Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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Epidermal growth factor (EGF) is involved in acute regulation of Na+/H+ exchangers (NHEs), but the effect of chronic EGF administration on NHE gene expression is unknown. The present studies showed that EGF treatment increased NHE2-mediated intestinal brush-border membrane vesicle Na+ absorption and NHE2 mRNA abundance by nearly twofold in 19-day-old rats. However, no changes were observed in renal NHE2 mRNA or intestinal and renal NHE3 mRNA abundance. To understand the mechanism of this regulation, we developed the rat intestinal epithelial (RIE) cell as an in vitro model to study the effect of EGF on NHE2 gene expression. EGF increased functional NHE2 activity and mRNA abundance in cultured RIE cells, and this stimulation could be blocked by actinomycin D (a transcriptional inhibitor). Additionally, NHE2 promoter reporter gene assays in transiently transfected RIE cells showed an almost twofold increase in promoter activity after EGF treatment. We conclude that rat NHE2 activity can be stimulated by chronic EGF treatment and that this response is at least partially mediated by gene transcription.
gene regulation; rat intestine; sodium-hydrogen exchanger isoform-2; rat intestinal epithelial cells
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INTRODUCTION |
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SODIUM IS A MAJOR BLOOD PLASMA electrolyte that plays important roles in body volume and acid-base regulation. The Na+/H+ exchangers (NHEs) are plasma membrane-bound antiporters that mediate the movement of extracellular Na+ into cells in exchange for intracellular H+. Six NHE isoforms have been identified from mammalian cells (15, 42, 59). These proteins play important roles in regulating intracellular pH (40, 46), cell volume and cellular proliferation (53, 57), and vectoral Na+ absorption across epithelia (14, 45, 54, 56, 58).
Many physiological factors regulate NHE activity. Osmotic changes stimulate or inhibit NHE activity (3, 55, 56, 60). Na+ content of diet also affects NHE activity (30). Additionally, glucocorticoids stimulate NHE activity (1, 5, 8, 33, 61), and parathyroid hormone inhibits NHE activity (2, 10, 21, 62).
Epidermal growth factor (EGF), as an important physiological regulator, also regulates NHE activity. EGF is a 53-amino acid polypeptide secreted predominantly by salivary glands, with lower-level secretion by the kidney and many other tissues. EGF receptors are expressed along the intestinal tract (7), and EGF has broad physiological effects on cell division, DNA synthesis, tissue proliferation, cellular differentiation, and electrolyte and nutrient absorption (29, 43, 44). Furthermore, EGF was shown to stimulate NHE activity in the gastrointestinal tract and other organs (24-26, 28, 31, 38, 47, 49, 50). These observations suggest that EGF may play an important role in the functional regulation of NHEs. Several of these studies showed that the increase in NHE activity induced by acute exposure to EGF is mediated by protein phosphorylation mechanisms (18, 24, 25, 32, 34, 52). However, there is no evidence showing direct EGF regulation of NHE gene expression.
As reported here, we initially detected a significant increase in intestinal brush-border membrane vesicle (BBMV) Na+ uptake mediated by NHE2 and NHE2 mRNA abundance in rats after 3 days of EGF treatment. These results suggested a possible role for EGF in transcriptional regulation of the NHE2 gene. Therefore, to understand the role of EGF in the regulation of NHE2 gene expression in the intestine, we extensively characterized NHE2 expression and function in rat intestinal epithelial (RIE) cells and used this cell line as an in vitro model to determine the molecular mechanism of gene regulation. These are the first studies that exemplify transcriptional effects on NHE2 gene expression induced by EGF.
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MATERIALS AND METHODS |
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Animals. Suckling Sprague-Dawley rats (16 days old) received subcutaneous injections of human recombinant EGF (1 µg/g body wt; Austral Biologicals, San Ramon, CA) or saline twice a day for 3 days. At 15 h after the last injection, rats were killed, and jejunal mucosa and kidney were harvested and used for mRNA purification and BBMV isolation. All animal work was approved by the University of Arizona Institutional Animal Care and Use Committee.
Cell culture. RIE cells were a gift from Dr. Raymond DuBois (Dept. of Medicine, Vanderbilt University School of Medicine, Nashville, TN). RIE cells were cultured in DMEM supplement with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were cultured at 37°C in a 95% air-5% CO2 atmosphere and passaged every 48-72 h. Media and other reagents used for cell culture were purchased from Irvine Scientific (Irvine, CA). In the EGF treatment experiments, the cells were incubated in the presence of human recombinant EGF (100 ng/ml) for 4 h before they were harvested. For transcriptional assays, cells were pretreated with actinomycin D (5 µg/ml) (Calbiochem-Novabiochem, San Diego, CA) for 2 h and then treated with EGF for 4 h in the presence of actinomycin D before they were harvested. To study the time course of NHE2 gene expression, RIE cells were seeded at 1 × 104 cells/cm2 surface area. Cells were harvested at different time points (days 1, 3, and 5 after seeding) for mRNA purification.
RNA purification and Northern blot analyses. mRNA was isolated from RIE cells, rat jejunal mucosa, and renal cortex using the Fast-Track mRNA purification kit (Invitrogen, Carlsbad, CA). Under high-stringency washing conditions (11), mRNA (5-10 µg) was utilized for Northern blot analyses with rat NHE2- and NHE3-specific cDNA probes [BamHI- and BglII-digested fragment for NHE2 or DraI- and KpnI-digested fragment for NHE3 (14)]. 1B15 [encoding rat cyclophilin (17)]-specific cDNA probes were used as internal standards for quantitating NHE2 and NHE3 gene expression. Blots were exposed to a phosphor imaging screen, and band intensities were determined with Quantity One Software (FX Molecular Imager, Bio-Rad, Hercules, CA).
Na+ uptake analysis in BBMVs. BBMVs were prepared from rat jejunal mucosa, and 22Na+ uptake was determined as previously described (12, 14). The contribution of NHE2 to total Na+ uptake was calculated by subtracting the uptake observed in the presence of 50 µM HOE-694 (the concentration used to inhibit NHE2 activity) from the total uptake in the absence of HOE-694 (12, 14). HOE-694 [(3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate] was kindly provided by Dr. H. J. Lang (Hoechst Marion Roussel Pharmaceuticals, Frankfurt am Main, Germany). HOE-694 selectively inhibits NHE1 with an inhibition constant (Ki) of 0.16 µM in PS120 fibroblast cells, NHE2 with a Ki of 5 µM in PS120 fibroblast cells, and NHE3 with a Ki of 650 µM in PS120 fibroblast cells (16).
PCR analysis to detect NHE expression in RIE cells.
mRNA was purified from RIE cells cultured in normal medium. RT-PCR
conditions were identical to those described in a previous publication
(3). The primers used to detect NHE and -actin mRNA
expression and the expected PCR product sizes are listed in Table
1.
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Semiquantitative RT-PCR analysis of NHE2 gene expression. mRNA was purified from RIE cells treated with standard or EGF-containing medium (100 ng/ml). RT-PCR conditions were described previously (3). Subsaturation levels of cDNA templates that were needed to produce a dose-dependent amount of PCR product were defined in initial experiments by testing a range of template concentrations. Subsequent PCR was carried out with subsaturation levels of RT reactions with identical amplification parameters.
Characterization of Na+ dependence of intracellular pH recovery in RIE cells. Na+-dependent pH recovery in RIE cells was analyzed by measuring the rate of intracellular pH (pHi) recovery in HEPES-buffered saline solution (HBSS; in mM: 5 KCl, 0.3 KH2PO4, 138 NaCl, 0.2 NaHCO3, 0.3 Na2HPO4, 10 HEPES, 1.3 CaCl2, 0.4 MgSO4, 5.6 glucose, and 5 glutamine, pH 7.4) after an acid load in the presence or absence of Na+. Na+-free HBSS contains 138 mM methylglucamine, instead of NaCl, NaHCO3, and Na2HPO4. The pHi recovery was measured as described below.
Calculation of NHE activity in RIE cells by measuring
pHi changes.
Functional NHE activity was monitored by following the
Na+-dependent recovery of pHi after acid
loading in HCO Rmin)/(Rmax
R)], where R represents
the experimentally measured ratio of 640 nm/570 nm, Rmin is
the ratio measured at the most acidic pH, and Rmax is the
ratio measured at the most basic pH. At the conclusion of each
experiment, Rmin and Rmax were assessed for
each individual cell by adding media with nigerin and valinomycin in
high K+ (36) and measuring the SNARF-1 ratio
at pH 6.8 and 8.2, respectively. This procedure allows for
normalization of estimated pH between individual cells.
Construction of reporter plasmids.
Reporter plasmids used in this study were derived from pGL3/basic
(Promega), which contains the firefly luciferase gene. The promoter
reporter constructs pGL3/2,630 bp, pGL3/
1,271 bp, pGL3/
110 bp,
pGL3/
36 bp, and pGL3/+2 bp were made by restriction enzyme digestion
(39) and by DNA deletion methods and PCR (3,
4). The 3' end of all constructs was at +116 bp of the rat NHE2
gene. The pGL3/+2 bp construct is a negative control that does not
contain the transcriptional start site. All constructs were confirmed by sequencing on both strands.
Transient transfection and functional promoter analysis. RIE cells were cultured in 24-well plates. When cells reached 60% confluence, liposome-mediated transfection was performed as follows: 0.5 µg of promoter construct DNA, 30 ng of pRL-CMV (Renilla luciferase reporter construct used as an internal standard; Promega), and 5 µl of Lipofectamine (GIBCO/BRL, Grand Island, NY) were mixed with 200 µl of Opti-MEM (GIBCO/BRL) for 30 min at room temperature. The mixture was added to the cells, the cells were incubated for 5 h, and then an equal volume of DMEM containing 20% FBS was added. On the next day, the medium was removed and replaced with standard medium with 10% FBS. After 24 h, cells were harvested for reporter gene assays. For EGF treatment, 100 ng/ml human recombinant EGF (Austral Biologicals) was added for 4 h before the cells were harvested. Promoter reporter assays were performed using the Dual Luciferase Assay Kit according to the manufacturer's instructions (Promega).
Statistical analysis. The Student's t-test was used to compare values of the experimental data. P < 0.05 was considered significant.
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RESULTS |
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Effect of EGF treatment on NHE2 activity in rat jejunum.
pH-dependent uptake of Na+ was assayed in rat jejunal BBMV
in EGF- or saline-injected groups in the presence of 0 or 50 µM HOE-694 (Fig. 1). Na+ uptake
with 0 µM HOE-694 (representing total uptake) was increased ~60%
by EGF treatment. Na+ uptake with 50 µM HOE-694
(representing NHE3-mediated uptake) was also increased ~30% by EGF
treatment. However, the increase in total Na+ uptake
induced by EGF was significantly larger than the increase in
NHE3-mediated Na+ uptake (Fig. 1A;
P < 0.04, n = 3). By subtracting the
uptake observed in the presence of 50 µM HOE-694 from the total
uptake observed in the absence of HOE-694, NHE2-mediated uptake was
calculated. Data showed that the NHE2 activity increased by almost
twofold after EGF administration in rat small intestine (Fig.
1B; P = 0.026, n = 3).
Therefore, the vast majority of the observed increase in BBMV
Na+ uptake induced by EGF could be ascribed to NHE2
(~80% of the increase was from NHE2 and 20% from NHE3).
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Effect of EGF treatment on NHE2 and NHE3 mRNA levels in rat jejunum
and kidney.
Hybridization with rat NHE2-specific cDNA probes clearly showed that
intestinal NHE2 mRNA abundance was increased approximately twofold
after EGF administration (Fig.
2A; n = 3, P = 0.013), while there was no change in renal NHE2
abundance (Fig. 2B; n = 3). Furthermore,
hybridization with rat NHE3-specific cDNA probes showed that NHE3 mRNA
abundance was not changed by EGF administration in intestine or kidney
(Fig. 2, C and D; n = 3).
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Characterization of NHE2 expression in RIE cells.
Northern blot was performed by loading 10 µg of mRNA isolated from
RIE cells on the gel. Hybridization with rat NHE2-specific cDNA probes
did not show a hybridization signal in untreated or treated cells.
However, -actin mRNA hybridization signal was easily detectable
(data not shown). This result suggested that NHE2 message levels were
too low to be detected by Northern analyses. Therefore, an alternative
method was selected.
RT-PCR analysis of NHE expression in RIE cells.
Endogenous NHE expression in RIE cells was confirmed by RT-PCR
using rat NHE isoform-specific primers (Fig.
3). These results showed that NHE1 mRNA,
but not NHE3 and NHE4 mRNAs, could be detected in RIE cells after
24 h of culture (Fig. 3A). Even after 72 h of
culture, NHE3 and NHE4 mRNAs still could not be detected by RT-PCR
(data not shown). NHE2 mRNA expression could be detected 24 h
after seeding (Fig. 3B). These results indicated that RIE cells endogenously express the NHE1 and NHE2 genes and that expression of NHE2 does not require postconfluent culture and cellular
differentiation.
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Characterization of NHE activity in RIE cells.
NHE activity in RIE cells was assessed by measuring the recovery of
pHi after an acid load. The pHi recovery after
acid load in RIE cells is Na+ dependent (Fig.
4A), which
suggested the involvement of NHEs (n = 10 cells,
P < 0.001). Further studies showed that NHE activity could be inhibited by HOE-694 (Fig. 4B). HOE-694 at 1 µM,
which was used to inhibit NHE1 activity, inhibited total NHE activity by ~60% in RIE cells. HOE-694 at 20 µM, which was used to inhibit NHE1 and NHE2 activity, almost completely blocked pH recovery in RIE
cells (n = 15-20 cells, P < 0.001).
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EGF treatment increases NHE2 mRNA abundance in RIE cells.
NHE2 mRNA expression in RIE cells, after exposure to normal or
EGF-containing medium, was assessed by semiquantitative RT-PCR using
rat NHE2-specific and -actin primers (Fig.
5). Data showed that NHE2 gene expression
increased by almost twofold in EGF-treated RIE cells compared with
untreated cells (n = 5, P < 0.001).
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EGF treatment increases NHE2 activity in RIE cells.
NHE2 activity in RIE cells, after exposure to standard or
EGF-containing medium, was studied by measuring the recovery rate of
pHi after an acid load in the presence of 1 µM HOE-694
(Fig. 6). NHE2 activity was increased by
twofold in EGF-treated RIE cells compared with untreated cells
(n = 15-20 cells, P < 0.002).
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Actinomycin D treatment blocks NHE2 mRNA increase induced by EGF
treatment in RIE cells.
To test whether the EGF effect on NHE2 gene expression is due to
transcriptional regulation, RIE cells were first treated with 5 µg/ml
actinomycin D for 2 h and then with 100 ng/ml EGF for 4 h in
the presence of actinomycin D before they were harvested. NHE2 mRNA
abundance was determined by semiquantitative RT-PCR using rat
NHE2-specific and -actin primers (Fig.
7A). Results showed that the
increase in NHE2 mRNA abundance induced by EGF treatment was abolished
by actinomycin D treatment (n = 5, P = 0.008).
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Actinomycin D blocks NHE2 activity increase induced by EGF treatment in RIE cells. To test whether the EGF effect on NHE2 activity is mediated by increased mRNA synthesis, RIE cells were treated with 5 µg/ml actinomycin D for 2 h and then with 100 ng/ml EGF for 4 h in the presence of actinomycin D before measurement of pHi recovery. NHE2 activity was determined by measuring the pHi recovery rate after acid load (Fig. 7B). Results showed that the increase in NHE2 activity induced by EGF was blocked in actinomycin D-treated cells (n = 20-30 cells, P = 0.009).
Rat NHE2 gene promoter analysis in RIE cells.
To determine whether the 5'-flanking region of the NHE2 gene is a
functional promoter in RIE cells, five constructs (pGL3/2,630 bp,
pGL3/
1,271 bp, pGL3/
110 bp, pGL3/
36 bp, and pGL3/+2 bp) were
transfected into RIE cells. Promoter reporter gene assays were
performed 48 h after transfection (Fig.
8A). The promoter assay data
showed that all promoter constructs except pGL3/+2 bp and pGL3/
36 bp
were functional in RIE cells. Compared with controls, these promoter
constructs resulted in five- to sevenfold stimulation of reporter gene
activity (n = 6, P < 0.002).
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DISCUSSION |
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EGF plays an important role in many physiological and pathophysiological processes, such as cell growth and recovery from tissue injury. EGF may also influence basic cellular homeostatic processes. For example, intestinal Na+ absorption can be enhanced by EGF treatment (26, 29), and therefore EGF may play a role in the regulation of Na+ homeostasis via increases in functional expression of NHEs. Other studies indicated that EGF increases NHE activity by posttranslational modification of NHE proteins (18, 24, 25, 32, 34, 52). However, it is not clear whether EGF stimulates NHE gene expression. In the present study, we demonstrate that EGF treatment increases NHE2-mediated Na+ absorption and NHE2 mRNA abundance in rat small intestinal tissue and cultured RIE cells. To decipher the molecular mechanism, we first characterized NHE2 expression in RIE cells and used RIE cells as an in vitro model to study NHE2 gene regulation. Then we characterized NHE2 gene promoter activity in RIE cells and studied EGF regulation of the NHE2 promoter in transiently transfected RIE cells. Our data suggest for the first time that EGF increases NHE2 mRNA abundance and activity at least partially through a gene transcription-mediated mechanism.
Although acute exposure to EGF clearly activates NHE function via protein phosphorylation (18, 24, 25, 32, 34, 52), the role of chronic exposure to EGF on NHE expression is unknown. Our NHE functional analyses demonstrated that long-term EGF treatment significantly increased NHE2-mediated Na+ uptake in rat small intestine. Furthermore, our Northern blot analyses showed that long-term EGF treatment did not alter NHE3 mRNA abundance in intestine and kidney or NHE2 mRNA abundance in kidney, but it specifically increases NHE2 mRNA abundance in intestine. This demonstrates that the EGF effect on NHE gene expression is isoform and tissue specific.
To understand the relationship between EGF and the regulation of intestinal NHE2 gene expression, we extensively characterized the RIE cell line as a model to study EGF-mediated regulation of NHE2 gene expression. RIE cells are epithelium derived and were isolated from rat small intestinal tissue (6). Our results showed that NHE1 and NHE2, but not NHE3 and NHE4, mRNAs could be detected in RIE cells, indicating that only NHE1 and NHE2 are expressed in RIE cells. NHE activity in RIE cells, as analyzed by measuring the recovery of cytosolic pH after an instantaneous acid load, is Na+ dependent and can be inhibited 60% by 1 µM HOE-694 and completely inhibited by 20 µM HOE-694. At 1 µM, HOE-694 effectively inhibits NHE1 activity (Ki = 0.16 µM in PS120 cells), and at 20 µM, HOE-694 effectively inhibits NHE2 activity (Ki = 5 µM in PS120 cells) (16). These results demonstrate that NHE1 and NHE2 are functional in RIE cells and that their activities can be differentiated by varying the concentration of HOE-694 (9, 41). Thus RIE cells are a suitable in vitro model to study intestinal NHE2 gene expression.
In vivo studies in rats and in vitro studies in RIE cells showed that EGF treatment increases NHE2 mRNA abundance by approximately twofold. Therefore, transcriptional regulation by EGF on NHE2 gene activity seems likely. Activation of NHE2 gene expression by EGF in RIE cells could be abolished by 5 µg/ml actinomycin D, a transcriptional inhibitor. Actinomycin D also inhibited the increase in NHE2 activity induced by EGF in RIE cells. These results suggest that the increase in NHE2 mRNA abundance and activity induced by EGF likely involves synthesis of new NHE2 mRNA. Furthermore, transfection studies with NHE2 promoter constructs showed that EGF increased NHE2 gene promoter activity by approximately twofold in transiently transfected RIE cells. When considered together, these data indicate that the effect of EGF on rat intestinal NHE2 activation is at least in part due to an increase in NHE2 gene transcription.
Transfection of cells with three longer NHE2 gene promoter constructs
(pGL3/2,630 bp, pGL3/
1,271 bp, and pGL3/
110 bp) significantly stimulated reporter gene expression, whereas the pGL3/
36 bp and pGL3/+2 bp promoter constructs were ineffective. This finding suggests
that the basal promoter region of the NHE2 gene is located within the
110- to
36-bp region. Interestingly, the promoter construct
pGL3/
36 bp was functional in transfected mIMCD3 cells, a mouse renal
inner medullary cell line (4), but it was not functional
in transfected RIE cells. This observation suggests that regulation of
NHE2 gene expression is different between renal and intestinal cells,
which suggests that NHE2 gene expression may be tissue or cell type
specific. Furthermore, the two larger promoter constructs (pGL3/
2,630
bp and pGL3/
1,271 bp) were responsive to EGF treatment, but the
smaller construct (pGL3/
110 bp) was unresponsive. This observation
suggests that EGF regulation of this gene is not mediated by the basal
transcriptional machinery, which is likely located within the first 110 bp upstream of the transcription initiation site. These data place the
putative EGF response element(s) between 110 and 1,271 bp upstream from
the transcriptional initiation site.
Several EGF-responsive elements have been previously identified,
including in the c-fos gene (22), the rat
preprothyrotropin-releasing hormone gene (48), the rat
prolactin gene (19), and the human gastrin gene (23,
27, 37). These EGF-responsive elements (EREs) include a serum
response element and AP-1 binding sequences in the c-fos
gene and SP-1 binding sequences in the rat preprothyrotropin-releasing hormone gene and human gastrin genes. By searching the rat NHE2 gene
promoter region from 110 to
1,271 bp for these known EREs, we found
only one sequence (
237 bp GGGCGGG
231 bp) that has high homology
with the ERE known to bind Sp1 from the rat preprothyrotropin-releasing hormone and human gastrin genes. This sequence may be responsible for
EGF regulation of the rat NHE2 gene, although further experimentation is required to make this determination.
Previous studies from our group showed that NHE2 and NHE3 each account for ~50% of intestinal Na+ absorption in suckling rats, which demonstrates that NHE2 makes an important contribution to intestinal Na+ absorption in young rats (13, 14). It is also a well-known fact that rat milk contains high levels of EGF. Therefore, our present studies suggest that EGF plays a critical role in regulating intestinal Na+ absorption via its effect on intestinal NHE2 gene expression in the suckling period.
In adult mice that underwent small bowel resection, EGF treatment increased NHE3, but not NHE2, mRNA and immunoreactive protein expression in the ileal mucosa (20). The relevance of these findings to the present investigation is, however, difficult to assess, inasmuch as no functional studies were performed. Furthermore, the two studies used different species (rats vs. mice), intestinal segments (jejunum vs. ileum), ages (sucklings vs. adults), and physiological states (normal vs. small bowel resection), which makes correlation of the data problematic.
In summary, we demonstrate that RIE cells are a suitable in vitro model to study the regulation of NHE2 gene expression. We also observed that EGF treatment increased NHE2 mRNA abundance and activity in rat intestine and RIE cells and also increased NHE2 gene promoter activity in transfected RIE cells. Because actinomycin D treatment blocked the EGF-induced increases in NHE2 gene expression and function in RIE cells, a transcriptional mechanism is likely involved in EGF regulation of NHE2 gene expression in the mammalian intestine. Further studies will focus on identification of the ERE(s) and the transcription factors involved in EGF regulation of the rat NHE2 gene.
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ACKNOWLEDGEMENTS |
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We thank Michael S. Inouye and Kevin D. Nullmeyer for help with transfection assays and pHi measurements, respectively.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-DK-41274 and the W. M. Keck Foundation.
Address for reprint requests and other correspondence: F. K. Ghishan, Dept. of Pediatrics, Steele Memorial Children's Research Center, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.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 5 February 2001; accepted in final form 19 March 2001.
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