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J Biol Chem, Vol. 274, Issue 40, 28256-28263, October 1, 1999
From the ¶ Laboratory of Molecular Genetics, Department of
Biotechnology, Graduate School of Engineering, Osaka University,
Yamadaoka 21-1, Suita City, Japan and the Dietary phosphate (Pi) is a
most important regulator for renal Pi reabsorption. The
type II sodium-dependent phosphate (Na/Pi) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal Pi reabsorption. The consumption of a
low-Pi diet induces the synthesis of NPT2, whereas a high
Pi diet decreases it. The molecular mechanisms of
regulation by dietary Pi are not yet known.
In this report, in weaning mice fed a low-Pi diet for 4 days, the NPT2 mRNA level was increased 1.8-fold
compared with mice fed a normal Pi diet. This increase was
due to an elevation of the transcriptional activity. In the
NPT2 gene promoter, the DNA footprint analysis showed that
six regions were masked by the binding protein, but at the position
The regulation of inorganic phosphate
(Pi)1 in the
human body is controlled mainly by reabsorption in the proximal tubules of the kidneys (1-3). Apical Na+-dependent
phosphate (NaPi) cotransport is central to the renal proximal tubular reabsorption of Pi (2, 3). Studies of
isolated kidney tubules and of brush-border membranes have demonstrated that the physiological regulation of proximal tubule Pi
transport involves complex hormonal and metabolic factors that affect
the activity or expression of the transporter molecules (1-3). A major
regulator of the NaPi cotransporter is dietary
Pi (4-7). Dietary Pi restriction is associated
with an adaptive increase of the overall proximal tubular capacity to
reabsorb Pi (4-7). Alterations of the dietary intake of
Pi lead to an adaptation of renal Pi transport
activity independent of extrarenal factors such as parathyroid hormone,
growth hormone, and vitamin D (1-3, 9, 10). The molecular mechanisms
of this adaptation are unknown.
Three types of Na+-dependent Pi
cotransporter have been isolated from a kidney library (11, 12). Recent
studies suggest that the type II transporters (NPT2) may play an
important role in Pi homeostasis in the kidney, that they
are controlled by parathyroid hormone, and by the dietary intake of
Pi (11-13). In a previous study, we investigated the
cellular mechanism of the up-regulation of the NaPi
cotransporter in mice induced by the intake of a low Pi
diet and found that the administration of a low Pi diet to the mice clearly stimulated the elevation of NPT2 mRNA and protein (7). We have been studying the NPT2 gene expression using
dietary Pi feeding in mice. The NPT2 genes
respond at the transcriptional and post-transcriptional level to an
increased Pi concentration in the diet (6, 7). The DNA
sequences responsible for the Pi response in the mouse
kidney, which we have designated the phosphate response elements (PRE),
have been mapped. The PRE of the NPT2 gene promoter shares a
region with 9 of 10 bp identity to yeast phosphate-responsive
transcription factor Pho4 binding element. At the center of this region
is a CACGTG motif, the core recognition site for the helix-loop-helix
of transcription factors. This segment contains a 5'-CACGTG-3' motif
that is sufficient to confer the transactivation by dietary
Pi deprivation. We also isolated a transcription factor (a
helix-loop-helix protein), which has structural features very similar
to those of yeast Pi regulon Pho 4.
Animals and Diets--
Male ICR mice (3 weeks after birth) were
purchased from SLC (Shizuoka, Japan). They were housed in plastic cages
and the animals were fed standard mouse chow (Oriental, Osaka, Japan)
ad libitum. For the first week, they were fed the diet.
After the period, they received a diet containing 1.2% calcium, 0.6%
phosphorus and 4.4 IU vitamin D3/g for 5 days. Thereafter,
they were fed a diet with a normal Pi level (0.6%) for 7 days, given between 11:00 a.m. and 1:00 p.m. (6, 7). On the 8th day,
the following three groups of six mice each were established the normal
Pi group, mice that were chronically fed a diet containing
0.6% Pi; the low Pi group, mice that received
a diet containing a low percentage (0.02%) of Pi; and the
high Pi group, in which the mice received a high percentage
(1.2%) Pi diet. After 4 days of the given diet, all of the
mice were anesthetized with intraperitoneal pentobarbital, and the
kidneys were rapidly removed. One-half of each kidney was used for RNA
isolation and the other half was used for the isolation of brush-border
membrane vesicles (7).
Northern Blot Analysis--
Total RNA was isolated from the
kidney by extraction with acid guanidine thiocyanate/phenol/chloroform
using the method of Chomczynski et al. (13). A
NaPi-7 cDNA probe (2.4 kilobase pairs) was obtained
from a mouse kidney cDNA library (5). The internal standard was
GAPDH cDNA.
Cloning of DNA-binding Proteins Using the Yeast One-hybrid
System--
A reporter gene, NPT2 PRE-CYC1-HIS3,
for yeast one-hybrid study (14) was constructed as follows. The
Saccharomyces cerevisiae HIS3 coding region connected
downstream of the UAS (upstream activating sequence)-less S. cerevisiae CYC1 promoter was constructed on a pUC19-based plasmid
containing the S. cerevisiae ADE2 gene fragment. This
plasmid was designed as pCHNaPi0. The five tandem copies of 36-bp
double-stranded oligonucleotide, which originates from the sequences
corresponding to the nucleotide positions from
More than 2 × 106 colonies of YBH5 transformants by
mouse kidney cDNA library (mouse kidney MATCHMAKER cDNA,
CLONTECH Inc., Palo Alto, CA) were screened by
their growth on the histidine omission plates with 30 mM of
3-amino-1,2,4-triazole as described in the CLONTECH
manual. Eighty-five of positive colonies were obtained on the original
plates, and two colonies out of them showed reproducible phenotypes on
the above condition. Plasmids carrying the cDNA clones were
obtained by extraction from the two yeast transformants as described
(15) followed by transformation of E. coli.
Reporter Plasmid Construction--
A 2450-bp
BamHI-EcoRI DNA fragment containing the
5'-flanking region of the NPT2 gene was subcloned into
pBluescript II SK(+) (16). A BamHI-HindIII
fragment from the resulting plasmid was then subcloned upstream of the
coding region of the luciferase gene in the vector Pica-Basic (Toyo
Ink, Tokyo, Japan) to generate the reporter plasmid p3P2400. Mouse
transcription factor µE3 (TFE3) expression vectors were constructed
by subcloning on an EcoRI DNA fragment containing the
full-length TFE3 cDNA into pcDL-SR Cell Culture and Transient Transfection--
COS-7 cells (Riken
Cell Bank, Tokyo) were cultured at 37 °C and under 5%
CO2 in Dulbecco's modified Eagle's medium (Sanko-junyaku, Tokyo) with 10% fetal bovine serum (Sigma). OK cells (ATCC:CRL1840) were maintained in F-12/Dulbecco's modified Eagle's medium (1:1, v/v)
containing 10% fetal bovine serum (16). Cells were transfected using
the DEAE-dextran method with 5 µg of the NPT2 gene
promoter-luciferase reporter plasmid, 0.5 µg of mouse TFE3L, or TFE3S
expression plasmid and 5 µg of pCMV Electrophoretic Mobility Shift Assay (EMSA) and DNase Footprint
Assays--
Nuclear extracts were prepared from kidney or COS-7 cells
as described previously (9). The 32P-labeled Shift-Western Blotting--
Shift-Western blotting was performed
according to the method of Demczuk et al. (21). This method
was developed for identification and analysis of protein and DNA
components of gel-shift assays. Approximately 20 µg of nuclear
protein was used in a preparative gel shift. After transfer onto DE81
paper, the protein-DNA complexes were identified by autoradiography.
Proteins were eluted from excised filter pieces with 0.4 M
acetic acid, 1 M NaCl at 65 °C for 40 min and then
precipitated for 30 min with 10% cold CCl3COOH, washed
once in acetone and twice in cold 100% ethanol, vacuum-dried, resuspended, denatured in sample buffer, and subjected to SDS/10% polyacrylamide gel electrophoresis and Western blotting (21).
Nuclear Run-on Transcription Assay--
Nuclei were prepared
from the kidney of mice fed a low Pi diet, and nuclear
run-on transcription assays were performed as described previously
(22). RNA was extracted and resuspended in 300 µl of hybridization
buffer (7% SDS, 10% polyethylene glycol (8,000), 1.5% saline/sodium
phosphate/EDTA). Aliquots of RNA from treated and untreated samples
were counted in a scintillation counter, and an equal number of counts
from each condition (1-2 × 106 cpm) were hybridized
to linearized cDNAs (5 µg) for NPT1, NPT2, GAPDH, pBluescript II
DNA, which were immobilized to Hybond filters using a slot blot apparatus.
Stastical Analysis--
Data are expressed as the mean ± S.E. Differences between experimental groups were determined by
analysis of variance, and p values < 0.05 were
accepted as indicating a significant difference.
Effects of Low Pi Diet on the Expression of the Type II
Transporter Gene--
The brush-border membrane vesicles isolated from
renal proximal tubules of mice fed a diet low in Pi for 4 days were prepared and used for the assay of Pi transport
activity. The analysis of the Na+-dependent
Pi uptake at 1 min revealed an approximately 1.7-fold increase in these mice compared with mice that received a control Pi diet (p < 0.01) (Fig.
1A). As shown in Fig.
1B, the NPT2 mRNA levels were significantly increased
(by about 1.8-fold) in the mice fed the low Pi diet for 4 days. In addition, the amounts of the NPT2 protein (the 80-90-kDa
bands) were significantly increased (by about 2.5-fold for the 90-kDa
band) compared with those in mice fed the control diet (Fig.
1C). In contrast, the high Pi diet significantly
suppressed these three parameters (transport activity, mRNA, and
protein).
We studied the in vitro transcription in isolated nuclei of
renal cortex cells from mice fed the low Pi diet. As shown
in Fig. 1D, the transcriptional activity in the
NPT2 is significantly increased in the mice fed the low
Pi diet compared with those in the mice fed the control
Pi. The transcriptional activity of the type I
NaPi cotransporter NPT1 gene was not
significantly different between the normal Pi and low
Pi groups. Thus, the elevation (1.8-fold) of the NPT2
mRNA levels in the mice fed the low Pi diet
was, at least in part, due to the increase in the transcription of the
NPT2 gene.
An E Box at Mutation Analysis of the PRE Sequence of the NPT2 Gene
Promoter--
Next, we used the oligonucleotide of PRE (
Interestingly, the sequence of PRE was very similar to those of the
Pi responsible element for the promoter of the
Pi transporter gene PHO84 and acid phosphatase
gene PHO5 in yeast (23). This element is known to be a Pho4
binding site. Pho4 is a helix-loop-helix transcription factor for the
genes associated with yeast Pi metabolism and binds the E
box sequence 5'-CACGTG-3'. To confirm that the E box sequence is the
target sequence of the putative binding protein, we performed EMSAs
with oligonucleotides containing specific mutations of this sequence
(Fig. 3B). The PRE-MT1 oligonucleotide, in which GG in the
5' half-site of the PRE sequence was changed to TT, showed binding
activity similar to that of the PRE-WT (Figs. 3B and
4B). The mutation of TG in the E-box to AA (PRE-MT2)
abolished the ability to interact with PRE-WT (Fig. 3B).
Mutation of the second and third nucleotides (PRE-MT3) of the E-box
also abolished the binding activity (Fig. 4B), suggesting
that the putative binding protein recognized the sequence 5'-CACGTG-3'
in the promoter of the NPT2 gene.
Functional Role of PRE in the NPT2 Promoter--
We investigated
the role of the PRE in the basic promoter activity of the
NPT2 gene. The vector (p3P1170) was constructed with the
NPT2 gene promoter ( Relationship between Plasma Pi Levels and PRE Binding
Activity--
To further analyze whether the binding activity in the
PRE is regulated by plasma Pi concentration, we measured
plasma Pi levels and PRE binding activity in the nuclei
isolated from mice fed a low Pi, normal Pi, or
a high Pi diet. As shown in Fig.
6, the binding activity in the PRE-WT
oligonucleotide decreased in parallel with the elevation of plasma
Pi levels. There was a good correlation with both
parameters (PRE binding activity and plasma Pi
concentration).
Cloning of PRE-binding Protein by the Yeast One-hybrid
System--
To begin to identify some of the proteins that bind this
segment, we used this DNA (nucleotide positions from
One cDNA contained an almost full-length coding sequence for TFE3
and the other was a shorter partial cDNA for TFE3. TFE3 is a
DNA-binding protein that activates transcription through the µE3 site
of the immunoglobulin heavy chain enhancer (17, 18). To isolate the
functional TFE3 full-length cDNA clone, we screened the mouse
kidney cDNA library (from mice fed a low Pi diet). We
isolated TFE3L and TFE3S cDNA clones. The full-length TFE3 was
termed TFE3L, and TFE3S is the isoform for the TFE3L and is truncated
at the N-terminal region (transactivation domain) of TFE3L (32) (Fig.
7A).
Functional Analysis of the Effects of TFE3L and TFE3S on the
Transactivation of the NPT2 Gene Promoter--
To determine whether
TFE3 can activate the transcription of the NPT2 gene, the
TFE3L cDNA was cloned in a mammalian expression vector, and the
vector was cotransfected with the NPT2 gene promoter ( Binding of TFE3 to the PRE--
To further confirm the specific
binding to TFE3L and TFE3S in the promoter of the NPT2 gene,
EMSAs were performed using a 32P-labeled
To further confirm that the DNA-binding protein is TFE3, we performed
the shift-Western analysis. The nuclear extract isolated from mice fed
a low Pi diet was incubated with 32P-labeled
PRE oligonucleotide and then performed the shift-Western analysis (Fig.
8B, lanes 1 and 2). This method was
developed for identification and analysis of protein and DNA components
of gel-shift assays. The protein-DNA complexes, separated in
polyacrylamide gels, were transferred onto stacked nitrocellulose and
anion-exchange membranes. The proteins bound to nitrocellulose were
identified by immunoblotting (Fig. 8B, lane 2),
while the DNA, which bound only to the anion-exchange membrane, was
detected by autoradiography (Fig. 8B, lane
1).
This experiment clearly indicated that the DNA binding protein is TFE3.
Moreover, we determined whether TFE3 antibody inhibits the binding of
PRE-WT and renal nuclear extract isolated from mice fed a low
Pi diet. As shown in Fig. 8C, TFE3 antibody
completely blocked the binding of PRE-WT and the nuclear protein (Fig.
8C, lane 2).
Expression of TFE3 mRNA--
To further clarify the role of
TFE3 on the expression of mouse NPT2, the amounts of TFE3 mRNA were
determined in the kidney of the mice fed a low Pi diet. The
levels of TFE3 mRNA were markedly increased 12 h after mice
were changed to the low Pi diet and had slightly decreased
by 72 h (Fig. 9A). At
72 h, the levels of TFE3 mRNA was 2.1-fold the control (zero
time) (Fig. 9B). In contrast, the levels of mouse NPT2
mRNA were increased at 48 h and significantly elevated at
72 h.
Chronic dietary Pi restriction leads to an increased
NaPi cotransport rate, along with increased NPT2 protein
and mRNA (3-8). In the present study, the transcription rate was
significantly increased in nuclei isolated from the kidney cortex of
mice fed the low Pi diet. Recent reports suggest that the
up-regulation is due to the elevation of the type II transporter
synthesis by Pi deprivation and is the elevation of the
stability for the type II transporter mRNA, but not transcription
(24, 25). However, in the present study, we concluded that the
elevation of the NPT2 mRNA level is due, at least in part, to an
increase in the transcription rate. The difference in the findings of
run-on assay may be based on the feeding schedule or animal age,
because our feeding schedule used meal feeding. In this schedule, the
animals can feed only during one period of 2 h in a day. This
schedule is useful to investigate the effect of dietary Pi
on the regulation of NPT2 synthesis (6). In the low Pi
group, the plasma Pi levels were suddenly decreased
compared with those in the normal Pi group. When the
animals were fed a low Pi diet ad libitum, the
plasma Pi levels were gradually decreased. The differences
of the feeding schedule might have affected the regulation of NPT2 in
the kidney.
In a nuclear run-on assay, we analyzed the four marker genes used:
neutral basic amino acid transporter (NBAT), peptide
transporters (PepT1), type I NaPi cotransporter
(Npt1), and GAPDH (data not shown). In these
conditions, we clearly found that all cDNA did not respond to
dietary Pi in a run-on assay. We also observed the increase
in the stability of the NPT2 transcripts in vitro assay (26). This step may also be an important regulatory point as
proposed by the Murer and Ghishan studies (24, 25).
The present DNA footprinting analysis showed that six regions of the
NPT2 gene promoter were masked by the nuclear protein isolated from the mice fed a low Pi diet. In addition, the
gel-shift mobility assay demonstrated that the binding for the element
was markedly increased in the nuclei isolated from the kidney cortex of
the mice fed the low Pi diet. This binding protein
recognized the consensus sequence 5'-CACGTG-3' known as the E box. The
PRE of the NPT2 gene was further investigated by EMSA with
various oligonucleotides as probes and competitors.
Interestingly, the sequence of the PRE was very similar to those in the
Pi-response element for the promoter of the Pi
transporter gene PHO84 and acid phosphatase gene
PHO5 (23, 27, 28) in the yeast S. cerevisiae.
This element is known to be a Pho4 binding site. Pho4 is a
helix-loop-helix transcription factor for the genes associated with
yeast Pi metabolism (23, 27, 28). An EMSA demonstrated the
formation of two complexes between oligonucleotides containing PRE and
nuclear extract. The binding of the sequence with isolated nuclei was
detected in the mice fed the low-Pi diet, but not in those
fed the normal diet. The formation of the protein-DNA complex was
inhibited in the presence of an oligonucleotide containing the Pho4
binding site of the yeast PHO84 gene promoter.
We isolated cDNAs for the protein TFE3L/S by the yeast one-hybrid
system. The coexpression of TFE3 markedly stimulated the promoter
activity in the NPT2 gene, but not in the NPT2
gene with the mutation sequence in the E box. This suggested the
possibility that TFE3L/S might bind specifically to the In addition, we identified a similar PRE in the NPT2 gene of
opossum kidney (29) cells. The similar sequences are located at
position What is the nature of the Pi-responsive factor? Factors
binding to the CACGTG motif have been shown to belong to the c-Myc family. While many members of this family have been identified, TFE3 is
the predominant factor in renal extracts that binds to the PRE in
vitro. A recent study demonstrated that the TFE3 gene was a candidate for papillary cell carcinoma (34), suggesting that the
gene may function as the tumor repressor in renal cells. In
addition, Hua et al. (35) reported that TFE3 is an important transcription factor in at least one TGF- Finally, we used the yeast one-hybrid system to clone a transcription
factor (TFE3) that binds to a specific sequence in the promoter of the
NPT2 gene. TFE3 is known to activate transcription through
the µE3 site of the immunoglobulin heavy chain enhancer. The
characterization of TFE3L and TFE3S-interacting transcription factors
and investigations of their regulation may provide further insights
into the molecular mechanisms involved in the regulation of the
NPT2 gene by dietary Pi.
We thank Dr. Calame for providing TFE3
expression vectors and mouse TFE3 antibodies.
*
This work was supported by Grant 11557202 (to K. M.)
from the Ministry of Education, Science, Sports and Culture of Japan.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.
§
To whom correspondence should be addressed: Dept. of Clinical
Nutrition, School of Medicine, Tokushima University, Kuramoto-Cho 3, Tokushima City 770, Japan. Tel.: 81-886-33-7095; Fax: 81-886-33-7094; E-mail: miyamoto@nutr.med.tokushima-u.ac.jp.
The abbreviations used are:
Pi, inorganic phosphate;
bp, base pair(s);
nt, nucleotide number;
PRE, phosphate response element;
EMSA, electrophoretic mobility shift assay;
UAS, upstream activating sequence;
TFE3, mouse transcription factor
µE3;
GAPDH, glyceraldehye-3-phosphate dehydrogenase.
Identification of Regulatory Sequences and Binding Proteins in
the Type II Sodium/Phosphate Cotransporter NPT2 Gene
Responsive to Dietary Phosphate*
,§,,,
Department of
Clinical Nutrition, School of Medicine, Tokushima University,
Kuramoto-Cho 3, Tokushima City 770, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1010 to 985 upstream of the transcription start site, the binding
clearly responded to the levels of dietary Pi. The
phosphate response element (PRE) of the NPT2 gene was found
to consist of the motif related to the E box, 5'-CACGTG-3'. A yeast
one-hybrid system was used to clone a transcription factor that binds
to the PRE sequences in the proximal promoter of the NPT2
gene. Two cDNA clones that encoded protein of the mouse
transcription factor µE3 (TFE3) were isolated. This is a DNA-binding
protein that activates transcription through the µE3 site of the
immunoglobulin heavy chain enhancer. TFE3 antibody completely inhibited
the binding to the PRE. The coexpression of TFE3 in COS-7 cells
transfected with the NPT2 gene promoter markedly stimulated
the transcriptional activity. The feeding of a low Pi diet
significantly increased the amount of TFE3 mRNA in the kidney.
These findings suggest that TFE3 may participate in the transcriptional
regulation of the NPT2 gene by dietary Pi.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1010 to 985 in human
NPT2 promoter, including PRE, were inserted into upstream of the
CYC1 promoter on pCHNaPi0 as its UAS sequences. The
resulting plasmid, pCHNaPi5, was integrated into a ade2
locus on chromosome of S. cerevisiae strain HYP100
(MATa ura3-52 leu2-3,112
trp1
his3 ade2-101 lys2-801; our
stock strain), and this integrant strain was designated as YBH5. The
other yeast strain YBH0 was constructed by the similar integration
using pCHNaPi0 instead of pCHNaPi5.
-296 (kindly provided by N. Arai) (17). The internal control vector pCMV
, which expresses
-galactosidase, was obtained from CLONTECH. Each
plasmid was purified with a plasmid kit (Qiagen, Hilden, Germany).
per 5 × 105
cells, as described previously (16-18). After transfection, the cells
were incubated under standard conditions for 48 h and then exposed
to various agents for 15 h. Cells were then harvested in cell
lysis buffer, and the lysate was assayed for luciferase activity,
-galactosidase activity, and protein concentration (19).
1045 to
1035 probe and wild-type and mutant promoter probes were purified
from a nondenaturing acrylamide gel. EMSA was carried out as described
previously (9) with the following modifications. The binding buffer for
the probes was 4 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 50 µM EDTA, and 0.1 mM
dithiothreitol, and the gel was run in a low ionic strength buffer (6.4 mM Tris, pH 7.5, 3.3 mM sodium acetate, and 1 mM EDTA) at room temperature. Supershift assays were
carried out as described previously (9) using available antibodies to
TFE3 from Dr. K. Calame (20). A DNase I footprinting analysis was
performed using a commercial kit (SureTrac Footprinting Kit, Amersham
Pharmacia Biotech, Uppsala, Sweden).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of dietary Pi on
Pi transport activity, type II transporter mRNA, and
protein levels in mouse kidney. brush-border membrane vesicles
were isolated from mice fed the test diet (a low Pi diet
(LP), a normal Pi diet (CP), or a
high Pi diet (HP)) for 4 days.
Na+-dependent Pi transport activity
(A) and the amounts of NPT2 protein (C) were
determined as described under "Experimental Procedures." The levels
of NPT2 mRNA were determined using Northern blot analysis
(B). The density of the signals was measured using an
imaging analyzer (Fuji-BAS2000). The relative intensity was based on
the control mRNA (GAPDH). *, p < 0.01. The data are mean ± S.E. of six mice per group. D,
in vitro transcription in isolated nuclei of renal cells
from mice fed the low Pi diet. Nuclei were isolated from
the renal cortex of mice fed a normal Pi diet
(CP), or 4 days after the change to a low Pi
diet (LP), and were assayed for transcription in
vivo. 32P-Labeled transcripts were hybridized to NPT1,
NPT2, and GAPDH cDNA. Lane CP, the normal Pi
diet; lane LP, the low Pi diet.
1010 to 985 Plays an Important Role in the Response
to Dietary Pi--
To clarify the protein binding region
in the NPT-2 gene promoter induced by the feeding of a low
Pi diet, we performed DNase footprint assays using nuclear
extracts from the renal cortex isolated from mice fed a low
Pi diet (Fig. 2). The
footprints in the nuclear extract isolated from mouse renal proximal
tubular cells had a series of hypersensitive sites in common and a
protected region extending from 2018 to 1996 nt (FP-1), 1556 to
1535 nt (FP-2), 1010 to 985 nt (FP-3) nt relative to the
transcription start site of the NPT2 gene that may indicate
the binding of a factor in mouse kidneys (Fig. 2). There was also
additional protection within the regions of 779 to 757 nt (FP-4),
635 to 612 nt (FP-5) and 321 to 298 nt (FP-6), indicating that
an additional factor binds the DNA in mice kidney (data not shown). In
the comparison of these elements between the mice fed the low
Pi and high Pi diets, we found that the extent
of the protection at 1010 to 985 nt (FP-3) was significantly
different between these two groups of animals (Fig.
3A). However, the other
protected regions were not changed by the deprivation of dietary
Pi (data not shown). We designed the FP-3 position as
PRE.
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Fig. 2.
In vitro DNase I footprint
analysis in NPT2 promoter. The NPT2
gene fragments were incubated with DNase I and 20 µg of kidney
extracts isolated from mice fed a low Pi diet. Nucleotide
sequence of a portion of NPT2 and exon 1. Footprints are
underlined by a black bar (FP-1 to FP-6). The
major transcription start site is marked by an arrow.
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Fig. 3.
Effect of dietray Pi on DNase I
footprinting with renal cortex nuclear extracts. A, the
NPT2 gene fragment (FP-3) was incubated with DNase I and 20 µg of kidney extracts isolated from mice fed either a low
Pi diet (lanes 1 and 2), those fed a
normal Pi diet (lane 4), or those fed a high
Pi diet (lanes 5 and 6). The number
is the location of the protected regions by DNase footprinting
analysis. Lane 3, no nuclear extract. B, the
proximal NPT2 promoter sequences and the location of PRE.
The lower panel indicates the sequences of oligonucleotides
for EMSA in Fig. 4.
1010/985
nt) and performed EMSA. In nuclear extract isolated from mice fed a low Pi diet, the increased DNA-protein complex was observed in
EMSA (Fig. 4), but the increase in the
DNA/protein complex was not observed with FP-1, FP-2, FP-4, FP-5, and
FP-6 probes (data not shown). The protein-DNA complex was completely
inhibited unlabeled PRE oligonucleotide.
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Fig. 4.
Characterization of nuclear protein binding
to PRE. A, EMSA of the 25-bp PRE-WT sequence. Nuclear
extracts were prepared from the kidneys of mice fed a low
Pi (LPD) or normal Pi diet
(CPD). The competitor used was a wild-type sequence of 25 bp
of PRE oligonucleotide (PRE-WT). B, EMSA of wild-type PRE
and mutations (PRE-MT1, PRE-MT2, and
PRE-MT3). Mice kidney nuclear extracts were used in these
assays to assess the effects of mutations in the PRE (the E box)
sequence on DNA-protein interaction. Oligonucleotides that corresponded
to the E box element were synthesized as described under
"Experimental Procedures." The results of competition experiments
are shown in the lanes with the triangles, with the
competing oligonucleotide indicated above the triangle. The
competitors were added 25-(×25), 50-fold (×50),
and 100-fold (×100) in the EMSA. The data presented are
representative of three independent experiments.
1289 to +54 nt), which contains the PRE (110/985 nt), linked to the luciferase reporter gene in OK
cells. Transfection of p3P1170 into OK cells showed a 6-fold increase
in the activity of control vector-transfected OK cells. Mutation of the
E box in the PRE (p3P1170MT) markedly decreased the basic promoter
activity, suggesting that the PRE is important for the expression of
the NPT2 gene in OK cells (Fig.
5).
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Fig. 5.
Mutation of the PRE in the NPT2 gene promoter. OK cells were transfected with luciferase
reporter genes constructed with the wild-type (p3P1170WT) or the
indicated mutation (p3P1170MT) of the NPT2 promoter.
Luciferase activity was determined as described previously (19). Each
bar represents the mean ± S.E. of five independent
transfections.
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Fig. 6.
Relationship between the PRE binding activity
and serum Pi concentration. The PRE binding activity
were measured in nuclei isolated from mice fed a high Pi, a
normal Pi, or a low Pi diet. Nuclear extracts
were prepared from the kidneys of mice fed a low Pi for 3 days, control Pi diet for 5 days, or a high Pi
diet for 8 days. Serum Pi concentration was measured as
described previously (7). The binding activity was measured by
densitometric analysis. Data represent a relative activity for the
binding activity of the nuclei isolated from normal diet fed mouse
(serum Pi = 6.4 mg/dl).
1010 to 985) as a UAS of the reporter gene's promoter in the yeast one-hybrid system and screened a mouse kidney cDNA library. The yeast strain YBH5 is histidine auxotroph and 3-amino-1,2,4-triazole-sensitive phenotypes depending on the expression of the reporter gene, NPT2 PRE-CYC1-HIS3 on its chromosome. More than 2 × 106 colonies of YBH5 transformants by mouse kidney cDNA
library which produced fusion proteins between cDNA-encoding
protein and the transcriptional activation domain of GAL4 were
screened. Two positive colonies reproducibly showed histidine
prototroph and 30 mM 3-amino-1,2,4-triazole resistance
phenotypes. The plasmids obtained from the two colonies didn't give
histidine prototrophy phenotype to YBH0 strain which has a UAS-less
CYC1-HIS3 reporter gene on its chromosome instead of NPT2
PRE-CYC1-HIS3. It was speculated that the cDNA on the two plasmids encode the binding proteins to the NPT2 PRE sequence on
the reporter gene.
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Fig. 7.
Transfection analysis of TFE3L and
TFE3S. A, schematic representation of TFE3L and TFE3S.
We isolated clones that encoded two isoforms of mTFE3. The mTFE3S
mRNA encodes a polypeptide of 291 amino acids, whereas the mTFE3L
mRNA encodes a polypeptide of 326 amino acids. AAD, acid
activation domain; HLH-LZip, a helix-loop-helix region and
DNA binding region; Pro-rich, proline-rich domain.
B, COS-7 cells expressing TFE3L or TFE3S were cotransfected
with the luciferase reporter gene constructed with the wild-type
(p3P1170WT) or the indicated mutation (p3P1170MT;
the position and nucleotide in the mutation are identical to the MT-3)
of the NPT2 gene promoter. Results are expressed as relative
activity of p3P1170WT vector or p3P1170MT vector, respectively. The
data presented are the means ± S.E. of five experiments.
1289
to +54) linked to the luciferase reporter gene (p3P1170WT) in COS-7
cells. TFE3L stimulated the luciferase activity 6-fold compared with
that of the control vector (Fig. 7B). TFE3S also stimulated
the transcription (2.8-fold). TFE3L induced the luciferase activity in
the COS-7 cells transfected with the NPT2 gene promoter with
the mutation of the PRE (p3P1170MT), but the induction was lower
compared with that in the p3P1170WT clone (Fig. 7B).
1010 to 985
oligonucleotide as a probe (PRE-WT) and several competitor DNA
oligonucleotides (PRE-MT1, PRE-MT2, and PRE-MT3) corresponding to the E
box (Fig. 8). In this experiment, the
mouse TFE3L expression vector was transfected into COS-7 cells, and the
nuclear extract expressing mouse TFE3L was used for EMSA. The PRE-WT
oligonucleotide was in competition for the binding. In addition, the
PRE-MT1 oligonucleotide partially prevented the binding. The PRE-MT2
and PRE-MT3 oligonucleotides were unable to compete for the DNA-protein
binding (Fig. 8, lanes 4 and 5). In addition,
TFE3-specific antibodies completely prevented the DNA-protein binding
in an EMSA (Fig. 8, lane 6). It suggests that TFE3 protein
bound to the 1010 to 985 oligonucleotide.
View larger version (47K):
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Fig. 8.
Effect of TFE3 antibody on the binding of PRE
in the nuclear extract isolated from the kidney cortex of the mice fed
a low Pi diet. A, EMSA with various PRE
sequence as shown in Fig. 3B. EMSA with nuclear extracts
from COS-7 cells transfected with expression vector for mouse TFE3L.
The PRE-MT1, PRE-TM2, and PRE-MT3 oligonucleotides were the same in
those shown Fig. 4, and their sequences are given in Fig.
3B. The antiserum of mouse TFE3 (2 µl) was added to the
binding reaction mixture before the addition of the radiolabeled
oligonucleotide (20). Lane 1, PRE-WT; lane 2,
added cold PRE-WT (×25); lane 3, added PRE-MT1
oligonucleotide (×25); lane 4, added PRE-MT2
oligonucleotide (×25); lane 5, added PRE-MT3
oligonucleotide (×25); lane 6, 2 µl of TFE3 antibody.
B, shift-Western analysis of the protein-DNA complex. The
protein-DNA complexes, separated in polyacrylamide gels, were
transferred onto stacked nitrocellulose and anion-exchange membranes.
The proteins bound to nitrocellulose were identified by immunoblotting
by TFE3-specific antibody (lane 2), while the DNA, which
bound only to the anion-exchange membrane, was detected by
autoradiography (lane 1). Nuclear proteins were prepared
from the kidneys of mice fed a low Pi diet. C,
effect of TFE3 antibody on nuclear protein binding to PRE. The nuclear
extract from kidney cortex of mice fed a low Pi diet
incubated with 32P-labeled PRE-WT oligonucleotide. The TFE3
antibody was added to EMSA reaction medium. Lane 1, DNA
protein complex in EMSA; lane 2, added TFE3 antibody.
View larger version (35K):
[in a new window]
Fig. 9.
Time course of TFE3 and
NPT2 mRNA expression in mice fed a low
Pi diet. A, mice were fed a diet with a
normal Pi level (0.6%) for 7 days, given between 11:00
a.m. and 1:00 p.m. On the 8th day, mice received a diet containing a
low percentage (0.02%) of Pi. At 0, 12, 24, 48, and
72 h after receiving the diet, total RNA (20 µg) was prepared as
described under "Experimental Procedures." Northern blot analysis
was performed using mouse TFE3 and NPT2 cDNA probes. The intensity
of each hybridization was normalized to the GAPDH mRNA.
Data represent means ± S.E. of five animals. B,
Northern blot analysis of TFE3 mRNA. Lane 1, 0 h;
lane 2, 12 h; lane 3, 24 h; lane
4, 36 h.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1010 to
985-bp segment of the human NPT2 gene in Pi
deprivation. Indeed, the incubation with antibodies for mouse TFE3
completely inhibited DNA/nuclear protein binding, suggesting that
protein-DNA complex in EMSA using the renal nuclear extract are
TFE3.
2453 to 2441 relative to the transcription start site of
the opossum NPT2 gene promoter. The sequence is
5'-CACNNTGC-3', and TFE3 can bind this E box sequence. In addition,
25-hydroxyvitamin D3 1-hydroxylase (1-hydroxylase)
catalyzes hydroxylation, mainly in the kidney, of 25-hydroxyvitamin
D3 into 1,25-dihydroxyvitamin D3, a hormonal
form of vitamin D, acting as a key enzyme of vitamin D biosynthesis
(30). Dietary Pi restriction increases 1-hydroxylase activity, while high Pi diet decreases it (31). In the
mouse and human 1-hydroxylase gene promoters, the similar sequence is located at position 660 to 655 relative to the transcription start site of the mouse 1-hydroxylase gene (32, 33). It is possible
that the PRE sequence in the 1-hydroxylase gene promoter may also be
important for dietary Pi regulation (data not shown). To
further clarify the role of the PRE in the NPT2 gene
promoter, we are now cloning mouse and rat NPT2 gene promoter.
-activated signal
transduction pathway. TGF- is known to have widespread regulatory
effects on extracellular matrix and has been implicated as a major
cause of increased extracellular matrix synthesis and buildup of
pathological matrix within glomeruli in experimental glomerulonephritis
(36). A mechanism of the rapid therapeutic effect of a low protein diet on experimental glomerulonephritis is through suppression of TGF- expression and prevention of the induction of extracellular matrix synthesis within the injured glomeruli (36). It is possible that TFE3
regulated the expression of TGF- in this model. Furthermore, in
uremic animals, a low Pi diet prevented
hyperparathyroidism, while a high Pi diet produced
hyperplasia of the parathyroid glands (37). These data suggest that the
Pi response factor TFE3 may regulate cell proliferation and
matrix synthesis.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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