Malcom Randall Veterans Affairs Medical Center and the Division of Gastroenterology, Hepatology and Nutrition, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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Mesalamine (5-ASA) is
effective in the treatment of inflammatory bowel diseases. However, the
mechanisms of action of 5-ASA remain unclear. IEC-6 and IRD-98,
nontransformed rat small intestinal epithelial cell lines, were used to
examine the effect of 5-ASA on the expression of manganese superoxide
dismutase (MnSOD). Rats were given 5-ASA enemas to determine the effect
on colonic MnSOD expression. Treatment with 5-ASA at 0.02 or 2 mg/ml
induced MnSOD mRNA levels 2.67-fold or 5.66-fold, respectively.
Inhibition of 5-lipoxygenase activating protein with MK-886 or
cyclooxygenase with indomethacin did not influence the level of MnSOD
mRNA. Nuclear run-on experiments demonstrated an increase in de novo
transcription following treatment with 5-ASA. MnSOD protein levels were
induced 2-fold at 24 h and 4.23-fold at 48 h following
treatment with 1 mg/ml 5-ASA. 5-ASA increased MnSOD 1.7-fold in vivo.
Pretreatment with 5-ASA significantly protected IRD-98 cells from tumor
necrosis factor- cytotoxicity. This is the first example of
transcriptional gene regulation by 5-ASA. The induction of MnSOD by
5-ASA may contribute to the therapeutic mechanism of 5-ASA.
5-aminosalicylic acid; transcriptional regulation; cytotoxicity
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INTRODUCTION |
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SULFASALAZINE AND MESALAMINE (5-ASA), the active ingredient in sulfasalazine (3), are effective first-line agents in the treatment of active inflammatory bowel disease (IBD) and in the maintenance of remission (19). Sulfasalazine has been used for over 50 years, but the mechanisms of action of sulfasalazine and 5-ASA have remained elusive. 5-ASA compounds are capable of multiple effects that may protect the colon from an inflammation-mediated damage. 5-ASA has been demonstrated to directly scavenge free radicals (1, 2), inhibit leukotriene production (43), inhibit the chemotactic response to leukotriene B4 (LTB4) (33), and inhibit cellular release of interleukin (IL)-1 in cultured mucosal biopsy specimens from ulcerative colitis patients (37). Sulfasalazine and 5-ASA inhibit the binding of formyl-methionyl-leucyl-phenylalanine to its receptor on neutrophils (15). Some authors believe that sulfasalazine and 5-ASA are effective because of the additive effects of their multiple actions on the immune system (15, 16).
The ability of 5-ASA and sulfasalazine to inhibit 5-lipoxygenase and LTB4 production (43), the chemotactic response to LTB4 (33), and the enhanced production of LTB4 in the inflamed colonic mucosa (42) resulted in clinical trials examining more potent inhibitors of LTB4 production. However, more potent 5-lipoxygenase inhibitors such as zileuton have not been effective (21). Although inhibition of leukotriene production may contribute to the mechanism of action of the 5-ASA compounds, the interest in this mechanism of action has waned.
In this study, 5-ASA at therapeutically relevant concentrations is shown to induce manganese superoxide dismutase (MnSOD) in vitro and in vivo. The following data examine, in detail, the regulation of MnSOD mRNA and protein levels in the rat intestinal epithelial cells and in the rat colon in vivo.
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METHODS |
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Cell culture and experimental treatments.
All chemicals including 5-ASA were purchased from Sigma (St. Louis, MO)
unless otherwise stated. IEC-6 (ATCC CRL-1592) is a nontransformed rat
small intestinal crypt cell line developed by Quaroni et al.
(36). IRD-98 is a fetal rat small intestinal epithelial
cell line that was established by Negrel et al. (32) and
was a gift from Patrick Rampal (Hospital de Cimiez, Nice, France). The
IEC-6 and IRD-98 cells were grown to confluence in DMEM (GIBCO,
Gaithersburg, MD) with 10 µg/ml insulin, 0.6 mg/ml L-glutamine, antibiotic/antimycotic solution, and 5% fetal
bovine serum at 37°C in 95% air and 5% CO2. The cells
were treated with 5-ASA at concentrations ranging from 0.002 to 2.0 mg/ml, Escherichia coli endotoxin [lipopolysaccharide
(LPS)] at 0.5 µg/ml, tumor necrosis factor (TNF)- at 10 or 100 ng/ml (R&D Systems, Minneapolis, MN), MK-886 (an inhibitor of
5-lipoxygenase activating protein; Merck, Malvern, PA) at 10 ng/ml, or
indomethacin (an inhibitor of cyclooxygenase) at 1 µM/l. The cells
were also cotreated with 1 mg/ml 5-ASA and 4 µM actinomycin D or 20 µM cycloheximide.
RNA isolation and Northern blot analysis. After 8 h of treatment, total cellular RNA was isolated from the cultured cells by a protocol modified from the guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (8) as previously described (47). Twenty micrograms of total cellular RNA from each sample was subjected to Northern blot analysis as has previously been described (47). The RNA was electrotransferred and cross-linked to nylon membrane (CUNO, Meriden, CT). The membranes were probed with a rat MnSOD, rat Cu/ZnSOD, and cathepsin D or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. All of the probes used were radiolabeled by random primer extension. Following an overnight hybridization at 62°C, the membranes were washed at 65°C in a 40 mM sodium phosphate, 0.1% SDS, 1 mM EDTA solution and exposed to X-ray film. The autoradiographs were analyzed with a Millipore video image densitometer (Millipore, Ann Arbor, MI).
Nuclear run-on experiments. Four plates each of control IEC-6 cells and cells treated for 3 h with 2 mg/ml 5-ASA or 0.5 µg/ml LPS were washed with PBS, trypsinized, and centrifuged for 5 min at 4°C and 300 g to pellet the cells. The pellets was resuspended in 6 ml of lysis buffer (10 mM Tris · HCl, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-40, pH 7.4) and Dounce homogenized to generate nuclei free of cell membrane debris. The nuclei were pelleted by centrifugation at 1,200 g for 5 min. The nuclei were resuspended in 2 ml of storage buffer [50 mM HEPES, 4 mM MnCl2, 1 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol (DTT), and 50% glycerol] and snap frozen in liquid N2.
Radiolabeled mRNA transcript analysis of control and treated cells were performed according to a modified procedure described by Laine et al. (26). The isolated nuclei were thawed on ice and centrifuged at 1,200 g. The pellet of each sample was resuspended in 175 µl of incubation buffer (75 mM HEPES, 100 mM KCl, 2.5 mM MgCl2, 0.05 mM EDTA, 25% glycerol, 5 mM DTT, 0.5 mM CTP, 0.5 mM GTP, 1.0 mM ATP, 22 U/ml creatine kinase, and 8.8 mM creatine phosphate) and 4 µl of RNase inhibitor (40U; stock 10 U/µl) and 250 µCi [32P]UTP. The reaction was incubated for 45 min at 30°C with agitation. RNA, including the radioactive nascent RNA, was then isolated according to our standard procedure (47). Nylon membranes were dot blotted with 2.5 µg of denatured MnSOD cDNA, and various cDNA fragments of constitutively expressed genes (Cu/ZnSOD and cathepsin B) were used to quantitate the assay. Linearized pUC19 DNA served as a negative control. One milliliter of hybridization solution was added to each radiolabeled RNA sample. A 2- to 5-µl aliquot was counted in a scintillation counter. The samples were normalized based on the total counts per million (cpm), and an equal volume of hybridization solution was added. The dot blot membranes were prehybridized for 1 h at 60°C. After a 48-h incubation at 60°C in 2 ml of hybridization solution containing 32P-labeled RNA (containing 1-4 × 107 cpm) from control and treated cells, the membrane strips were washed and autoradiographed.Protein isolation and Western blot analysis. Following a treatment period of 12-48 h, control and 5-ASA-treated cells were rinsed twice with sterile PBS and scraped off the plate in 2 ml ice-cold PBS. The cells were pelleted, and the cellular protein was isolated as previously described (47). In the in vivo experiments, after 4 days of 5-ASA enema administration (2 ml of a 4 g/60 ml 5-ASA suspension; Rowasa; Solvay Pharmaceuticals, Marietta, GA), the rats were killed and the colons were excised. The mucosa and submucosa were separated from the remaining distal colon, and protein was isolated as previously described (45). The protein concentrations of the samples were determined by a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). A 20-µg aliquot of each sample was denatured by boiling, loaded onto a stacking gel, and fractionated on a 15% acrylamide-SDS running gel and electrotransferred to a nitrocellulose membrane as has been previously described (45, 47). The membranes were probed by incubation for 1 h with a 1:1,000 dilution of rabbit anti-rat MnSOD polyclonal antibody (in blocking buffer). The bound MnSOD antibody was detected using a donkey anti-rabbit secondary antibody and an Amersham enhanced chemiluminescence detection system (Amersham, Arlington Heights IL). The autoradiographs were analyzed with a Millipore video image densitometer or with Scion Image software (Scion, Frederick, MD).
Protection from TNF--mediated cytotoxicity.
IRD-98 cells were plated in 96-well plates. Some wells were pretreated
with 2 mg/ml 5-ASA for 24 h before the addition of 5-ASA plus
TNF-
(100 ng/ml) and cycloheximide (0.5 µg/ml). Other wells were
treated with TNF-
(100 ng/ml) and cycloheximide (0.5 µg/ml) or
5-ASA (2 mg/ml) plus TNF-
(100 ng/ml) and cycloheximide (0.5 µg/ml). After 12-24 h, 10 µl/well of the WST reagent
(Roche Molecular Biochemicals, Indianapolis, IN) was added, and the
plates were read after 3 h at 450 nm in an ELISA plate reader (STL
Lab Instruments, Grodig, Austria).
Statistics.
Statistical significance was determined by the t-test, with
the value deemed significant at P 0.05. All data are
expressed as means ± SE.
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RESULTS |
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Effect of 5-ASA on MnSOD mRNA levels in cell culture.
Treatment of IEC-6 cells with 5-ASA results in an induction of MnSOD
mRNA levels. Figure 1A is a
Northern blot analysis of MnSOD and Cu/ZnSOD mRNA levels at an 8-h time
point, examining the effect of a concentration curve of 5-ASA. MnSOD
has five mature mRNA transcripts that contain the complete coding
sequence. MnSOD mRNA levels are induced by 5-ASA beginning at 0.02 mg/ml and further increase through 2 mg/ml. Cu/ZnSOD mRNA levels were
not affected by treatment with 5-ASA and are used as an internal
control. Figure 1B is the result of densitometry data from
five experiments resulting in a 2.67-fold induction of MnSOD mRNA at
0.02 mg/ml and reaching a 5.66-fold induction at 2 mg/ml.
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Actinomycin and cycloheximide experiments.
To elucidate the mechanism underlying the increase in MnSOD mRNA in
response to treatment with 5-ASA, the cells were cotreated with 5-ASA
and actinomycin D, an RNA synthesis inhibitor, or the protein synthesis
inhibitor cycloheximide. As shown in Fig.
2, treatment with actinomycin D alone
does not alter the basal level of MnSOD mRNA, but cotreatment with
5-ASA and actinomycin D inhibits the 5-ASA-dependent elevation in the
level of MnSOD mRNA. Similarly, treatment with cycloheximide alone has
little affect on the basal level of MnSOD mRNA; however, the induction
of MnSOD mRNA by 5-ASA is not inhibited by cotreatment with 5-ASA and
cycloheximide.
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Nuclear run-on experiments.
Nuclear run-on experiments are the gold standard for determining that
the induction of a gene is transcriptional (17). By this
method, newly synthesized RNA in isolated nuclei can be labeled with a
high specific activity. Figure 3 is a
representative nuclear run-on experiment from control cells and cells
treated for 3 h with 5-ASA or LPS. If Cu/ZnSOD levels are used for
the internal control, 5-ASA induces MnSOD mRNA production by twofold
and LPS induces MnSOD mRNA production by fourfold. If cathepsin signal is used as the control, 5-ASA induces MnSOD mRNA production by 2.8-fold
and LPS induces MnSOD mRNA production by 6-fold. Similar data has been
obtained from two additional experiments.
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Effect of lipoxygenase and cyclooxygenase inhibition on MnSOD mRNA
levels.
A proposed mechanism for the therapeutic action of 5-ASA is the
inhibition of 5-lipoxygenase (33). 5-ASA has also been
reported to inhibit cyclooxygenase (15). To learn if
inhibition of lipoxygenase and/or cyclooxygenase was involved in the
mechanism for 5-ASA induction of MnSOD, the cells were treated with
MK-886, an inhibitor of 5-lipoxygenase activating protein
(39), at 10 ng/ml, indomethacin, an inhibitor of
cyclooxygenase, at 1 µM/l (20), or a combination of
MK-886 and indomethacin. Figure 4 is a
representative Northern blot analysis of MnSOD mRNA levels following
treatment with 5-ASA, indomethacin, MK-886, or indomethacin plus
MK-886. Ethanol was the solvent for indomethacin and MK-886, and
therefore ethanol serves as an additional control. Inhibition of
5-lipoxygenase activating protein by treatment with MK-886 and/or
inhibition of cyclooxygenase by treatment with indomethacin did not
affect MnSOD mRNA levels compared with control.
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5-ASA oxidation.
5-ASA readily oxidizes in most solutions. To attempt to determine if
the 5-ASA oxidation leads to induction of MnSOD, IRD-98 cells were
treated at a final concentration of 2 mg/ml 5-ASA in the form of Rowasa
enema, a 5-ASA suspension in an antioxidant carrier containing carbomer
934P, edetate disodium, potassium acetate, potassium metabisulfite,
sodium benzoate, purified water, and xanthan gum. 5-ASA in the form of
Rowasa has a shelf life of several months and thus is effective in
inhibiting 5-ASA oxidation. Figure 5
shows that 5-ASA in the form of Rowasa induces MnSOD mRNA levels
similar to that observed with nonproprietary 5-ASA. Similar results
were observed in IEC-6 cells (data not shown).
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In vitro induction of MnSOD protein levels.
To verify that the induction of MnSOD mRNA by 5-ASA is followed by an
increase in MnSOD protein levels, IEC-6 cells were treated with 1 mg/ml
5-ASA and MnSOD protein levels were determined by Western blot
analysis. In Fig. 6A, MnSOD
protein levels were determined at 12, 24, and 48 h after treatment
with 5-ASA. Densitometry revealed that MnSOD protein levels are induced
1.99-fold at 24 h and 4.23-fold at 48 h and are shown in Fig.
6B.
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In vivo induction of MnSOD by 5-ASA.
To confirm that the induction of MnSOD observed in cell culture
reflects the in vivo effects, 150-g Sprague-Dawley rats were treated
with a 2-ml enema of a 4 mg/60 ml 5-ASA suspension (Rowasa enema) on
four consecutive days. The results of the Western blot analysis of the
distal colonic mucosal MnSOD levels are shown in Fig.
7. Densitometry indicated that treatment
with 5-ASA results in a 1.7-fold induction of MnSOD protein levels
compared with controls.
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Protection from TNF--mediated cytotoxicity.
An assay using the conversion of WST was used to demonstrate protection
from TNF-
-mediated cytotoxicity by 5-ASA. WST reagent is converted
by functional mitochondrial dehydrogenase to tetrazolium and thus is a
marker of viable cells. Figure 8 shows
the results of an experiment in IRD-98 cells performed in triplicate.
Treatment for 24 h with 2 mg/ml 5-ASA before the addition of 2 mg/ml 5-ASA plus 100 ng/ml TNF-
and 0.5 µg/ml cycloheximide
resulted in 96.7% cell survival at 12 h and 88.7% survival at
24 h. However, only 32.7% of cells treated with
TNF-
/cycloheximide alone survived at the 24-h time point. The
addition of 5-ASA at the same time as the addition of
TNF-
/cycloheximide did not afford protection from TNF-
-mediated
cytotoxicity.
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DISCUSSION |
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5-ASA, the active constituent in sulfasalazine, is poorly absorbed in the colon, and ~50% is metabolized to acetyl 5-ASA by the intestinal epithelium and luminal bacteria (16). However, acetyl 5-ASA is even more poorly absorbed and is therapeutically inactive (16). Although the mechanism of action of 5-ASA remains ill defined, it may be therapeutically effective due to multiple effects that may protect the colon from an inflammatory response (15, 16). The induction of MnSOD may contribute to the therapeutic action of 5-ASA in the treatment of IBD.
In eukaryotes, three SODs have been identified. MnSOD is a
nuclear-encoded mitochondrial matrix protein (4).
Unrelated to the MnSOD is Cu/ZnSOD, a cytoplasmic protein
(4), and the extracellular SOD (EC-SOD) is a secreted form
with 22.6% amino acid sequence homology with Cu/ZnSOD
(22). SOD catalyzes the reaction 2O H2O2 + O2, thus eliminating oxygen free radicals.
H2O2 is then inactivated by catalase and
glutathione peroxidase to form water. In IBD, the production of oxygen
free radicals by cytokines and activated neutrophils may overwhelm the
intestinal defense mechanisms. This is particularly relevant since the
colonic mucosa, submucosa, and muscularis mucosa have been found to
contain low levels of SOD, catalase, and glutathione peroxidase
activity (18). Evidence of direct oxidant-induced colonic
epithelial cell injury in IBD has been established by examination of
the oxidation of GAPDH in freshly isolated colonic epithelial cells
(31). Treatment with SOD has been shown to reduce
inflammation in acetic acid (13) and
trinitrobenzenesulfonic acid (52) models of colitis and in
Crohn's disease (12). Cytoprotective properties of MnSOD have been demonstrated in other tissues as well. Overexpression of
MnSOD in the lung in transgenic mice reduces the toxic effects and
improved survival in mice exposed to 95% oxygen (50).
The induction of MnSOD mRNA by 5-ASA is dose dependent and within the concentration range of 0.5-3.5 mM interstitial concentrations (15) to 7 mM luminal concentrations (34) that have been documented in patients taking 2-3 g of sulfasalazine orally. Oral 5-ASA is frequently given in doses of 2.4-4.8 g/day (equivalent to 6-12 g of sulfasalazine), and the concentration of 5-ASA in enema form (4 g/60 ml) is 433 mM (19). Consequently, newer forms of 5-ASA delivery provide even higher concentrations of 5-ASA to the colon and result in even higher interstitial and luminal levels of 5-ASA (14). The induction of MnSOD may contribute to the free radical scavenging activity of 5-ASA that has been observed in cellular systems and in vivo; however, 5-ASA also has free radical scavenging activity independent of MnSOD in cell-free systems (2).
The induction of MnSOD by 5-ASA is not limited to IEC-6 and IRD-98 cells. Similar results were obtained in rat lung pulmonary epithelial L2 cells and the rat intestinal epithelial cell line FRI-1 (unpublished data). 5-ASA did not induce MnSOD in the human colon carcinoma cell line T84 (unpublished data); however, we have not been able to induce MnSOD with any stimulus in this cell line, which is consistent with the finding of abnormal MnSOD regulation in many carcinoma cell lines (5, 9). Other investigators have shown that overexpression of MnSOD results in a reduction of the malignant phenotype in multiple cell lines, including breast cancer (28), prostate cancer (29), and melanoma cell lines (9). Therefore, the induction of MnSOD may contribute to the chemopreventive properties of 5-ASA (7, 38).
The induction of MnSOD by 5-ASA is eliminated by actinomycin D but is
unaffected by cotreatment with cycloheximide, implicating de novo
transcription but not translation as a requirement for the induction of
MnSOD mRNA levels by 5-ASA. This finding is similar to the regulation
of MnSOD in IEC-6 cells by LPS, TNF-, and IL-1
(44).
Nuclear run-on experiments confirmed the transcriptional nature of the
regulation of MnSOD mRNA levels by 5-ASA. This procedure results in the
elongation of the transcripts initiated at the time of nuclei
isolation. Therefore, the rate of RNA synthesis can be compared between
the control and treated cells.
Translation was confirmed by the 4.23-fold increase in MnSOD protein levels following treatment of IEC-6 cells with 5-ASA and the 1.7-fold increase in the colonic mucosa in vivo. The lesser degree of induction observed in vivo may be the result of differences between cell culture and the in vivo environment or poor retention and penetration of 5-ASA through the mucus layer in the colon. We are not aware of other publications reporting gene induction by 5-ASA. 5-ASA has been reported to enhance the induction of heat shock protein expression in intestinal epithelial cells, but 5-ASA alone did not affect heat shock protein expression (6). Stevens et al. (44) found that 5-ASA and sulfasalazine reduced IL-2 expression in cultured T cells by a largely posttranscriptional mechanism. However, sulfasalazine and 5-ASA treatment of these cells also resulted in 63 and 37% cytotoxicity, respectively.
We have hypothesized that MnSOD is functioning as a cytokine-inducible
acute-phase protein that functions to protect the cell from cytokine
toxicity and increased intracellular free radical production
(46). Recently, gene array technology has identified MnSOD
as a gene that is induced fivefold in surgical specimens from patients
with active ulcerative colitis (10). In an animal model of
acute colitis, we have found that MnSOD mRNA is induced as early as
4 h after the colonic insult (45). Other
investigators have demonstrated that MnSOD is both induced by TNF-
and IL-1 and required for protection from TNF-
and IL-1 cytotoxicity
(49, 22). Treatment with 5-ASA protected IEC-6 cells from
TNF cytotoxicity. TNF-
results in a free radical leak from the
ubisemiquinone step (complex III) of the mitochondrial electron
transport chain (41), and thus the induction of MnSOD may
protect the cell from TNF-induced free radical damage. We used the
TNF/cycloheximide cytotoxicity system with which Wong et al.
(49) had established a cytoprotective role for MnSOD;
however, IRD-98 and IEC-6 cells appear more sensitive to the effects of
cycloheximide (data not shown). For MnSOD to protect cells from TNF
cytotoxicity, MnSOD levels must be elevated before exposure to
TNF/cycloheximide, and our data is consistent with this finding.
The mechanism of 5-ASA induction of MnSOD is not clear but likely
involves a redox-sensitive transcription factor. Neither inhibition of
cyclooxygenase with indomethacin nor inhibition of the 5-lipoxygenase
pathway with MK-886 resulted in changes in MnSOD mRNA levels, making
this mechanism unlikely for the induction by 5-ASA. Several
transcription factors, such as nuclear factor-B (NF-
B) and AP-1
are activated by TNF-
, IL-1, or oxidants (40, 35).
5-ASA readily oxidizes; however, once oxidized, 5-ASA will not induce
MnSOD (unpublished data). Treatment with 5-ASA in an antioxidant
carrier still resulted in induction of MnSOD; however, we are unable to
confirm that intracellular oxidation was prevented. 5-ASA, through
radical scavenging activity and by inducing MnSOD, may prevent the
activation of NF-
B. In cancer cell lines, overexpression of MnSOD
has been reported to inhibit the activation of the transcription factors AP-1, NF-
B, and c-jun (25, 27). The
data on inhibition of NF-
B activation by 5-ASA is conflicting. Two
reports describe inhibition of NF-
B activation by sulfasalazine but
not 5-ASA (30, 48), whereas others have reported that
5-ASA does inhibit the activation of NF-
B (11, 24, 51).
The induction of MnSOD by 5-ASA at therapeutically relevant concentrations is the first demonstration of transcriptional gene regulation by 5-ASA. Our results do not address the signal transduction mechanism that leads to the induction of MnSOD by this poorly absorbable compound. It remains to be determined whether increasing the intracellular concentration of 5-ASA is required for the induction of MnSOD. The induction of MnSOD by 5-ASA may contribute to the mechanism of action of 5-ASA by reducing the cytotoxicity of cytokines and oxygen free radicals generated in the gut and by altering the activity of redox-sensitive transcription factors.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1 DK-54919 and by the Medical Research Service of the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. F. Valentine, Univ. of Florida, College of Medicine, Box 100214, Gainesville, FL 32610 (E-mail: valenjf{at}medicine.ufl.edu).
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.
Received 31 July 2000; accepted in final form 9 May 2001.
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