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 intestinal sodium-phosphate cotransporter (NaPi-IIb) plays a major role in intestinal Pi absorption. Epidermal growth factor (EGF) is involved in the regulation of Pi homeostasis. However, the role of EGF in intestinal NaPi-IIb regulation is not clear. The current studies showed that EGF decreased NaPi-IIb mRNA abundance by 40-50% in both rat intestine and Caco-2 cells. To understand the mechanism of this regulation, we cloned the human NaPi-IIb gene and promoter region and studied the effect of EGF on NaPi-IIb gene transcription. The human NaPi-IIb gene has 12 exons and 11 introns. Two transcription initiation sites were identified by primer extension. Additionally, 2.8 kb of the 5'-flanking region of the gene was characterized as a functional promoter in human intestinal (Caco-2) and human lung (A549) cells. Additional studies showed that EGF inhibited promoter activity by 40-50% in Caco-2 cells and that actinomycin D treatment abolished this inhibition. EGF had no effect on promoter activity in lung (A549) cells. We conclude that the human NaPi-IIb gene promoter is functional in Caco-2 and A549 cells and that the gene is responsive to EGF by a transcriptionally mediated mechanism in intestinal cells.
Caco-2 cells; A549 cells; human intestine; sodium-phosphate cotransporter; transcriptional regulation
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INTRODUCTION |
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PHOSPHATE (Pi) plays a major role in growth, development, bone formation, and cellular metabolism. The kidney and small intestine are important sites that regulate body phosphate homeostasis. In both organs, sodium-coupled phosphate transport is the major form of Pi absorption. The human and mouse intestinal sodium-dependent phosphate cotransporter (NaPi-IIb) cDNAs were cloned recently (9, 17, 34), and the human NaPi-IIb cotransporter gene (SCLA34A2) was mapped to human chromosome 4p15.1-15.3 (33, 34).
Intestinal sodium-dependent phosphate absorption occurs predominantly through the NaPi cotransport system, while renal sodium-dependent phosphate absorption is mainly mediated by another closely related isoform, NaPi-IIa. In both tissues, Pi absorption across the brush-border membrane through NaPi cotransporters is the rate-limiting step. Furthermore, Pi absorption is tightly regulated to maintain body Pi homeostasis. Many nutritional and endocrine factors regulate Pi absorption in the intestine and kidney. For instance, a low-phosphate diet stimulates renal NaPi-IIa (5, 25, 28, 29) and intestinal NaPi-IIb cotransporter activity (16). Also, vitamin D3 and glucocorticoids affect renal and intestinal Pi absorption (7, 27).
An important physiological regulator of renal Pi absorption is epidermal growth factor (EGF), which acts through modulation of NaPi cotransporter activity (1, 2). EGF is a 53-amino acid polypeptide, which is secreted predominantly by salivary glands, with lower levels excreted by kidney and many other tissues. EGF receptors are expressed along the intestinal tract (4), and EFG has broad effects on cell division, DNA synthesis, tissue proliferation, cellular differentiation, and eletrocyte and nutrient absorption (24). More specifically, EGF was shown to inhibit the activity of several intestinal brush-border membrane proteins including sucrase, maltase, and alkaline phosphatase (21). Other studies showed that EGF inhibited renal Pi uptake by modulating NaPi-IIa cotransporter protein and mRNA levels (1, 2). Additionally, in some disease states, such as hyperphosphatemia induced by intestinal ischemia/injury, serum Pi levels are elevated (10, 19), and EGF production and utilization are increased (30). These observations suggest that EGF plays an important role in Pi homeostasis by regulating renal and possibly intestinal Pi absorption.
As reported in the current communication, we initially detected a significant decrease in intestinal NaPi-IIb mRNA abundance in EGF-treated rats and in human intestinal cells (Caco-2). These results suggested a possible role for EGF in transcriptional regulation of the NaPi-IIb gene. Therefore, to further understand the role of EGF in the regulation of intestinal Pi homeostasis, we cloned the human NaPi-IIb gene and characterized in vitro regulation of the promoter 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 Biological, San Ramon, CA) or saline twice a day for 3 days. Fifteen hours after the last injection, rats were killed, and jejunal mucosa was harvested and used for mRNA purification. All animal work was approved by the University of Arizona Institutional Animal Care and Use Committee.
Cell culture. Human lung cells (A549) and human intestinal cells (Caco-2) 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. Media and other reagents used for cell culture were purchased from Irvine Scientific (Irvine, CA).
RNA purification and Northern blot analyses. mRNA was isolated from A549 cells, Caco-2 cells, and rat jejunal mucosa with the FastTrack mRNA purification kit (Invitrogen, Carlsbad, CA). Part of the rat NaPi-IIb cDNA was amplified utilizing standard PCR conditions from rat jejunal cDNA, with highly conserved primers previously utilized for cloning of the human intestinal NaPi-IIb cDNA (34). This rat cDNA fragment was subcloned and sequenced on both strands. Ten micrograms of mRNA were utilized for Northern blot analysis with rat or human NaPi-IIb cDNA-specific probes under high stringency washing conditions (6). 1B15 (encoding cyclophilin) was utilized as a constitutive probe (6). Blots were exposed to a phosphorimaging screen, and band intensities were determined with Quantity One software (FX Molecular Imager; Bio-Rad, Hercules, CA).
Semiquantitative RT-PCR analysis.
mRNA was purified from Caco-2 cells treated with standard or
EGF-containing medium (100 ng/ml). The primers used for detecting NaPi-IIb expression were the same as those previously
described for cloning of the human intestinal NaPi-IIb cDNA
(34). The primers used for detecting -actin and RT-PCR
conditions were described previously (3). Subsaturation
levels of cDNA templates that were needed to produce a dose-dependent
amount of PCR products 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 parameters.
Isolation and characterization of genomic clones. A human genomic bacterial artificial chromosome (BAC) DNA library was screened by Research Genetics (Huntsville, AL) using a 32P-labeled human intestinal NaPi-IIb 5' cDNA fragment (nt 175-350). Library screening resulted in three putative-positive clones, and one was selected for further analysis. Two strategies were undertaken to determine the human NaPi-IIb gene structure: 1) PCR amplification of BAC DNA with cDNA-specific primer pairs, and 2) direct sequencing of BAC DNA via primer walking. PCR products were then subcloned into pGEM-T vector (Promega, Madison, WI) and sequenced on both strands. For primer walking, BAC DNA was isolated from bacterial cultures by use of a standard plasmid DNA purification method. BAC DNA was treated with RNase A (Sigma, St. Louis, MO) and NotI restriction enzyme (Promega), and then extracted with phenol-chloroform, precipitated with EtOH, and resuspended in water. The BAC DNA was sequenced on both strands.
Primer extension.
The transcription initiation site of the human NaPi-IIb
gene was determined by primer extension. Briefly, a primer designed complementary to the human NaPi-IIb cDNA at nt 43-62
(in exon II) was end labeled with [-32P]ATP and
T4 polynucleotide kinase. Labeled primer (200 fmol) was
annealed to 50 ng mRNA isolated from Caco-2 or A549 cells in a 12-µl
reaction by heating at 90°C for 2 min and then cooling to 58°C at
1°C/min. The annealing reaction was then held at 58°C for 30 min
and snap chilled on ice. Annealed primers were extended at 42°C for
2 h by adding 200 units of SuperScript II Reverse Transcriptase
(Life Technologies, Bethesda, MD), 1 µl RNasin, 1 µl 10 mM dNTPS,
and 4 µl 5× first-strand reaction buffer in a 20-µl reaction
volume. The reaction was terminated by adding 3 µl 0.2 M EDTA (pH 8),
and the RNA was degraded by adding 0.9 µg DNase-free RNase A followed
by incubation at 37°C for 30 min. The primer extension product was
then EtOH precipitated, and the pellet was resuspended in 5 µl
loading dye/10 mM Tris-1 mM EDTA buffer, pH 8.0 (1:1). Samples
were heated at 75°C for 10 min before loading on a sequencing gel.
The gel was subsequently dried and exposed to Kodak Xomat Blue XB-1
film overnight at
70°C.
Construction of reporter plasmids.
Luciferase reporter plasmids used in this study were derived from
pGL3 Basic (pGL3b; Promega). A 1,103/+15 promoter reporter construct
(pGL3-1,103) was made by subcloning a
SacI/XmaI-digested pZero-4.5 fragment into pGL3b.
A
181/+15 promoter reporter construct (pGL3-181) was made by
digesting pGL3-1,103 construct with
SacI/ApaI and then blunting both ends with Klenow
fragment of DNA polymerase I and ligating them together. A
380/+15
promoter reporter construct (pGL3-380) was made by inserting a
SacI/XmaI-digested PCR product, amplified with
forward primer 5'-TATGAGCTCTTGAGCCTCACAGGAG-3' at nt
361
to
380 (underlined nucleotides represent SacI site and overhanging bases) and reverse primer
5'-TATAGATCTCCGCAGGTGCTGGGCT-3' in intron 1 (underlined
nucleotides indicate overhanging bases originally inserted for
BglII subcloning not used for this construct), into pGL3b
vector (note that the XmaI site is in exon I). A
2,783/+15 promoter reporter construct (pGL3-2,783) was made by inserting a
NotI/SacI-digested fragment (~1.7 kb) into the
pGL3-1,103 construct. This 1.7-kb fragment was originally
amplified from BAC DNA with a high fidelity thermostable DNA polymerase
using forward primer 5'-CGATCTTGGCTCAGTTGCAA-3' (nt-2783 to
2764) and
reverse primer 5'-TTGTCTCCAATGTGCACACG-3' (nt
873 to
864) and
cloned into the pTarget vector (Promega). In this strategy,
SacI cut the human NaPi-IIb 5'-flanking region,
and NotI cut the vectors. All constructs were sequenced to
confirm the splice sites.
Transient transfection and functional promoter analysis. A549 and Caco-2 cells were cultured in 24-well plates. When cell density reached 60-70%, liposome-mediated transfection was performed as indicated. Promoter construct DNA (0.5 µg), 30 ng pRL-CMV (Renilla luciferase reporter construct used as an internal standard; Promega), and 5 µl Lipofectamine (GIBCO BRL, Grand Island, NY) were mixed with 200 µl OptiMEM medium (GIBCO BRL) for 30 min at room temperature. The mixture was then added to the cells, and they were incubated for 5 h, followed by the addition of an equal volume of medium that contained 20% fetal bovine serum (FBS). The next day, the medium was removed and replaced with standard medium with 10% FBS. After 24 h, the cells were harvested for reporter gene assays. For EGF treatment, 100 ng/ml human recombinant EGF (Austral Biological) were added for 8 h before harvesting cells. To study the effect of actinomycin D on NaPi-IIb promoter activity, transiently transfected cells were pretreated with actinomycin D (5 µg/ml) for 2 h before EGF treatment for 8 h in the presence of actinomycin D. Promoter reporter assays were performed using a 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 values of <0.05 were considered significant.
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RESULTS |
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Effect of EGF treatment on NaPi-IIb mRNA levels in rat
jejunum.
Northern blot analysis of mRNA isolated from rat jejunal mucosa
is shown in Fig. 1. Hybridization with a
757-bp rat intestinal NaPi-IIb cDNA probe clearly showed
that NaPi-IIb mRNA abundance was decreased 50% by EGF
administration (n = 3; P = 0.008).
However, no change was observed for 1B15 mRNA abundance, which was used as an internal standard to normalize the NaPi-IIb signal.
Furthermore, the rat NaPi-IIb cDNA fragment (GenBank
accession no. AF247725) exhibited 93% nucleotide sequence homology
with the mouse intestinal NaPi-IIb cDNA.
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Effect of EGF treatment on NaPi-IIb mRNA levels in Caco-2 cells. Northern blot analysis of mRNA isolated from Caco-2 cells was performed by loading 10 µg mRNA per gel lane. Hybridization with radiolabeled, 757-bp human intestinal NaPi-IIb cDNA-specific probes showed no signal from untreated or treated cells. However, the 1B15 hybridization signal was readily apparent (data not shown, n = 3). These results suggested that the NaPi-IIb message was expressed below the Northern blot detection limits in Caco-2 cells under these culture conditions.
RT-PCR analysis of cells.
Endogenous expression of human NaPi-IIb mRNA in human
intestinal cells (Caco-2) and human lung cells (A549) was confirmed by
RT-PCR using human intestinal NaPi-IIb primers (Fig.
2). These results showed that an
~760-bp band was amplified from both cells. The PCR product was
subcloned and sequenced, and it was 100% identical to the human
NaPi-IIb cDNA. This indicated that these cells endogenously express the NaPi-IIb gene.
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Effect of EGF treatment on NaPi-IIb mRNA levels in
Caco-2 cells.
The expression of human intestinal NaPi-IIb
mRNA in Caco-2 cells after exposure to standard or EGF-containing
medium was assessed by semiquantitative RT-PCR using human intestinal
NaPi-IIb and -actin primers (Fig.
3). Data showed that human
NaPi-IIb gene expression was significantly reduced by 40%
in EGF-treated Caco-2 cells, compared with untreated cells
(n = 3; P = 0.02). This
observation is in agreement with the in vivo observation
that showed a ~50% decrease in NaPi-IIb mRNA
abundance in EGF-treated rat intestine.
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Genomic organization of the human NaPi-IIb gene.
We focused our studies on one BAC clone. Direct sequencing of this BAC
DNA identified intron 1 and intron 2. PCR amplification with different
sets of primers identified introns 3-11 (Fig.
4). The introns ranged in size from 91 to
~6,800 bp, whereas the exons ranged in size from 97 to 613 bp. The
translation start site was located in exon II, and the translation stop
site was located in exon XII. The human NaPi-IIb gene spans
24 kb and has 11 introns and 12 exons. DNA sequences at the intron/exon
boundaries conformed to the general GT/AG rule for intron donor and
acceptor splice sites. The intronic sequences determined on both
strands have been deposited in GenBank with accession nos.
AF234236-234245.
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Mapping of the transcription initiation site of the human
NaPi-IIb gene.
The transcription initiation site was determined by primer
extension with a reverse primer at cDNA nt 43-62. Figure
5A shows the primer extension
results with mRNA isolated from both human lung cells (A549) and human
intestinal cells (Caco-2). Two primer extension products were detected
from both mRNA samples that aligned 106 bp and 119 bp upstream of the
translation start site of human NaPi-IIb cDNA. This
experiment was repeated three times, and identical results were
obtained.
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Sequence of the 5'-flanking region of the human
NaPi-IIb gene.
The primer walking technique was used to directly sequence about 2,800 bp of BAC DNA upstream of exon I. Figure 5B shows ~500 bp
from the 5'-flanking region of the human NaPi-IIb gene.
Additionally, 1.1 kb of the proximal 5'-flanking region was examined
for the presence of typical eukaryotic promoter elements by use of
Omiga sequence analysis software (version 2.0; Oxford Molecular,
Oxford, UK) and TRANSFAC promoter analysis software (32).
Search results showed that there was no TATA or CAAT box in the first
500 bp upstream of the transcription initiation site of the human
NaPi-IIb gene. However, several other putative cis
elements were identified, including transcriptional factors
AP1, AP2, AP4, C/EBP, GATA1, and Sp1 binding sites.
Furthermore, a putative glucocorticoid receptor binding site was
identified further upstream (1,082 bp).
Promoter characterization of the human NaPi-IIb gene.
To determine whether the 5'-flanking region of the human
NaPi-IIb gene contained a functional promoter, four
constructs (pGL3 181/+15,
380/+15,
1,103/+15, and
2,783/+15)
were transfected into A549 and Caco-2 cells. Reporter gene assays were
performed 48 h after transfection (Fig.
6). The promoter assay data showed that
all the promoter constructs were functional in both cell lines.
Compared with the negative control transfections, these promoter
constructs resulted in 10- to 17-fold stimulation of reporter gene
activity (n = 4-10; P < 0.002).
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Effect of EGF on human NaPi-IIb gene promoter activity.
To test the EGF effect on human NaPi-IIb gene promoter
activity, A549 and Caco-2 cells were first transfected with promoter constructs and then treated with 100 ng/ml EGF before harvesting cells.
Promoter activity was 40-50% decreased in Caco-2 cells after
8 h of EGF treatment (n = 4-10;
P < 0.02), but no change was seen in A549 cells
(n = 4-10) (Fig.
7).
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Effect of actinomycin D on human NaPi-IIb gene promoter
activity induced by EGF.
To determine whether the EGF effect on human NaPi-IIb gene
promoter activity is due to transcriptional regulation, Caco-2 cells
were first transfected with promoter constructs and then treated with 5 µg/ml actinomycin D for 2 h followed by treatment with 100 ng/ml
EGF in the presence of actinomycin D. Results showed that the
downregulation of NaPi-IIb promoter activity by EGF
treatment in the 1,103 bp construct transfected cells was blocked by
actinomycin D (n = 4; P < 0.03) (Fig.
8). Furthermore, there was no effect of
either EGF or EGF/actinomycin D treatment in pGL3b or the
118 bp
construct transfected cells (n = 4).
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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 injury. Additionally, it has been shown that the plasma Pi level and EGF levels were increased with intestinal injury (10, 19, 30). Thus EGF may play a role in regulation of Pi homeostasis in response to intestinal injury. However, the precise relationship between EGF and intestinal Pi absorption is not clear. In this study, we demonstrated that EGF treatment reduced intestinal NaPi-IIb mRNA abundance by 40-50% in rat and human intestinal cells (Caco-2). To decipher the molecular mechanism, we first cloned the human NaPi-IIb gene and promoter. We then characterized the gene promoter function and studied EGF regulation of promoter activity in transfected human lung and intestinal epithelial cells. Our data suggest for the first time that EGF reduces NaPi-IIb mRNA abundance via a gene transcription-mediated mechanism.
Although EGF is clearly involved in regulating renal Pi absorption, the role of EGF in modulating intestinal Pi absorption is unknown. To understand the relationship between EGF and intestinal Pi absorption, we cloned the complete human NaPi-IIb gene. Interestingly, the human NaPi-IIb gene showed structural differences when compared with the human NaPi-IIa gene (15). The human NaPi-IIb gene is larger than the human NaPi-IIa gene (24 kb vs. 16 kb), although it has fewer exons and introns (12 exons/11 introns in NaPi-IIb vs. 13 exons/12 introns in NaPi-IIa). Also, in the human NaPi-IIb gene, intron 1 is the largest (~6,800 bp), whereas in NaPi-IIa, intron 8 is the largest (~5,000 bp). These differences may be the result of evolutionary divergence.
Furthermore, the human NaPi-IIb and NaPi-IIa genes showed differences in the 5'-flanking regions. Unlike the human NaPi-IIa gene promoter (15, 18), the typical TATA box was absent in the proximal region of the human NaPi-IIb gene. However, in the TATA-less human NaPi-IIb gene promoter, several GATA1 binding sites are predicted. Previous studies showed that GATA transcription factors are involved in the regulation of cell type and tissue-specific gene expression (14, 20, 23, 31). This finding is in agreement with the observation that this gene is expressed in only a few tissues [i.e., in human intestine, lung, and several glands (9, 34)].
Because the NaPi-IIb gene was highly expressed in human
intestine and lung, we chose a human intestinal cell line (Caco-2) and
a human lung cell line (A549) for promoter characterization. RT-PCR
studies demonstrated that NaPi-IIb mRNA was endogenously expressed in both Caco-2 and A549 cell lines, which suggested that they
were appropriate in vitro models for promoter analyses. All four human
NaPi-IIb gene promoter constructs (as described in
MATERIALS AND METHODS) significantly stimulated reporter
gene expression in both cell lines. This finding suggested that the basal promoter region of the gene is within the first 181 bp upstream of the transcription initiation site, since the shortest construct (181/+15) was active.
In vivo and in vitro studies showed that EGF treatment decreased intestinal NaPi-IIb mRNA abundance by ~50%, suggesting possible transcriptional regulation. In transient transfected Caco-2 cells, EGF also decreased NaPi-IIb gene promoter activity by ~40%. This promoter activity decrease could be abolished by actinomycin D, a transcriptional inhibitor. These data indicated that the effect of EGF on intestinal NaPi-IIb mRNA expression was due, at least in part, to reduced gene transcription. This reduction in promoter activity induced by EGF treatment was observed only in intestinal cells (Caco-2), but not in lung cells (A549), which suggested that the EGF effect on human NaPi-IIb gene expression was tissue specific.
Furthermore, the two larger promoter constructs (1,103/+15 and
2,783/+15) were responsive to EGF treatment, but the two smaller ones
(
181/+15 and
380+15) were not responsive. These data suggested that
the putative EGF response element(s) was located between 380 and 1,103 bp upstream of the transcriptional unit. This observation also suggests
that EGF regulation of this gene is not mediated by the basal
transcriptional machinery, which is likely located within the first 200 bp upstream of transcription initiation site.
Several EGF responsive elements have been previously identified
from the c-fos gene (11), the rat
preprothyrotropin-release hormone gene (26), the rat
prolactin gene (8), and the human gastrin gene (12,
13, 22). These EGF responsive elements include a serum-response
element and AP1 binding sequences in the c-fos gene and Sp1
binding sequences in the rat preprothyrotropin-release hormone gene and
the human gastrin gene. We searched the human NaPi-IIb gene promoter region (1,103 bp
to
380 bp) for these known EGF response elements from these other
genes, and we found two sequences (
792 bp GGGAAGG
786 bp and
479
bp GGGCGC
474 bp) that have high homology with the EGF response
element of rat preprothyrotropin-release hormone gene. These sequences
may be responsible for EGF regulation in the human NaPi-IIb
gene, although further experiments will be required to make this determination.
In summary, we cloned the complete human NaPi-IIb gene from a human genomic BAC DNA library. This gene contains 12 exons and 11 introns. Approximately 2.8 kb of the 5'-flanking region of the human NaPi-IIb gene was sequenced and confirmed to be a functional promoter. EGF treatment decreased NaPi-IIb mRNA abundance in rat intestine and in human intestinal cells and also reduced the NaPi-IIb gene promoter activity in transfected human intestinal cells. Actinomycin D treatment blocked the promoter activity decrease induced by EGF treatment, confirming transcriptional regulation. These novel findings suggest that transcriptional mechanisms are involved in EGF regulation of intestinal NaPi-IIb cotransporter gene expression. Further studies will focus on identification of the EGF responsive element(s) and the transcription factors involved in EFG regulation of the human NaPi-IIb gene.
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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-33209-17 and the W. M. Keck Foundation.
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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.
Received 18 July 2000; accepted in final form 11 October 2000.
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