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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The type IIb sodium-phosphate
(NaPi-IIb) cotransporter mediates intestinal phosphate
absorption. Previous work in our laboratory has shown that EGF
inhibited NaPi-IIb cotransporter expression through
transcriptional regulation. To understand this regulation, progressively shorter human NaPi-IIb promoter constructs
were used to define the EGF response region, and gel mobility shift assays (GMSAs) were used to characterize DNA-protein interactions. Promoter analysis determined that the EGF response region was located
between 784 and
729 base pair (bp) of the promoter. GMSAs and
overexpression studies revealed an interaction between this promoter
region and c-myb transcription factor. Inhibition of EGF receptor
activation restored promoter function. Further studies suggested that
MAPK, PKC, and/or PKA pathways are involved in this regulation. In
conclusion, these studies suggest that EGF decreases human
NaPi-IIb gene expression by modifying the c-myb protein
such that it inhibits transcriptional activation. We further conclude
that this downregulation of promoter function is mediated by
EGF-activated PKC/PKA and MAPK pathways. This is the first study that
demonstrates involvement of c-myb in the regulation of intestinal
nutrient absorption.
type IIb sodium-phosphate cotransporter; epidermal growth factor
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INTRODUCTION |
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PHOSPHATE (Pi) plays a major role in growth, development, bone formation, and cellular metabolism. Pi (re)absorption occurs in the small intestine and the kidney, mainly through sodium-dependent pathways. Type II sodium-dependent Pi (NaPi-II) cotransporters are the major proteins involved in these processes. The type IIb sodium-dependent Pi (NaPi-IIb) cotransporter is responsible for intestinal Pi absorption, whereas the type IIa sodium-dependent Pi (NaPi-IIa) cotransporter is critical for renal Pi reabsorption. Many physiological factors regulate Pi homeostasis via modulating Pi absorption in the kidney and intestine. Specifically in the small intestine, low-Pi diet, as well as 1,25(OH)2 vitamin D3, stimulate Pi absorption by elevating NaPi-IIb cotransporter activity (18, 44), whereas glucocorticoids reduce Pi absorption by decreasing NaPi cotransporter function (4, 10, 39).
Epidermal growth factor (EGF), a growth hormone, also regulates renal
and intestinal Pi absorption. EGF has been shown to inhibit
renal Pi reabsorption by modulating NaPi-IIa
mRNA expression (2, 3). Our previous study also showed
that EGF reduced NaPi-IIb mRNA synthesis in rat intestine
and in human intestinal epithelial (Caco-2) cells by a transcriptional
mechanism (46). Furthermore, a putative EGF response
element(s) was shown to be in the region of 1,103 to
380 base pairs
(bp) of the human NaPi-IIb (hNaPi-IIb) gene promoter.
EGF response elements have been identified as a serum-response element
and AP1 binding sequences from the c-fos gene
(15) and as Sp1 binding sequences from the rat
preprothyrotropin-releasing hormone (33) and the human
gastrin genes (16, 17, 25). In the hNaPi-IIb
gene promoter region (1,103 to
380 bp), two sequences at position
792 to
786 bp (GGGAAGG) and
479 to
474 bp (GGGCGC) were found
to have high homology with the EGF response elements identified as Sp1
binding sequences from the rat preprothyrotropin-releasing hormone gene
(33). However, it is unclear whether these sequences are
involved in EGF regulation of hNaPi-IIb cotransporter gene expression.
To determine whether these DNA sequences are involved in EGF regulation of hNaPi-IIb gene expression, we made a series of promoter constructs that contain different lengths of the 5'-flanking region of the hNaPi-IIb gene upstream of a luciferase reporter gene and transfected these plasmids into Caco-2 cells to test the promoter response to EGF. DNA gel mobility shift assay (GMSA) was then used to detect interactions between DNA and nuclear proteins. From these results, we have shown for the first time that EGF regulation of hNaPi-IIb gene expression involves modulation of c-myb binding affinity as mediated by EGF activated PKC/PKA and MAPK pathways.
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MATERIALS AND METHODS |
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Cell culture. Human intestinal epithelial (Caco-2) cells were purchased from American Type Culture Collection (ATCC) and cultured according to ATCC guidelines. Cells were cultured at 37°C in a 95% air-5% CO2 atmosphere and passaged every 72 h. In EGF treatment experiments, cells were incubated with 50 or 100 ng/ml EGF for 8 h before being harvested. Media and other reagents used for cell culture were purchased from Irvine Scientific (Irvine, CA). Cells between passages 40 and 46 were used in this study.
Assembly of reporter gene constructs.
A series of progressively shorter hNaPi-IIb promoter
constructs in the pGL-3/basic luciferase reporter vector (Promega,
Madison, MI) were made by restriction enzyme digestion or PCR
(46). Briefly, pGL3/1,103 bp construct was made by
subcloning a SacI-XmaI fragment of the
hNaPi-IIb promoter into the pGL3/Basic vector. For other deletion constructs, pGL3/
784 bp, pGL3/
729 bp, pGL3/
646 bp, pGL3/
624 bp, pGL3/
563 bp, and pGL3/
380 bp, different lengths of
hNaPi-IIb promoter were PCR amplified, utilizing the same
reverse primer and different forward primers containing sequences for SacI. These PCR products were then ligated into
SacI-XmaI digested pGL3/Basic plasmid and
sequenced. All deletion constructs end on the 3' end at +15 bp of the
hNaPi-IIb gene.
Transient transfection, EGF treatment, and luciferase assays. Caco-2 cells were seeded in 24-well plates and maintained in a defined medium. When cells were 70% confluent, lipofectamine (Invitrogen, Carlsbad, CA)-mediated transfection was performed as previously described (46). Cells were treated with EGF (50 or 100 ng/ml; Austral Biological, San Ramon, CA) or vehicle for 8 h before harvest. Promoter reporter gene assays were performed using a dual luciferase assay kit according to the manufacturer's instructions (Promega). For EGF-signaling pathway studies, various inhibitors or vehicles were added to the transfected cells 2 h before EGF was added. A mouse monoclonal antibody against the human EGF receptor (Ab-3) was purchased from Oncogene Research Products (Boston, MA). Tyrphostin AG-1478 (called AG-1478 throughout) and PD-098059 were purchased from Sigma (St. Louis, MO). H7 was purchased from Calbiochem (San Diego, CA).
Preparation of nuclear extracts for GMSA.
Nuclear extracts were prepared by a previously described method
(38) from Caco-2 cells treated with EGF (100 ng/ml) or
vehicle for 8 h. Synthetic double-stranded oligonucleotides were
designed to cover the promoter region 799 to
727 bp. DNA
oligonucleotides were end-labeled with [
-32P]ATP, and
4 µg of nuclear extract were incubated with 1 ng of labeled probe in
GMSA binding buffer containing 10 mM HEPES (pH 7.5), 1 mM EDTA, 50 mM
NaCl, 1 mM dithiothreitol, and 50 µg/ml poly[d(I-C)]. After
incubation at room temperature for 20-30 min, the mixture was
electrophoresed on a 6% polyacrylamide gel in 0.25× Tris-boric
acid-EDTA buffer. Gels were dried and exposed to X-ray film. For
competition experiments, a 100-fold molar excess of unlabeled probe was
added to the reaction mixture before the labeled probe was added. For
supershift assays, 4 µg of a rabbit polyclonal antibody raised
against a recombinant protein corresponding to amino acids 500-640
from the COOH terminus of human c-myb or nonspecific rabbit IgG (Santa
Cruz Biotechnology, Santa Cruz, CA) were added to the reaction mixtures.
Cellular protein preparation.
Cells were rinsed twice with PBS buffer and incubated in cell lysis
buffer containing 0.45 × PBS (pH 7.4), 0.5% Na-deoxycholate, 1%
NP-40, 0.1% SDS, 2 mM PMSF, and protease inhibitor cocktail (Boehinger
Mannheim, Indianapolis, IN) for 30 min at 4°C. Cells were scraped and
passed through a 21-gauge needle several times. The cell lysates were
then spun down at 10,000 g for 10 min at 4°C. The
supernatant, containing cellular protein, was stored at 70°C.
Western blotting. Nuclear protein (20 µg) or cellular protein (40 µg) was subjected to SDS-PAGE (7.5%). The proteins were electroblotted to a membrane and reacted with an affinity-purified rabbit c-myb polyclonal antibody raised against an immunogenic peptide corresponding to amino acid residues 2-16 of the human c-myb protein (Active Motif, Carlsbad, CA). Immunodetection was performed using the BM chemiluminescence Western blotting kit (Roche Molecular Biochemicals, Mannheim, Germany). Nuclear proteins from the human chronic myelogenous leukemia (K562) cells (Active Motif) were used as a positive control for c-myb immunoblotting.
mRNA purification and PCR amplification.
mRNA was purified from Caco-2 cells using the Micro FastTrack mRNA
purification kit (Invitrogen). A reverse transcription reaction was
performed in the presence of oligo(dT)15 primers and AMV
reverse transcriptase. The primers used to amplify NaPi-IIb and -actin gene products were the same as described previously (46). The primers used to detect the c-myb gene product
were designed to cover the human c-myb cDNA (accession no. NM005375)
region from 439 to 701 bp. Semiquantitative RT-PCR, as previously
described (46), was used to determine the
NaPi-IIb and c-myb gene expression levels in the absence or
presence of EGF.
Overexpression of human c-Myb in Caco-2 cells.
Human c-myb cDNA was PCR amplified from Caco-2 cells between bp 110 to
2,037 bp. PCR products were cloned into the pCR2.1 vector (Invitrogen)
and confirmed by sequencing. The PCR insert was then moved into the
mammalian expression vector pTarget (Promega) by restriction enzyme
digestion. Recombinant plasmids were transfected into Caco2 cells
by Lipofectamine (Invitrogen/GIBCO). Forty-eight hours after
transfection, G418 was added (1 mg/ml) to the standard culture medium
for 3 days, and the cells were lysed in 20 mM HEPES (pH 7.9), 0.4 M
NaCl, 25% glycerol, 1 mM EDTA, 2.5 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride on ice for 20 min. Cell extracts were
then frozen, thawed, and centrifuged for 10 min at 13,000 g
at 4°C. Supernatants were collected and stored at 80°C until use
(40).
Statistical analysis. ANOVA post hoc tests (StatView 5.0.1; SAS Institute, Cary, NC) were used to compare values of experimental data. P values of <0.05 were considered significant.
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RESULTS |
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Identification of the EGF responsive region in the
hNaPi-IIb promoter.
A series of 5' deletion constructs was made between 1,103 and
380
bp of the hNaPi-IIb promoter region. Caco-2 cells were transfected with these promoter constructs and treated with EGF (100 ng/ml) or vehicle for 8 h. Promoter reporter gene assays showed
that deletion constructs pGL3/
563, pGL3/
624, pGL3/
646, and
pGL3/
729 did not respond to EGF treatment, whereas the constructs pGL3/
784 and pGL3/
1,103 were suppressed by EGF (Fig.
1).
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GMSA identification of DNA sequences involved in the EGF response
of the hNaPi-IIb promoter.
To identify the precise DNA sequences involved in the EGF response,
four DNA oligos were designed to overlap the promoter region 799 to
727 bp. As shown in Fig.
2A, a low
molecular weight DNA-protein interaction band was obtained from all the
oligos used, whereas a higher molecular weight DNA-protein interaction band was observed with only the probe covering the promoter region
751 to
727 bp. Furthermore, the higher molecular weight-shifted band, but not the lower molecular weight-shifted band, was reduced by
EGF administration. Therefore, the lower molecular weight-shifted band
was considered nonspecific, and we felt that it was most likely not
involved in EGF regulation of hNaPi-IIb cotransporter gene
expression.
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Mutation at promoter region 739 to
734 bp abolished nuclear protein binding.
To further define the DNA-protein interaction at
751 to
727 bp,
mutant DNA oligos (Fig. 2B) covering different parts of this
region were used. Nuclear protein (4 µg) from Caco-2 cells was
incubated with labeled probe 751/727 in the presence or absence of a
100-fold excess of mutant or wild-type DNA oligos. As shown in Fig.
2B, wild-type DNA oligos completely competed the binding of
nuclear proteins in shifted complex a. Mutant oligos (m2 and m2a)
containing the wild-type sequence (739 bp -AACTGG- 734 bp) also
competed binding of nuclear proteins in shifted complex a. Other mutant
oligos (m1, m3, and m4), which have mutations of the -AACTGG- sequence,
had no effect on shifted complex a. The mutant oligo m5, which has
upstream bps mutated, reduced the binding in complex a.
Functional confirmation of the DNA sequence involved in EGF
regulation.
To determine whether the DNA sequence (-AACTGG-) identified by GMSAs
plays a functional role in EGF regulation of the hNaPi-IIb gene expression, the wild-type sequence in the promoter construct pGL3/784 was replaced by mutant sequence 5'-TCTGTT-3'. The mutant promoter construct pGL3/
784m was then transfected into Caco-2 cells,
and promoter activity was measured by promoter-reporter assays after
EGF treatment. As shown in Fig. 3, EGF
treatment did not affect promoter activity in constructs pGL3/
380 or
pGL3/
729, but it reduced promoter activity in promoter construct
pGL3/
784-transfected cells (as also shown in Fig. 1). This reduction
by EGF was abolished in mutant promoter construct
pGL3/
784m-transfected cells. Promoter activity was similar in
pGL3/
784m DNA-transfected cells as in other promoter
construct-transfected cells in the absence of EGF (data not shown).
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Identification of the protein interacting with the DNA sequence
involved in EGF regulation by supershift assay.
GMSA and functional studies suggested that the DNA sequence (bp 739 5'-AACTGG-3' bp 734) in the hNaPi-IIb promoter is the potential cis-acting element involved in EGF regulation. A
search for transcription factor binding motifs within this region
suggested a potential consensus binding site (-AACT/GG-) for the c-myb
transcription factor. To determine whether this DNA sequence could be
bound by c-myb protein, supershift GMSAs were performed. As shown in Fig. 4A, the nuclear protein
bound to the probe (751/727) caused a specific band shift, and this
band could be partially supershifted by an anti-c-myb antibody. As a
control to demonstrate specificity of the c-myb antibody, we showed
that the addition of rabbit IgG to the reaction did not produce a
supershift. To further exemplify c-myb binding to the
hNaPi-IIb promoter, human c-myb cDNA was overexpressed in
Caco-2 cells, and the cytoplasmic extracts were used for GMSAs. As
shown in Fig. 4B, the cell lysate from c-myb cDNA-transfected cells (pT-myb) generated an identical band shift to
that seen with Caco-2 cell nuclear extracts. Conversely, cell lysate
from vector DNA-transfected cells (pT) did not produce a band shift.
These data, when considered together, indicate that the protein bound
to this hNaPi-IIb gene promoter region is c-myb.
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Quantitation of endogenous c-myb gene expression in Caco-2 cells.
Our studies suggested that c-myb protein is possibly involved in
regulation of hNaPi-IIb gene expression by EGF. To further confirm that Caco-2 cells endogenously express the c-myb gene, mRNA was
purified from EGF- or vehicle-treated Caco-2 cells, and semiquantitative RT-PCR was used to quantitate c-myb gene expression. As shown in Fig. 5, c-myb mRNA could be
detected in Caco-2 cells. Furthermore, EGF treatment reduced
hNaPi-IIb mRNA expression but had no effect on c-myb or
-actin (used as a constitutive control) mRNA expression levels.
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Quantitation of endogenous c-myb protein expression by Western
blotting.
To determine endogenous c-myb protein distribution and expression
levels, total cellular proteins and nuclear proteins were prepared from
Caco-2 cells treated with EGF or vehicle. As shown in Fig.
6, a protein at ~80 KDa was detected by
the antibody against the NH2 terminus of the human c-myb
protein from both nuclear and cellular protein samples. EGF treatment
did not alter c-myb protein expression levels or its cellular
distribution in Caco-2 cells.
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c-Myb involvement in hNaPi-IIb gene regulation is
mediated through EGF receptor tyrosine kinase signaling transduction
pathway.
It is known that EGF can exert its effects on gene expression
through various signal transduction pathways. To further elucidate the
pathway involved in the regulation of hNaPi-IIb gene
expression, various EGF receptor-signaling pathway blockers were used.
Caco-2 cells were transfected with promoter construct pGL3/784 and
pretreated with various inhibitors for 2 h before EGF was added.
As shown in Fig. 7, hNaPi-IIb
gene promoter activity was reduced 32% by 50 and 100 ng/ml EGF in
transfected Caco-2 cells. Administration of 50 nM monoclonal EGF
receptor antibodies blocked the response of hNaPi-IIb
promoter to EGF treatment (50 ng/ml; Fig. 7A). AG-1478, a
specific inhibitor of EGF receptor tyrosine kinase activity, also
abolished the EGF effect on the hNaPi-IIb promoter at
concentrations of 1 and 5 µM (Fig. 7B). Furthermore,
administration of inhibitors PD-098059 (25 µM) and H7 (10 µM)
inhibited 30 and 60% of the promoter response, respectively. The
combination of PD-098059 and H7 completely blocked the response of
hNaPi-IIb promoter to EGF (Fig. 7C).
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Tyrphostin AG-1478 inhibited the response of hNaPi-IIb
gene promoter to EGF by restoring the interaction between nuclear
protein and DNA.
Functional promoter analysis results showed that AG-1478 completely
blocked the effect of EGF on hNaPi-IIb promoter activity. We therefore treated Caco-2 cells with 1 µM AG-1478 and purified nuclear protein for GMSA to determine whether the DNA-protein interaction was restored. As shown in Fig.
8, EGF treatment reduced the specific
binding of nuclear protein to the probe 751/727. However, in the
presence of 1 µM AG-1478, binding levels were restored. AG-1478
itself had no effect on this DNA-protein interaction.
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DISCUSSION |
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Pi is an essential element of the body, and its
homeostatic regulation is thus important. EGF, as a growth hormone,
plays an important role in modulating intestinal Pi
absorption in certain pathophysiological conditions, such as
hyperphosphatemia induced by intestinal ischemia/injury
(14, 20, 41). Previous work in our laboratory has shown
that EGF reduced NaPi-IIb gene expression in the rat
intestine and in human intestinal epithelial (Caco-2) cells. EGF
affected NaPi-IIb gene expression by inhibiting
transcriptional activation in Caco-2 cells. The DNA region involved in
this regulation was hypothesized to be located between bp 1,103 and
380 of the hNaPi-IIb gene promoter (45). In
the current studies, we further narrowed the EGF response region to bp
784 to
729 of the hNaPi-IIb promoter. This DNA region
has no homology with known EGF response elements identified from the
c-fos (15), the rat preprothyrotropin-releasing hormone (33), the rat prolactin (13), or the
human gastrin genes (16, 17, 25). These findings suggest
that a novel EGF response element may be present in the
hNaPi-IIb promoter.
The EGF-responsive region was further refined to bp 751 to
727 by
GMSA and functional analysis in Caco-2 cells. In GMSAs, the DNA-protein
complex (called a) was decreased by EGF treatment. Competition studies
with mutant oligos from the
751 to
727 region showed that the
sequence at bp
739 to
734 (5'-AACTGG-3') was critical for this
DNA-protein interaction. When this wild-type DNA sequence in the
hNaPi-IIb promoter was replaced by a mutant DNA sequence
(5'-TCTGTT-3'), this DNA-protein interaction and the functional
response of the hNaPi-IIb promoter to EGF were abolished.
These results strongly suggest that this region is involved in EGF
regulation of hNaPi-IIb gene expression.
Transcription factor binding motif searches suggested that the DNA sequence (5'-AACTGG-3') identified in the hNaPi-IIb promoter could be recognized by the c-myb transcription factor. Additionally, supershift studies with an anti-c-myb antibody indicated that the c-myb transcription factor could bind to this EGF responsive sequence in the hNaPi-IIb promoter, suggesting a novel role for c-myb in EGF regulation. The fact that only a portion of the DNA-protein complex could be supershifted by c-myb antibody might be due to insufficient antibody affinity for c-myb protein or to low specific antibody concentration. Furthermore, we used cellular extracts from Caco-2 cells transfected with human c-myb cDNA in GMSAs and demonstrated that overexpressed human c-myb can bind to the hNaPi-IIb promoter oligo probe and produce an identical band shift to that seen with Caco-2 cell nuclear extracts. Overall, these data strongly suggest that the protein interacting at the EGF responsive site is c-myb.
c-Myb is a phosphorylated nuclear protein, which plays important roles in regulating cell growth, differentiation, and apoptosis (30). c-Myb protein has three distinct regions, which are individually responsible for DNA binding, protein-protein interactions, and negative regulatory functions (26). The COOH-terminal negative regulatory region of c-myb interacts with its DNA binding domain at the NH2-terminal region to influence trans interactions with transcriptional coactivators, cooperating proteins, or DNA (11, 29). Studies have shown that c-myb function is regulated at the posttranslation level (28). MAPK phosphorylates chicken c-myb protein at Ser-11/12, resulting in reduced DNA-binding activity (23, 24). MAPK also phosphorylates homologous serines in mouse (Ser-528) and human (Ser-532) c-myb proteins, which also interferes with c-myb activity (5, 6, 43). c-Myb is highly expressed in immature hemopoeitic cells and in the human intestine (35, 42). It regulates the expression of many genes, such as myb-induced myeloid protein-1 (mim-1) (12), T cell surface markers CD4 (27, 37), and CD34 (19) in immature hemopoeitic cells. However, little is known about the target genes of c-myb in intestinal epithelial cells.
Our results demonstrated that mutation of the c-myb-binding site abolished the EGF response of the hNaPi-IIb promoter. Further data suggested that c-myb binding to the promoter under basal conditions has no effect on transcriptional activation because mutation of the putative binding site did not alter basal promoter activity. But, upon EGF stimulation, c-myb is likely modified in some way that then leads to an inhibitory effect on transcriptional activation and decreased binding affinity on the hNaPi-IIb promoter. In fact, our data (as discussed in detail later) show that inhibition of MAPK or PKC activity could partially restore the activity of the hNaPi-IIb promoter, which suggests that MAPK- and/or PKC-mediated phosphorylation of c-myb induced by EGF signaling may be this proposed modification of c-myb. Furthermore, we found that inhibition of EGF receptor tyrosine kinase activity and EGF-EGF receptor interaction also abolished the effect of EGF on hNaPi-IIb promoter activity, presumably by blocking this modification of c-myb, which leads to its inhibitory properties on promoter function. Additionally, the findings that EGF did not alter c-myb mRNA abundance, c-myb protein expression levels, or distribution between cytosol and nucleus further suggested that the c-myb protein was modified by EGF treatment in Caco-2 cells. This possible posttranslational modification might involve changing protein-protein interaction(s) between c-myb and basal transcription factor(s) (28, 30), which together may mediate the EGF response of the hNaPi-IIb gene.
EGF receptor signaling transduction pathways have been extensively studied. EGF receptor activation by EGF binding initiates multiple cellular signaling pathways. Ab-3, a mouse monoclonal antibody against the human EGF receptor, inhibits EGF binding to the EGF receptor and thus inhibits EGF-dependent tyrosine protein kinase activity. In preliminary experiments, we determined that 50 ng/ml EGF led to a maximal decrease in hNaPi-IIb gene expression and that 50 nM Ab-3 (the maximal concentration provided by the manufacturer) completely abolished this downregulation. Furthermore, we used an inhibitor of EGF receptor tyrosine kinase activity (tyrphostin AG-1478), which has been previously used at concentrations between 1 and 10 µM (21, 34, 36). These experiments showed that the response of the hNaPi-IIb promoter to EGF treatment could be completely blocked by treating transfected cells with 1 or 5 µM tyrphostin AG-1478. Overall, these findings indicated that EGF downregulation of hNaPi-IIb gene expression involves EGF binding to its receptor, followed by EGF receptor tyrosine kinase activation.
Further experiments were performed to identify downstream pathways after EGF receptor activation by PD-098059, which is a highly specific inhibitor of MAPK activation and has been previously used at concentrations between 10 and 50 µM (1, 21, 34). We chose 25 µM for our experiments on the basis of these previous investigations. We also utilized H7, a potent inhibitor of both protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). In previous PKC-signaling pathway studies, the H7 concentration used was between 10 and 50 µM (22, 31, 32). We used the minimal dose of 10 µM H7 for our experiments to avoid toxicity and nonspecific inhibition of other protein kinases. Our results with these inhibitors showed that administration of 10 µM H7 reduced 60% of the promoter response to EGF and that 25 µM PD-098059 reduced 30% of the promoter response to EGF. When used in combination, PD-098059 and H7 fully restored hNaPi-IIb promoter activity in the presence of EGF. These data suggest that both PKC and MAPK pathways are involved in EGF regulation of hNaPi-IIb gene expression, with less impact from the MAPK pathway. Because 10 µM H7 could also inhibit PKA activity, PKA pathway involvement is possible.
PKC activation is known to be one of the downstream signals for
EGF-induced receptor tyrosine kinase activation (9). There are at least 12 PKC isoforms grouped into three subtypes identified to
date, and each isoform has a unique, nonredundant role in signal transduction (8). As an intestinal epithelial cell model,
Caco-2 cells express at least five different PKC isoforms encompassing the three subtypes (8). PKC activation by EGF has been
shown to be related to protecting intestinal cells from
oxidants/ethanol damage through PKC-1 (9) and PKC-
(8) isoforms. Here, we observed that the downregulation of
hNaPi-IIb gene promoter function by EGF could be partially
restored by use of the PKC inhibitor H7, but this inhibitor is not
isoform-specific, so it is currently unknown which PKC isoforms may be involved.
In summary, this study has identified the DNA sequence in the hNaPi-IIb gene promoter likely responsible for EGF downregulation of hNaPi-IIb gene expression. Supershift analysis and overexpression studies indicated that the transcription factor c-myb was responsible for binding to this sequence and that EGF treatment reduced binding affinity and concomitantly reduced promoter activity. Further work showed that EGF receptor tyrosine kinase-activated MAPK, PKC, and/or PKA pathways are involved in EGF modulation of hNaPi-IIb gene expression. These studies, for the first time, directly demonstrate c-myb regulation of a gene involved in intestinal nutrient absorption. Moreover, our data further exemplify a novel target gene (the NaPi-IIb cotransporter) of EGF receptor signaling in the mammalian intestine.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-33209.
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FOOTNOTES |
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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.
First published January 15, 2003;10.1152/ajpcell.00456.2002
Received 30 September 2002; accepted in final form 12 January 2003.
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