse macrophages were harvested for total RNA 12 h following treatment with vehicle, LPS + IFN-{gamma}/vehicle, orLPS + IFN-{gamma}/Dist A. A representative Northern blot (10 µg of RNA per lane) is shown using a radiolabeled probe for NOS2 and an 18S probe (to confirm equal loading). B, RAW 264.7 mouse macrophages were harvested for total RNA 12 h following treatment with vehicle, LPS + IFN-{gamma}/vehicle, or LPS + IFN-{gamma}/Dist A. A representative Northern blot (10 µg of RNA per lane) is shown using a radiolabeled probe for NOS2 and an 18S probe (to confirm equal loading). This experiment was performed two separate times. C, RAW 264.7 mouse macrophages were treated for 48 h with vehicle, LPS + IFN-{gamma}/vehicle, or LPS + IFN-{gamma}/Dist A, following which medium was assessed for nitrite levels normalized for total cellular protein. Fold increase in nitrite levels was determined relative to vehicle-treated cells. This experiment was performed two separate times (*, p < 0.05 compared with vehicle-treated cells and compared with LPS + IFN-{gamma}/Dist A-treated cells).

 
Dist A attenuates induction of NOS2 promoter activity through the IRF-1 binding site in LPS/IFN-{gamma}-treated RAW 264.7 mouse macrophages

As NOS2 is a gene that is highly transcriptionally regulated (26, 27), we hypothesized that Dist A was acting at the transcriptional level. We therefore examined the effect of Dist A on NOS2 promoter activity in mouse macrophages treated with LPS/IFN-{gamma}.

RAW 264.7 cells were transiently transfected with the WT murine NOS2 luciferase promoter-reporter plasmid, then harvested for luciferase activity 24 h following treatment with vehicle, LPS +IFN-{gamma}/vehicle, or LPS + IFN-{gamma}/Dist A (Fig. 5A). Exposure of the cells to vehicle produced low basal NOS2 promoter activity (first bar), but marked induction of promoter activity occurred with addition of LPS + IFN-{gamma} (second bar, 20-fold induction, p < 0.05 compared with vehicle). Induction of NOS2 promoter activity was significantly attenuated with the addition of Dist A to LPS + IFN-{gamma} (third bar, mean 63% reduction, p < 0.05 compared with LPS + IFN-{gamma}/vehicle-treated cells). Furthermore, the LPS + IFN-{gamma}-induced NOS2 promoter activity could be attenuated when Dist A was added up to 6 h following LPS + IFN-{gamma} treatment (data not shown).



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  		FIGURE 5. Dist A attenuates induction of NOS2 promoter activity through the IRF-1 binding site in LPS + IFN-{gamma}-treated RAW 264.7 mouse macrophages. A, RAW 264.7 mouse macrophages were transfected with 0.5 µg of the NOS2 luciferase WT promoter-reporter construct, then treated with vehicle, LPS + IFN-{gamma}/vehicle, or LPS + IFN-{gamma}/Dist A. Cells were harvested for luciferase activity, which was normalized for {beta}-galactosidase, then compared with vehicle-treated cells to determine fold induction (*, p < 0.05 compared with vehicle-treated cells and compared with LPS + IFN-{gamma}/Dist A-treated cells). This experiment was repeated three separate times, with each condition performed in duplicate in each experiment. B, Schema of murine NOS2 promoter, demonstrating the enhancer region critical for synergistic induction of NOS2 promoter activity by LPS +IFN-{gamma}. C, RAW 264.7 murine macrophages were transfected with 0.5 µg of mNF-{kappa}B, mGAS, or mISRE luciferase promoter-reporter constructs, then treated with LPS + IFN-{gamma}/vehicle, or LPS + IFN-{gamma}/Dist A. Cells were harvested for luciferase activity, which was normalized for {beta}-galactosidase, then compared with vehicle-treated cells transfected with the WT plasmid to determine fold induction (*, p < 0.05 compared with LPS + IFN-{gamma}/Dist A-treated cells). This experiment was repeated three separate times, with each condition performed in duplicate in each experiment.

 
We next examined whether a particular transcription factor consensus binding site in the NOS2 promoter is important for the reduction of LPS/IFN-{gamma}-induced NOS2 expression by Dist A. Other investigators have shown that the enhancer region of the NOS2 promoter (–985 to –910) is critical for the synergistic induction of NOS2 by LPS/IFN-{gamma} in murine macrophages (26, 27, 28). Mutation of each of three critical transcription factor consensus binding sites in the enhancer (p50/p65 binding to an NF-{kappa}B site, Stat-1{alpha} binding to GAS, and IFN regulatory factor (IRF)-1 binding to an ISRE site) reduces the synergistic induction of NOS2 by LPS/IFN-{gamma} (29, 30, 31, 32) (schema, Fig. 5B). Additionally, there exist numerous AT-rich DNA sequences within or near these consensus binding sites to which Dist A might bind. We therefore generated promoter-reporter constructs with mutations at each of these sites to determine whether they were required for the effect of Dist A in attenuating the LPS/IFN-{gamma}-induced NOS2 expression.

RAW 264.7 cells were transiently transfected with the mNF-{kappa}B, mGAS, and mISRE luciferase promoter-reporter plasmids, then harvested for luciferase activity 24 h following treatment with LPS +IFN-{gamma}/vehicle or LPS + IFN-{gamma}/Dist A (Fig. 5C). As described by others, the luciferase activity for each of the mutated constructs was reduced (~5-fold) compared with the LPS/IFN-{gamma}-treated WT plasmid (29, 30, 31, 32). Similar to the WT plasmid, the mNF-{kappa}B and mGAS constructs exhibited a significant reduction in luciferase activity when Dist A was added to LPS/IFN-{gamma} (mean 89 and 75%, respectively, p < 0.05 for LPS/IFN-{gamma}/vehicle vs LPS/IFN-{gamma}/Dist A). In contrast, the mISRE plasmid showed no significant reduction in luciferase activity when Dist A was added to LPS/IFN-{gamma} (mean 0.08% reduction, p = NS for LPS/IFN-{gamma}/vehicle vs LPS/IFN-{gamma}/Dist A). Thus, the IRF-1 binding site in the NOS2 enhancer is critical for this attenuated induction by Dist A.

Dist A attenuates IRF-1 binding to the NOS2 promoter

We next directly assessed the effect of Dist A on IRF-1 binding to the NOS2 enhancer using nuclear extract from LPS/IFN-{gamma}-treated murine macrophages and rIRF-1 protein.

EMSA was performed using nuclear protein harvested from RAW 264.7 cells 2 h following treatment with LPS + IFN-{gamma} (Fig. 6A). Nuclear extracts were incubated with radiolabeled probes spanning the IRF-1 (ISRE) consensus binding site in the NOS2 enhancer, as well as increasing concentrations of Dist A (0–15 µM). The reaction mixtures were then subjected to electrophoresis. Nucleoprotein complexes were present following LPS + IFN-{gamma} stimulation, as has been reported previously by others (Fig. 6A, lane 1) (32). Specificity of the complexes was shown by using identical and nonidentical competitors (data not shown). With addition of increasing concentrations of Dist A, binding of the nuclear protein complex to the IRF-1 site decreased in a dose-dependent manner (Fig. 6A, lanes 2 and 3).



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  		FIGURE 6. Dist A attenuates IRF-1 binding to the NOS2 promoter. A, EMSA was performed using nuclear protein harvested from RAW 264.7 macrophages 2 h following treatment with LPS + IFN-{gamma}. Nuclear protein (10 µg per reaction) and increasing concentrations of Dist A (0–15 µM) were incubated with a reaction mixture containing a radiolabeled probe spanning the IRF-1 (ISRE) consensus binding site in the enhancer region of the murine NOS2 promoter (lanes 1-3). Reaction mixtures were then subjected to electrophoresis (*, specific nucleoprotein complexes; arrow represents free probe). This experiment was repeated three separate times. B, EMSA was performed using rIRF-1 protein. Equal amounts of recombinant protein and increasing concentrations of Dist A (0–10 µM) were incubated with a reaction mixture containing radiolabeled probes spanning the IRF-1 (ISRE) binding site in the enhancer region of the murine NOS2 promoter (lanes 2–5). Reaction mixtures were then subjected to electrophoresis. As a control for the probe, the reaction mixture was incubated without the addition of recombinant protein (lane 1) (*, nucleoprotein complex representing IRF-1; arrow represents free probe). This experiment was repeated three separate times.

 
Similarly, EMSA was performed using rIRF-1 protein and radiolabeled probes spanning the IRF-1 (ISRE) consensus binding site in the NOS2 enhancer (Fig. 6B). Probes were incubated with increasing concentrations of Dist A (0–10 µM), then reaction mixtures were subjected to electrophoresis. IRF-1 binding was observed at baseline (Fig. 6B, lane 2), and a dose-dependent reduction in this binding was seen with addition of increasing concentrations of Dist A (Fig. 6B, lanes 3–5). Thus, decreased induction of NOS2 message and promoter activity by Dist A during endotoxemia correlates with reduced binding of IRF-1 to AT-rich regions of DNA within the NOS2 enhancer in murine macrophages and with reduced binding of rIRF-1 in vitro.

Discussion

This study highlights three important new concepts. First, use of a drug to reduce NOS2 induction, rather than completely eliminate its expression, results in a significant survival advantage and attenuation of hypotension during murine endotoxemia. Second, administration of a drug that decreases transcription factor binding to AT-rich regions of DNA can decrease NOS2 expression in vivo. Third, this in vivo effect can be correlated with alteration of AT-rich region DNA binding in a sequence/conformation-specific manner in vitro, thus providing a powerful tool for altering gene expression.

The effect of NOS2 expression on murine mortality from endotoxemic shock has received significant attention in the literature, with varying results reported (5, 6, 7, 8, 9). More recent data have suggested that there is no consistent difference in survival between nonanesthetized, noninstrumented WT, and NOS2-deficient animals subjected to endotoxemic shock (8, 9, 21). Our current findings support this concept, in that both WT and NOS2-deficient animals exhibited nearly 100% mortality within 30 h of high-dose i.p. LPS administration (Fig. 1). Complete elimination of NOS2 expression (in NOS2-deficient animals) therefore does not appear to improve murine mortality from endotoxemic shock (10). Although administration of chemical NOS inhibitors might allow titration of NOS inhibition, studies of these agents in animal experiments and human trials of sepsis have not proven consistently effective in providing mortality benefit, perhaps related to the nonspecific inhibition of NO production from all three NOS isoforms (NOS1 and NOS3, in addition to NOS2) (22). Although more selective NOS2 isoform inhibitors have shown some promise in improving survival from murine endotoxemic shock, it is likely that nonspecific inhibition of NO production from all three NOS isoforms occurs with increasing doses of these agents (33). We did not find a protective effect of the NOS2-specific inhibitor, 1400W (14, 15), on the LPS-induced mortality of WT mice in our model (Fig. 1).

We now report the beneficial effect of an inhibitor of AT-rich region DNA binding, Dist A, on survival and blood pressure during murine endotoxemic shock (Figs. 1 and 2). Dist A is a member of a class of drugs termed minor-groove binders whose hallmark is sequence- and conformation-specific binding to AT-rich sequences in the minor groove of DNA (34). We have previously used Dist A in vitro to inhibit binding of high mobility group I/Y (17, 35, 36), which is one of a select group of architectural transcription factors that binds exclusively to the minor groove of DNA (37). Interestingly, Speight et al. (38) showed that Dist A, through binding the minor groove of DNA, interferes with in vitro binding of a transcription factor to the major groove of DNA. These authors proposed that when NF-{kappa}B binds to certain distinct DNA sequences, the minor groove of DNA is exposed such that Dist A can interfere with NF-{kappa}B-DNA binding through altering DNA conformation (38). Furthermore, they reported that Dist A does not generically inhibit all NF-{kappa}B-DNA binding in vitro, requiring specific DNA sequences for binding inhibition. In fact, Dist A does not mediate its effects through the NF-{kappa}B binding site in the NOS2 enhancer (Fig. 5C). Although Dist A and other similar compounds have been used as vehicles to bind chemotherapeutic agents to DNA in human studies (11), minor-groove binders have not been previously tested in sepsis nor in endotoxemic shock.

Although Dist A may have myriad effects on an organism, the lack of benefit of this agent in endotoxemic NOS2-deficient animals supports a significant role for NOS2 in mediating the beneficial effects of Dist A during endotoxemia in wild-type animals (Fig. 1). Furthermore, this beneficial effect of Dist A during endotoxemia correlates with decreased NOS2 induction in vivo (Fig. 3), as well as a significant attenuation of LPS-induced hypotension (Fig. 2). Numerous authors have reported a significant role for NOS2 in mediating LPS-induced vasodilation and hypotension (6, 10, 22, 23). Interestingly, we found that Dist A reduces, but does not eliminate LPS-induced NOS2 expression, both in vivo and in vitro. Thus, our data support the intriguing concept that Dist A, through decreasing LPS-induced NOS2 expression, attenuates hypotension and improves survival from endotoxemia.

To further investigate the mechanism by which Dist A attenuates LPS-induced NOS2 expression, we chose to extend our studies in murine macrophages. This cell type plays a critical role in endotoxemia, and the regulation of NOS2 expression has been extensively examined in macrophages. Other authors have reported that the full synergistic induction of NOS2 expression in RAW 264.7 cells by LPS and IFN-{gamma} (a downstream mediator of LPS in vivo) (24, 25) requires the enhancer region of the NOS2 promoter (–985 to –910) (26, 27, 28). Within this region exist consensus-binding sites for three transcription factors critical in the synergistic induction of NOS2: p50/p65 binding to an NF-{kappa}B site (29), Stat-1{alpha} binding to GAS (30), and IRF-1 binding to an ISRE binding site (31, 32). We now report that within this NOS2 enhancer region, which contains numerous AT-rich DNA regions in and around the critical consensus binding sites, Dist A selectively interferes with IRF-1 binding (Figs. 5 and 6). Interestingly, IRF-1 binds in a unique manner to the murine NOS2 promoter, in which sequential binding of IRF-1 molecules results in a dimeric complex extending from the major groove of DNA to occupy additional contacts in the minor groove of DNA (39). Therefore, Dist A might disrupt IRF-1 binding to the NOS2 enhancer region through interfering directly with both major- and minor-groove DNA binding. It is well known that macrophages from IRF-1-deficient mice do not synthesize NOS2 message nor produce NO in response to LPS + IFN-{gamma} stimulation (31). Furthermore, IRF-1-deficient mice are protected from LPS-induced mortality (40). Thus, the ability to decrease IRF-1-DNA binding provides a potential powerful tool for modulating NOS2 expression and for improving outcomes from endotoxemia.

In conclusion, we now report for the first time that a DNA minor-groove binding drug can dramatically improve murine survival and attenuate hypotension from endotoxemia. This beneficial effect correlates with reduced NOS2 induction in response to endotoxemic stimuli in vivo and in vitro and with decreased IRF-1-DNA binding in murine macrophages. Although further investigations will be required to determine the broader effects of these drugs and their effects in other models, our data raise the possibility for novel treatment approaches to critical illness.


   Acknowledgments
 
We are grateful to Drs. Shaw-Fang Yet and Richard Riese for helpful review of the manuscript, and to Dr. Qi Xue for technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants AI054465 (to R.M.B.), GM53249 (to M.A.P.), and HL60788 (to M.A.P.), all from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Mark A. Perrella, Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: mperrella{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: NOS, NO synthase; Dist A, distamycin A; GAS, IFN-{gamma} activation site; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; m, mutated; MAP, mean arterial pressure; WT, wild type. Back

Received for publication March 3, 2004. Accepted for publication July 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 References
 

  1. Wheeler, A. P., G. R. Bernard. 1999. Treating patients with severe sepsis. N. Engl. J. Med. 340:207.[Free Full Text]
  2. Riedemann, N. C., R.-F. Guo, P. A. Ward. 2003. The enigma of sepsis. J. Clin. Invest. 112:460.[Medline]
  3. Moncada, S., R. M. Palmer, E. A. Higgs. 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43:109.[Medline]
  4. Ejima, K., M. D. Layne, I. M. Carvajal, P. A. Kritek, R. M. Baron, Y.-H. Chen, J. Vom Saal, B. D. Levy, S.-F. Yet, M. A. Perrella. 2003. Cyclooxygenase-2 deficient mice are resistant to endotoxin-induced inflammation and death. FASEB J. 17:1325.[Abstract/Free Full Text]
  5. Laubach, V. E., P. L. Foley, K. S. Shockey, C. G. Tribble, I. L. Kron. 1998. Protective roles of nitric oxide and testosterone in endotoxemia: evidence from NOS-2 deficient mice. Am. J. Physiol. 275:H2211.
  6. MacMicking, J. D., C. Nathan, G. Hom, N. Chartrain, D. S. Fletcher, M. Trumbauer, K. Stevens, Q. W. Xie, K. Sokol, N. Hutchinson, et al 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641.[Medline]
  7. Wei, X., I. G. Charles, A. Smith, J. Ure, G. Feng, F. Huang, D. Xu, W. Muller, S. Moncada, F. Y. Liew. 1995. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375:408.[Medline]
  8. Laubach, V. W., E. G. Shesely, W. Smithies, P. A. Sherman. 1995. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc. Natl. Acad. Sci. USA 92:10688.[Abstract/Free Full Text]
  9. Zingarelli, B., P. W. Hake, J. A. Cook. 2002. Inducible nitric oxide synthase is not required in the development of endotoxin tolerance in mice. Shock 17:478.[Medline]
  10. Nathan, C.. 1997. Perspective series: nitric oxide and nitric oxide synthases: inducible nitric oxide synthase: what difference does it make?. J. Clin. Invest. 100:2417.[Medline]
  11. Cozzi, P.. 2000. Recent outcome in the field of distamycin-derived minor groove binders. Il. Farmaco 55:168.
  12. Bustin, M., R. Reeves. 1996. High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function. Prog. Nucleic Acid Res. Mol. Biol. 54:35.[Medline]
  13. Wegner, M., F. Grummt. 1990. Netropsin, distamycin and berenil interact differentially with a high-affinity binding site for the high mobility group protein HMG-I. Biochem. Biophys. Res. Commun. 166:1110.[Medline]
  14. Garvey, E. P., J. A. Oplinger, E. S. Furfine, R. J. Kiff, F. Laszlo, B. J. Whittle, R. G. Knowles. 1997. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J. Biol. Chem. 272:4959.[Abstract/Free Full Text]
  15. Rudkowski, J. C., E. Barreiro, R. Harfouche, P. Goldberg, O. Kishta, P. D’Orleans-Juste, J. Labonte, O. Lesur, S. N. A. Hussain. 2004. Roles of iNOS and nNOS in sepsis-induced pulmonary apoptosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 286:L793.[Abstract/Free Full Text]
  16. Perrella, M. A., C.-M. Hsieh, W.-S. Lee, S. Shieh, J.-C. Tsai, C. Patterson, C. J. Lowenstein, N. C. Long, E. Haber, S. Shore, M.-E. Lee. 1996. Arrest of endotoxin-induced hypotension by transforming growth factor {beta}1. Proc. Natl. Acad. Sci. USA 93:2054.[Abstract/Free Full Text]
  17. Pellacani, A., P. Wiesel, S. Razavi, V. Vasilj, M. W. Feinberg, M. T. Chin, R. Reeves, M. A. Perrella. 2001. Down-regulation of high mobility group-I(Y) protein contributes to the inhibition of nitric-oxide synthase 2 by transforming growth factor-{beta}1. J. Biol. Chem. 276:1653.[Abstract/Free Full Text]
  18. Perrella, M. A., C. Patterson, L. Tan, S.-Y. Yet, C.-M. Hsieh, M. Yoshizumi, M.-E. Lee. 1996. Suppression of interleukin-1{beta}-induced nitric-oxide synthase promoter/enhancer activity by transforming growth factor-{beta}1 in vascular smooth muscle cells. J. Biol. Chem. 271:13776.[Abstract/Free Full Text]
  19. Chen, Y.-H., M. D. Layne, S. W. Chung, K. Ejima, R. M. Baron, S.-F. Yet, M. A. Perrella. 2003. Elk-3 is a transcriptional repressor of nitric-oxide synthase 2. J. Biol. Chem. 278:39572.[Abstract/Free Full Text]
  20. Perrella, M. A., M. Yoshizumi, Z. Fen, J.-C. Tsai, C.-M. Hsieh, S. Kourembanas, M.-E. Lee. 1994. Transforming growth factor-{beta}1, but not dexamethasone, down-regulates nitric-oxide synthase mRNA after its induction by interleukin-1{beta} in rat smooth muscle cells. J. Biol. Chem. 269:14595.[Abstract/Free Full Text]
  21. Nicholson, S. C., S. R. Grobmyer, M. U. Shiloh, J. E. Brause, S. Potter, J. D. MacMicking, M. C. Dinauer, C. F. Nathan. 1999. Lethality of endotoxin in mice genetically deficient in the respiratory burst oxidase, inducible nitric oxide synthase, or both. Shock 4:253.
  22. Cobb, J. P., R. L. Danner. 1996. Nitric oxide and septic shock. J. Am. Med. Assoc. 275:1192.[Abstract/Free Full Text]
  23. Boyle, W. A., III, L. S. Parvathaneni, V. Bourlier, C. Sauter, V. E. Laubach, J. P. Cobb. 2000. INOS gene expression modulates microvascular responsiveness in endotoxin-challenged mice. Circ. Res. 87:E18.
  24. Heremans, H., R. Dijkmans, H. Sobis, F. Vandekerckhove, A. Billiau. 1987. Regulation by interferons of the local inflammatory response to bacterial lipopolysaccharide. J. Immunol. 138:4175.[Abstract]
  25. Kiener, P. A., F. Marek, G. Rodgers, P. F. Lin, G. Warr, J. Desiderio. 1988. Induction of tumor necrosis factor, IFN-{gamma}, and acute lethality in mice by toxic and non-toxic forms of lipid A. J. Immunol. 141:870.[Abstract]
  26. Xie, Q., R. Whisnant, C. Nathan. 1993. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon {gamma} and bacterial lipopolysaccharide. J. Exp. Med. 177:1779.[Abstract/Free Full Text]
  27. Lowenstein, C. J., E. W. Alley, P. Raval, A. M. Snowman, S. H. Snyder, S. W. Russell, W. J. Murphy. 1993. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon {gamma} and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90:9730.[Abstract/Free Full Text]
  28. Alley, E. W., W. J. Murphy, S. W. Russell. 1995. A classical enhancer element responsive to both lipopolysaccharide and interferon-{gamma} augments induction of the iNOS gene in mouse macrophages. Gene 158:247.[Medline]
  29. Kim, Y.-M., B.-S. Lee, K.-Y. Yi, S.-G. Paik. 1997. Upstream NF-{kappa}B site is required for the maximal expression of mouse inducible nitric oxide synthase gene in interferon-{gamma} plus lipopolysaccharide-induced RAW 264.7 macrophages. Biochem. Biophys. Res. Commun. 236:655.[Medline]
  30. Gao, J., D. C. Morrison, T. J. Parmely, S. W. Russell, W. J. Murphy. 1997. An interferon-{gamma}-activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon-{gamma} and lipopolysaccharide. J. Biol. Chem. 272:1226.[Abstract/Free Full Text]
  31. Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, S. I. Koh, T. Kimura, S. J. Green, et al 1994. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263:1612.[Abstract/Free Full Text]
  32. Martin, E., C. Nathan, Q. Xie. 1994. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med. 180:977.[Abstract/Free Full Text]
  33. Liaudet, L., A. Rosselet, M.-D. Schaller, M. Markert, C. Perret, F. Feihl. 1998. Nonselective versus selective inhibition of inducible nitric oxide synthase in experimental endotoxic shock. J. Infect. Dis. 177:127.[Medline]
  34. Bell, A., L. Kittler, G. Löber, C. Zimmer. 1997. DNA binding properties of minor groove binders and their influence on the topoisomerase II cleavage reaction. J. Mol. Recognit. 10:245.[Medline]
  35. Pellacani, A., M. T. Chin, P. Wiesel, M. Ibanez, A. Patel, S.-F. Yet, S.-M. Hsieh, J. D. Paulauskis, R. Reeves, M.-E. Lee, M. A. Perrella. 1999. Induction of high mobility group-I(Y) protein by endotoxin and interleukin-1{beta} in vascular smooth muscle cells. J. Biol. Chem. 274:1525.[Abstract/Free Full Text]
  36. Foster, L. C., P. Wiesel, G. S. Huggins, R. Panares, M. T. Chin, A. Pellacani, M. A. Perrella. 2000. Role of activating protein-1 and high mobility group-I(Y) in the induction of CD44 gene expression by interleukin-1{beta} in vascular smooth muscle cells. FASEB J. 14:368.[Abstract/Free Full Text]
  37. Bewley, C. A., A. M. Gronenborn, G. M. Clore. 1998. Minor groove-binding architectural proteins: structure, function, and DNA recognition. Annu. Rev. Biophys. Biomol. Struct. 27:105.[Medline]
  38. Speight, R. E., D. J. Hart, J. M. Blackburn. 2002. Distamycin A affects the stability of NF-{kappa}B p50-DNA complexes in a sequence-specific manner. J. Mol. Recognit. 15:19.[Medline]
  39. Spink, J., T. Evans. 1997. Binding of the transcription factor interferon regulatory factor-1 to the inducible nitric-oxide synthase promoter. J. Biol. Chem. 272:24417.[Abstract/Free Full Text]
  40. Senaldi, G., C. L. Shaklee, J. Guo, L. Martin, T. Boone, T. W. Mak, T. R. Ulich. 1999. Protection against the mortality associated with disease models mediated by TNF and IFN-{gamma} in mice lacking IFN regulatory factor-1. J. Immunol. 163:6820.[Abstract/Free Full Text]



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