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Am J Physiol Gastrointest Liver Physiol 290: G1211-G1218, 2006. First published February 16, 2006; doi:10.1152/ajpgi.00078.2005
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MUCOSAL BIOLOGY

Suppression of early growth response factor-1 with egr-1 antisense oligodeoxynucleotide aggravates experimental duodenal ulcers

Tetyana Khomenko, Sandor Szabo, Xiaoming Deng, Martin R. Jadus, Hideki Ishikawa, Klara Osapay, Zsuzsa Sandor, and Longchuan Chen

Diagnostic and Molecular Medicine Health Care Group, Veterans Affairs Medical Center, and Departments of Pathology and Pharmacology, University of California-Irvine, Long Beach, California

Submitted 21 February 2005 ; accepted in final form 9 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously, we demonstrated that cysteamine releases endothelin-1 in the rat duodenal mucosa, followed by increased expression of early growth response factor-1 (egr-1). We hypothesized that egr-1 is a key mediator gene in the multifactorial mechanisms of duodenal ulcer development and healing because its protein, transcription factor product Egr-1, regulates the expression of angiogenic growth factors. We wanted to determine the effect of egr-1 antisense oligonucleotide on cysteamine-induced duodenal ulcers as well as on the expression of bFGF, PDGF, and VEGF, of which synthesis is modulated by Egr-1. An antisense oligonucleotide to egr-1 was used to inhibit the synthesis of Egr-1 and to determine its effect on ulcer formation in the rat model of cysteamine-induced duodenal ulceration. Real-time RT-PCR and Western blot analysis were used to assess the expression of Egr-1 mRNA and protein as well as ERK, bFGF, PDGF, and VEGF. The antisense Egr-1 oligonucleotide inhibited the expression of egr-1 mRNA and protein and increased the duodenal ulcer size from 8.1 ± 1.8 mm2 in controls to 20.7 ± 4.0 mm2 (P < 0.01). Cysteamine induced phosphorylation of ERK1/2 and enhanced the synthesis of bFGF, PDGF, and VEGF in the preulcerogenic stages of duodenal ulceration, whereas egr-1 antisense oligonucleotide markedly decreased the expression of these growth factors in the duodenal mucosa. We also demonstrated that Egr-1 expression relates to the ulcerogenic effect of cysteamine because these actions were not exerted by the toxic analog ethanolamine. Thus Egr-1 seems to play a critical role in duodenal ulceration because Egr-1 downregulation aggravates experimental duodenal ulcers, most likely through the transcriptional inhibition of bFGF, PDGF, and VEGF synthesis.

early growth response factor-1; cysteamine-induced duodenal ulcer; transcription regulation; angiogenic growth factors

DUODENAL ULCERS are two to four times more prevalent than gastric ulcers, and this form of "peptic ulcer" disease affects 5–10% of the United States population at least once in their lifetime (14, 38). In addition to the role of Heliobacteri pylori, about one-third of ulcers are H. pylori negative and are probably drug induced (7, 9). In any case, the molecular mechanisms of ulcer development and healing remain poorly understood. Animal models of duodenal ulcer disease [e.g., induced by cysteamine or propionitrile, because aspirin-like drugs produce only gastric erosions or ulcers in rodents (8, 39, 43)] are needed to gain insight into the early preulcerogenic molecular and biochemical changes in the pathogenesis of duodenal ulceration. Most of the pathogenesis research has been performed with the duodenal ulcerogen cysteamine, which produces perforating ulcers in the rat proximal duodenum in 24–48 h (8, 39, 43). The cysteamine-induced solitary or "kissing" duodenal ulcers usually heal in 2–3 wk. The repair process includes the proliferation of endothelial (i.e., angiogenesis), epithelial, and connective tissue cells, and it is regulated by growth factors (4, 41, 45–47). Our laboratory was the first to report that an intragastric administration of bFGF, PDGF, or VEGF or that gene therapy with either naked DNA or adenoviral vectors of VEGF and PDGF significantly accelerated chronic duodenal ulcer healing in rats (40, 41, 45, 46). One of the very early events in experimental duodenal ulceration is the rapid, local release of endothelin-1 (ET-1) followed by increased expression of transcription factor early growth response factor-1 (Egr-1) in the nuclear fraction of the duodenal mucosa (21, 42, 46). Our microarray gene expression studies (19, 21, 40) revealed the consistent activation of egr-1 and Sp1 as well as bFGF or PDGF genes after cysteamine administration.

The zinc finger transcription factor Egr-1 is an immediate-early gene product that interacts with consensus GC-rich promoter regions to regulate the transcription of a diverse set of genes in response to mitogenic and nonmitogenic stimuli such as growth factors, shear stress, mechanical injury, or hypoxia (1, 18, 23, 31, 33, 36). These stimuli induce the activation of ERK1/2, members of the MAPK family, leading to enhanced Egr-1 transcriptional function and activation of target genes (17, 27, 29). In turn, Egr-1 induces the expression of several target genes, including bFGF, PDGF-A, PDGF-B, and VEGF receptor-1/Flt-1 (3, 35, 50, 52).

Recently, we (20) demonstrated that cysteamine induced hypoxia in the proximal duodenum, stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) expression, enhanced HIF-1{alpha} transcriptional activity, and increased the transcriptional interaction of HIF-1{alpha} with Egr-1. HIF-1{alpha} is a hypoxia-inducible transcription factor that enhances promoter activity of VEGF gene and VEGF synthesis (22). Because Egr-1 regulates the expression of genes involved in the proliferative response to a variety of stimuli, targeting this transcription factor may reduce proliferation or repair after injury. An antisense egr-1 oligonucleotide abolished the stimulation of protein synthesis induced by ET-1 in myogenic cell line (24). Inhibition of Egr-1 synthesis in smooth muscle cells by antisense egr-1 oligonucleotide decreased serum-inducible [3H]thymidine incorporation as well as the stimulation of cell proliferation and repair after injury. Egr-1 induction was critical for PDGF-induced mitogenic signaling in mesangial cells, and inhibition of Egr-1 using antisense egr-1 oligonucleotide in vivo decreased cell proliferation in experimental glomerulonephritis (32). BKS-2 (B cell lymphoma) cells treated with antisense egr-1 oligodeoxynucleotide underwent growth arrest and apoptosis, suggesting that the expression of Egr-1 is important not only for the growth of but also for the survival of BKS cells (10).

In the present study, we tested the hypothesis that egr-1 plays a critical role in multifactorial duodenal ulcerogenesis because its protein, transcription factor Egr-1, regulates the expression of angiogenic growth factors like bFGF, PDGF, and VEGF. We investigated the specificity of the cysteamine effect compared with its chemical analog ethanolamine on the time-dependent expression of egr-1 gene and protein and synthesis and activity of bFGF, PDGF, VEGF, and ERK1/2 in the duodenal mucosa before the morphological development of ulcers. Furthermore, we studied whether inhibition of Egr-1 expression by egr-1 antisense oligodeoxynucleotide might affect the cysteamine-induced duodenal ulcer formation in vivo and the expression of bFGF, PDGF, and VEGF in the ulcerated rat duodenal mucosa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal experiments. This study was approved by the Animal Study Subcommittee of the Veterans Affairs Medical Center in Long Beach, CA. Duodenal ulcers were induced in female Sprague-Dawley rats (180–210 g) by an intragastric administration of cysteamine HCl (Aldrich; Milwaukee, WI). Rats had unlimited access to food and water. Randomized groups of rats (n = 5–7) were given either saline or cysteamine HCl (25 mg/100 g) by gavage once and euthanized 0.5, 1, or 2 h later. In other groups, cysteamine was given twice (4-h interval), and rats were euthanized 6 h later. Animals from the next group received three cysteamine treatments at 4-h intervals, and rats were euthanized 12, 24, or 48 h after the first dose of cysteamine. Because our previous structure-activity studies (42, 43) demonstrated that nonulcerogenic analogs could be used as appropriate controls to separate ulcerogenic and toxic effects, we performed additional experiments in which groups of rats were given the nonulcerogenic structural analog of cysteamine (HS-CH2-CH2-NH2)-HCl, i.e., ethanolamine (HO-CH2-CH2-NH2)-HCl (21.47 mg/100 g), once, twice, or three times (as above), and animals were euthanized. In the ulcer studies, all rats were given cysteamine three times at 4-h intervals, and rats were euthanized 48 h after first dose. Groups of rats (n = 7–10) were pretreated with saline, antisense egr-1 oligonucleotide (phosphothioate-modified and HPLC-purified oligodeoxynucleotides, 5'-GCGGGGTGCAGGGGCACACT-3', –118 to –90), or the respective scrambled control (5'-AGGCTGGCTGCCGGGAGCGA-3', Research Genetics; Huntsville, AL; or MoleculA; Sterling, VA) subcutaneously at 0.02 (n = 7) or 0.2 mg/rat (n = 10) 30 min before every dose of cysteamine. This sequence of egr-1 antisense oligonucleotide, according to other studies (2, 12), induced the prominent inhibition of Egr-1 synthesis and cell proliferation.

Gross and histological evaluations of duodenal ulcers. Rats were euthanized by CO2 inhalation and cervical dislocation. Duodenal ulcer crater dimensions were measured in millimeters, and ulcer areas were calculated using the ellipsoid formula. The opened stomachs with 2.5-cm duodenum were fixed in 10% formalin. Sections were embedded in paraffin and stained with hematoxylin and eosin. Mucosal scrapings of 2.5 cm of the proximal duodenum, glandular stomach, and jejunum from some animals were harvested and frozen (–80°C) for study of gene and protein expressions.

Real-time RT-PCR. The total RNA isolation kit (Clontech Laboratories; Palo Alto, CA) was used for total RNA extraction and purification. Real-time RT-PCR experiments were performed using TaqMan gene expression assays (Applied Biosystems; Forest City, CA) for selected genes such as egr-1. For a reaction volume of 20 µl, 1 µl of RT product was used with the TaqMan Universal PCR Master Mix and Gene Expression Assay Mix. The following cycles were used on a Bio-Rad iCycler Real-Time PCR machine: 2 min at 50°C and 10 min at 95°C and 40°C cycles of two steps: 15 s at 95°C and 1 min at 60°C. All assays were repeated with highly reproducible results using RNA from different rats. The level of target gene mRNA (egr-1) measured by threshold cycle number was compared with GAPDH, which was used as an internal control to correct variability in starting mRNA concentration. The fold changes of treated groups over control group were calculated.

RT-PCR. RT-PCR was performed with a SuperScript One-Step RT-PCR kit (GIBCO-BRL; Carlsbad, CA). From each sample, 1 µg of total RNA was amplified with the following primers (Sigma Genosys; Woodlands, TX): Egr-1 5'-GAGCCGAGCGAACAACCCTACGAGCACCTG-3' (sense) and 5'-GCGCCGAGGATGAAGAGGTCGGAGGATTGG-3' (antisense), producing a 253-bp product; and internal control GAPDH 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCCACCCTGTTGCTGTA-3' (antisense), producing a 452-bp fragment. The RT-PCR conditions were 1 cycle at 50°C for 30 min and 94°C for 2 min and 30 cycles at 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min. PCR products were separated on 1.5% agarose gels.

Western blot analysis. Equal amounts of proteins (25, 50, or 100 µg), which were extracted from duodenal, gastric, or jejunal mucosa in a buffer containing protease and phosphatase inhibitors (0.1 mM PMSF, 40 µg/ml leupeptin, 40 µg/ml aprotinin, 20 µg/ml pepstatin, and 1 mM sodium orthovanadate), were subjected to 7.5%, 10%, or 12% SDS-PAGE analysis. Proteins were transferred onto Hybond-ECL nitrocellulose membranes (Amersham Biosciences; Buckinghamshire, UK) and then incubated for 1–3 h at 20°C with rabbit polyclonal anti-Egr-1, ERK1, ERK2, FGF-2, PDGF-B, or VEGF or mouse monoclonal anti-phospho-ERK1/2 (Tyr204) antibodies (Santa Cruz Biotechnology; Santa Cruz, CA; dilution 1:500). The loading controls were performed by using a mouse monoclonal antibody to GAPDH (EnCor Biotechnology; Alachua, FL; dilution 1:2,000). Blots were treated with secondary antibodies and visualized using the ECL detection system (Amersham Biosciences). The density of protein bands in Western blots was assessed by a digital imaging system, Eagle Eye II (Stratagene; Austin, TX). Each experiment was repeated four times.

Statistical analysis. The statistical significance of changes was determined by the nonparametric Mann-Whitney U-test. Differences resulting in P values of <0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Early increase of egr-1 mRNA and protein expression in the duodenal mucosa of cysteamine-treated rats. The results demonstrated that the duodenal ulcerogen cysteamine increased egr-1 gene activity in the duodenal mucosa by 3.8- and 12.4-fold at 0.5 and 12 h after the administration of cysteamine, respectively (Fig. 1A). The time-dependent increased expression of the egr-1 gene was followed by elevated Egr-1 protein expression in the duodenal mucosa in the early stage of cysteamine-induced duodenal ulcers (Fig. 1B).


Figure 1
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  		Fig. 1. Early growth response factor (egr)-1 gene and protein expressions in the preulcerogenic stages of cysteamine-induced duodenal ulceration in rats. A: increased expression of egr-1 mRNA after cysteamine treatment by real-time RT-PCR. The level of target gene mRNA (egr-1) measured by threshold cycle number was normalized by GAPDH, which was used as an internal control to correct variability in starting mRNA concentration. The fold changes of treated groups over the control group were calculated. B and C: time-dependent expression of Egr-1 protein in the duodenal mucosa after cysteamine (B) and after its toxic analog ethanolamine (C). GAPDH detection was used as an additional control. Assays were repeated 3 times with highly reproducible results using RNA or protein from 3 different rats/group.

 
Comparison of Egr-1 expression induced by cysteamine and structurally related ethanolamine. Ethanolamine is a toxic analog of the duodenal ulcerogen cysteamine. To establish the specificity of Egr-1 in duodenal ulcers, we compared Egr-1 expression in the duodenal mucosa of rats treated with cysteamine or ethanolamine. These results demonstrated that the nonulcerogenic analog of cysteamine, ethanolamine, did not change the expression of Egr-1 in the early stage of ulcer development (Fig. 1C).

Effect of cysteamine on bFGF, PDGF, and VEGF expression. The first dose of cysteamine did not change the expression of bFGF in the duodenal mucosa. The expression of bFGF was slightly elevated at 6 h and significantly increased after three doses of cysteamine at 12 h. Enhanced bFGF expression was detected at the time of duodenal ulcer appearance (24–48 h) after cysteamine (Fig. 2A). The expression of PDGF and VEGF was enhanced soon after first dose of cysteamine and seemed to peak at 12 h after cysteamine (Fig. 2, B and C).


Figure 2
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  		Fig. 2. Representative Western blot analysis of bFGF, PDGF, and VEGF expressions in the rat duodenal mucosa after cysteamine. Time courses of the expressions of bFGF (A), PDGF (B), and VEGF (C) in the proximal duodenal mucosa of control rats and rats after cysteamine treatment are shown. Data depicted are means ± SE of 3 rats/group. *P < 0.05.

 
Antisense egr-1 oligonucleotide aggravated cysteamine-induced duodenal ulcers. To determine the role of Egr-1 in cysteamine-induced duodenal ulceration, rats were pretreated with saline or phosphothioate-modified antisense egr-1 or scrambled control oligodeoxynucleotides either at 0.02 or 0.2 mg/rat 30 min before each cysteamine administration. Scrambled control and the small dose of antisense egr-1 oligonucleotides did not alter the size of cysteamine-induced duodenal ulcers. Scrambled control oligodeoxynucleotides did not cause any change in duodenal ulcer appearance in cysteamine-treated rats compared with control saline-pretreated animals, implicating no toxicity of the oligodeoxynucleotide. The larger antisense egr-1 oligonucleotide dose aggravated duodenal ulcer formation and increased the size of ulcers from 8.1 ± 1.8 to 20.7 ± 4.0 mm2 (P < 0.01; Fig. 3, A–C). Histological evaluation revealed mostly superficial ulcers in the proximal duodenum of rats treated with scrambled control and cysteamine, whereas deep ulcers penetrating into the Brunner glands, often with perforation, were seen in rats given cysteamine and pretreated with egr-1 antisense oligonucleotide (Fig. 3C).


Figure 3
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  		Fig. 3. Effect of egr-1 antisense oligodeoxynucleotide on cysteamine-induced duodenal ulcers in rats. A: gross appearance of duodenal ulcers 48 h after cysteamine administration. Animals were pretreated with either saline (S; n = 10), scrambled control oligodeoxynucleotide (SC; n = 7), or antisense egr-1 (phosphothioate-modified) oligodeoxynucleotide at 0.2 mg/rat (AS; n = 10). B: light microscopy of duodenal ulcers in rats pretreated with either saline (showing superficial ulcer; left), scrambled control oligodeoxynucleotide (similar ulcer size; middle), or antisense egr-1 (phosphothioate-modified) oligodeoxynucleotide at 0.2 mg/rat before cysteamine (deep ulcer; right) with hematoxylin and eosin staining. C: duodenal ulcer crater dimensions were measured in millimeters, and the ulcer areas were calculated using the ellipsoid formula. Sizes of duodenal ulcers are expressed as means ± SE. **P < 0.01.

 
Time-dependent effect of antisense egr-1 oligonucleotide on Egr-1 protein expression in the rat duodenal mucosa. The Western blot results demonstrated that the expression of Egr-1 at 6 h after cysteamine in rats pretreated with saline or scrambled egr-1 oligodeoxynucleotide was 56% or 70% higher than in saline-treated control animals (Fig. 4, A and B). There were no differences in Egr-1 expression in rats that were given saline or scrambled oligonucleotide before cysteamine. Antisense egr-1 oligodeoxynucleotide inhibited Egr-1 protein expression by 70%, 35%, and 32% at 6, 12, and 24 h, respectively, after cysteamine.


Figure 4
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  		Fig. 4. Time-dependent expression of Egr-1 protein, ERK1 (p44) and ERK2 (p42) kinases, and phosphorylated ERK1/2 in the duodenal mucosa of rats pretreated with saline, scrambled control oligodeoxynucleotide, or antisense egr-1 oligodeoxynucleotide before cysteamine compared with rats treated with saline only. A: representative Western blots of the time course of duodenal Egr-1 protein expression in saline-treated animals and in rats pretreated with saline, scrambled control oligodeoxynucleotide, or egr-1 antisense oligodeoxynucleotide (0.2 mg/rat) 0.5 h before cysteamine. B: quantified data of time-dependent Egr-1 protein expression in control duodenal mucosa (saline treatment only) or in duodenal mucosa of rats pretreated 0.5 h before each dose of cysteamine with saline, scrambled control oligodeoxynucleotide, or egr-1 antisense (phosphothioate-modified) oligodeoxynucleotide at 0.2 mg/rat. Assays were repeated 3 times. Data depicted are means ± SE of 3 rats/group. *P < 0.05. C: representative Western blots of ERK1 (p44) and ERK2 (p42) kinase in the same groups of animals. D: detection of ERK1/2 activation in preulcerogenic duodenal mucosa of animals pretreated with saline, scrambled control oligodeoxynucleotide, or egr-1 antisense oligodeoxynucleotide before cysteamine compared with rats treated with saline only. ERK activation was measured using a phosphospecific anti-ERK antibody, which specifically recognizes ERK after catalytic activation by phosphorylation (Tyr204).

 
Cysteamine activates ERK1/2 in the rat duodenal mucosa. We measured ERK1/2 expression and activity in the same samples from the duodenal mucosa of rats treated with only saline and rats pretreated with saline, scrambled control oligonucleotide, or antisense egr-1 oligonucleotide before each cysteamine dose. Figure 4C demonstrates that the expression of ERK1 (p44) and ERK2 (p42) kinases was not changed in rats treated with saline or pretreated with saline, scrambled control oligodeoxynucleotide, or egr-1 antisense oligodeoxynucleotide before cysteamine. ERK activation was measured using a phosphospecific anti-ERK antibody, which specifically recognizes ERK after catalytic activation by phosphorylation (Tyr204). The results demonstrated that cysteamine activated ERK1/2 kinases at 6, 12, and 24 h in the duodenal mucosa of rats pretreated with saline or scrambled control compared with rats given saline only (Fig. 4D). Cysteamine treatment did not change the level of total ERK (Fig. 4C), suggesting that ERK activity in our model was the result of catalytic phosphorylation by MEK rather than by changes in total ERK protein expression. Interestingly, we detected an inhibition of ERK phosphorylation in the duodenal mucosa of all rats pretreated with egr-1 antisense oligonucleotide compared with saline-treated or scrambled control-pretreated animals (Fig. 4D).

Egr-1 antisense oligonucleotide decreased the expression of Egr-1 mRNA and protein in the duodenal mucosa of rats with formed ulcers. In our model of duodenal ulceration, cysteamine leads to the development of severe ulcers at 48 h after treatment. The egr-1 antisense oligonucleotide significantly aggravated duodenal ulcer formation, and we measured the changes in egr-1 mRNA and protein expression at 48 h after cysteamine treatment. To investigate the effect of antisense egr-1 oligonucleotide on egr-1 mRNA, we performed RT-PCR analysis. Scrambled oligodeoxynucleotide did not change egr-1 mRNA expression compared with saline-treated rats at 48 h after first dose of cysteamine, whereas the antisense egr-1 oligodeoxynucleotide inhibited egr-1 mRNA expression in the duodenal mucosa (Fig. 5A). The antisense egr-1 oligodeoxynucleotide inhibited Egr-1 protein expression in the duodenal mucosa by ~50%, whereas protein levels did not markedly change in the gastric and jejunal mucosa (Fig. 5B).


Figure 5
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  		Fig. 5. Effect of antisense egr-1 oligodeoxynucleotide on egr-1 mRNA and Egr-1 protein expressions in the duodenal mucosa of cysteamine-treated rats. A: RT-PCR transcript analysis of egr-1 gene expression in the rat duodenal mucosa 48 h after cysteamine. Animals were pretreated 0.5 h before each dose of cysteamine with saline, scrambled control oligodeoxynucleotide, or antisense egr-1 (phosphothioate-modified) oligodeoxynucleotide at 0.2 mg/rat. B: representative Western blot anaylsis of Egr-1 expression in the proximal duodenal, gastric, and jejunal mucosa 48 h after cysteamine administration. Animals were pretreated 0.5 h before each cysteamine dose with saline, scrambled control oligodeoxynucleotide, or antisense Egr-1 (phosphothioate-modified) oligodeoxynucleotide at 0.2 mg/rat.

 
Effect of antisense egr-1 oligonucleotide on expression of bFGF, PDGF, and VEGF in the duodenal mucosa of rats treated with cysteamine. Western blots of bFGF expression showed that scrambled control oligonucleotide (two different doses) and the low dose of antisense egr-1 oligonucleotide (0.02 mg/rat) did not alter bFGF protein expression in the rat duodenal mucosa at 48 h. Antisense egr-1 oligonucleotide, at 0.2 mg/rat, however, decreased bFGF expression in the duodenal mucosa by 2.3-fold (Fig. 6A). PDGF and VEGF protein levels in the duodenal mucosa in rats pretreated with antisense egr-1 oligonucleotide were also significantly decreased (Fig. 6, B and C), e.g., the expression of PDGF in the duodenum was decreased by antisense egr-1 oligonucleotide by 43–50%. The VEGF level in the ulcerated duodenal mucosa was only slightly decreased by 0.02 mg/rat antisense egr-1 oligonucleotide but was significantly decreased (55%) by the 0.2 mg/rat dose of antisense egr-1 oligonucleotide.


Figure 6
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  		Fig. 6. Representative Western blot analysis of bFGF (A), PDGF (B), and VEGF (C) expressions in the proximal duodenal mucosa 48 h after administration of the duodenal ulcerogen cysteamine. Animals were pretreated 0.5 h before the each dose of cysteamine with saline, scrambled control oligodeoxynucleotide, or antisense egr-1 (phosphothioate-modified) oligodeoxynucleotide at 0.02 or 0.2 mg/rat. Data depicted are means ± SE of 3 rats/group. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrated, for the first time, that egr-1 antisense oligonucleotide significantly aggravated experimental duodenal ulcers and inhibited the expression of egr-1 mRNA and protein and bFGF, PDGF, and VEGF synthesis in the rat duodenal mucosa. Ethanolamine, a nonulcerogenic analog of cysteamine, did not alter Egr-1 expression in the duodenal mucosa. Thus these are specific, duodenal ulcerogenic effects of cysteamine, because these are not shared by the toxic analog ethanolamine, underlining a critical role of Egr-1 in duodenal ulceration. Furthermore, Egr-1 antisense oligonucleotide did not affect Egr-1 protein expression in the stomach or jejunum. Our results also demonstrated that cysteamine activated ERK1/2 kinases without any changes in protein expressions in the rat duodenal mucosa.

The Egr-1 transcription factor belongs to hypoxia-inducible transcription factors, and recently we (20) demonstrated that cysteamine induced hypoxia only in the proximal duodenum but not in the stomach. The cysteamine-induced Egr-1 transcriptional activity, as we (19) demonstrated previously, might result in increased expression of bFGF and PDGF in the duodenal mucosa, because the promoter region of bFGF and PDGF genes contains the Egr-1 binding site (3, 18). Our results show that cysteamine enhanced bFGF and PDGF synthesis in preulcerogenic stages, and the decreased Egr-1 expression in the duodenal mucosa might be the cause for the inhibition of synthesis of these growth factors. Recently, we demonstrated that cysteamine enhanced the ability of HIF-1{alpha} to interact with Egr-1. The increase of HIF-1{alpha} binding to hypoxia-responsive elements of the promoter region of the VEGF gene was shown in response to hypoxia (22). Cysteamine increased the expression and transcriptional activity of HIF-1{alpha} (20) and enhanced the transcriptional interaction of HIF-1{alpha} with Egr-1. This increased interaction in preulcerogenic stages apparently might participate in mechanisms of enhanced VEGF expression in the duodenal mucosa, e.g., the downregulation of Egr-1 expression and its interaction with HIF-1{alpha} might suppress VEGF synthesis. Cysteamine is a reducing aminothiol and changed the redox state in the rat proximal duodenal mucosa (20). This ability of cysteamine might also participate in the activation of redox-sensitive transcription factors including Egr-1 and HIF-1{alpha}, increase their protein-protein interaction through reducing cysteine residues, and enhance the activation of their target genes bFGF, PDGF, and VEGF.

The transcription factor Egr-1 has nucleotide recognition elements in the promoters of many pathophysiologically relevant genes. The mechanisms underlying the inducible expression of Egr-1 are not clear. Egr-1 was expressed in endothelial cells from the wound edge within minutes of injury (34). Early Egr-1 expression in small intestinal epithelial IEC-6 cells, derived from fetal rat duodenal crypt cells after monolayer injury, was ERK dependent, and inhibition of ERK activity decreased Egr-1 mRNA expression (5). Overexpression of Egr-1 protein was prevented with the MAPKK inhibitor PD-98059, indicating that MAPK ERK is involved in the regulation of Egr-1 expression in renal cells (11). Hypoxia-reoxygenation induces ERK1/2 phosphorylation as well as transactivation of the transcription factors NF-{kappa}B and Egr-1 in pulmonary artery endothelial cells (6). Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of the pulmonary arterial wall and increases in tissue mass and total RNA, JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities. Hypoxia also upregulated VEGF mRNA and PDGF receptor mRNA levels in the pulmonary artery with a time course that correlated with the activation of ERK and upregulated egr-1 mRNA (16).

Cysteamine-induced ERK activation in the duodenal mucosa in the preulcerogenic stage may participate in the increased Egr-1 expression and DNA binding activity, resulting in enhanced synthesis of growth factors in the preulcerogenic duodenal mucosa. Our study also demonstrates that the inhibition of Egr-1 synthesis induced by antisense egr-1 oligonucleotide leads to a partial decrease in ERK phosphorylation at 6, 12, and 24 h after cysteamine administration. Egr-1 might participate in the early increase of growth factor synthesis in the preulcerogenic mucosa. Growth factors (e.g., PDGF and bFGF) activate ERK (26–28, 30). Thus the inhibition of Egr-1 expression might decrease growth factor synthesis and result in decreased ERK activity and profound inhibition of growth factor synthesis. This may explain our observation that egr-1 antisense oligonucleotide delayed not only growth factor synthesis but also inhibited ERK phosphorylation induced by cysteamine.

Studies (33, 34) with antisense egr-1 oligodeoxynucleotides have suggested that Egr-1 plays a key regulatory role in smooth muscle cell proliferation and repair after injury. These findings demonstrate that inducible Egr-1 expression after injury is dependent on the release and paracrine action of growth factors. A DNA enzyme targeting egr-1 mRNA has been shown to inhibit vascular smooth muscle proliferation and regrowth after injury (35). A protective function of inducible Egr-1 expression in cell survival was shown after ultraviolet irradiation and egr-1 antisense oligonucleotide reduced the cell growth rate (13). Damage of IEC-6 cell monolayers resulted in rapid and significant increases in Fos and egr-1 mRNA levels, and direct interference with egr-1 by the expression of a dominant negative mutant led to a significantly reduced in vitro monolayer restitution (5).

The angiogenic growth factors VEGF, PDGF, and bFGF may play a role in ulcer development and healing not only by regulating angiogenesis but also by promoting the survival of different cells, e.g., lowering the synthesis of growth factors may increase the susceptibility to apoptosis. VEGF significantly reduced the apoptotic chemotherapeutic damage in endothelial cells, and overexpression of a VEGF dominant-interfering mutant abrogated the protective effect of VEGF (48). VEGF and bFGF synergistically enhanced endothelial cytoprotection (25). bFGF, produced by hearts subjected to ischemia, is a protective factor against myocardial cell apoptosis (15). FGF-2 is a potent cell survival factor and protected human neuroblastoma cells from nitric oxide-mediated apoptosis without affecting cell proliferation (51).

The importance of growth factor synthesis in the proximal duodenum was demonstrated recently in mice lacking epidermal growth factor (EGF), transforming growth factor-{alpha}, and amphiregulin (49). Triple-null neonates displayed spontaneous duodenal lesions. Adult EGF–/– mice displayed more severe lesions in response to cysteamine treatment compared with wild-type counterparts.

Our study demonstrates that egr-1 is a key gene in the multifactorial mechanisms of cytoprotection and healing of duodenal ulcers because its transcription factor product Egr-1 regulates the expression of angiogenic growth factors. Namely, egr-1 antisense oligonucleotide significantly aggravated experimental duodenal ulcers and inhibited the expression of Egr-1 mRNA and protein as well as the synthesis of bFGF, PDGF, and VEGF in the rat duodenal mucosa. Thus the egr-1 gene and Egr-1 transcription factor seem to be important molecules in the pathogenesis of experimental duodenal ulceration.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
The present study was supported by a Department of Veterans Affairs Medical Research Service Merit Review grant.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Szabo, Diagnostic and Molecular Medicine Health Care Group, Veterans Affairs Medical Center, 5901 E. 7th St., Long Beach, CA 90822-5201 (e-mail: sandor.szabo{at}med.va.gov)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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