From the Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
Received for publication, February 21, 2001, and in revised form, April 18, 2001
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ABSTRACT |
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The molecular mechanism underlying the renal
expression localization of the thiazide-sensitive Na-Cl
cotransporter (TSC) gene was studied. The
TSC gene was localized to chromosome 19p12-14. In
cultured cells, tissue-specific transcription activity of the 5'-flanking region of the rat rTSC gene (5'FL/rTSC) was
demonstrated, and the major promoter region was located between
position Thiazide-sensitive Na-Cl cotransporter
(TSC)1 is an important
molecule for reabsorption of NaCl in the kidney and a target of
thiazide diuretics. TSC cDNAs were cloned, and the TSC function was
characterized (1-3). Mutations possibly leading to loss-of-function in
the human TSC gene have been shown to cause Gitelman's
syndrome, which is characterized by dehydration, hypokalemic
metabolic alkalosis, hypomagnesemia, and hypocalciuria (4, 5).
Recently, TSC-deficient mice were generated, and their phenotype was
shown to bear a good resemblance to Gitelman's syndrome (6). Thus, TSC
plays an important role not only in NaCl metabolism but in acid-base
balance and in the metabolism of other electrolytes such as potassium, magnesium, and calcium.
TSC mRNA expression has been shown to be localized to the distal
convoluted tubule (DCT) in the kidney by in situ
hybridization histochemistry (7, 8) as well as reverse transcription
and polymerase chain reaction (RT-PCR) with microdissected nephron segments (9). Immunohistochemistry using a specific antibody against
TSC has also shown that immunoreactive TSC is localized to DCT (10,
11). However, no study to uncover the molecular mechanism underlying
the renal tubule-specific TSC expression has yet been performed in any species.
Recently, a transcription factor, hepatocyte nuclear factor-3/folk head
homologue-3 (HFH-3), was isolated and partly characterized (12). HFH-3
belongs to an HFH/winged helix transcription factor family and is
identical to FREAC-6 (13). Members of the family have been shown to be
involved in tissue- or cell-specific gene expression, as well as
cellular differentiation during embryonic development (14, 15).
Interestingly, HFH-3 mRNA was reported to be localized to the
epithelium of the DCT, where TSC is also localized. We therefore
hypothesized that HFH-3 might be involved in the TSC gene expression.
In the present study, we studied the tissue-specific expression of the
TSC gene in terms of gene transcription by an in vivo as well as in vitro experiments. The structure of the
5'-flanking region of the rat TSC gene (5'FL/rTSC) was
revealed, and the rTSC gene was mapped to chromosome
19p12-14 by FISH. The transcription function was shown to be
tissue-specific in cultured cells. Moreover, we generated transgenic
rats harboring the 5'FL/rTSC fused upstream of the LacZ gene
to examine the tissue-specific transcription of 5'FL/rTSC.
Immunohistochemical analysis clearly demonstrated that
LacZ expression was localized to DCT in the
transgenic rat kidney, indicating that the 5'FL/rTSC regulates the
DCT-specific TSC localization in vivo. Furthermore, we
identified a functional HFH-3 binding site in the major promoter region
of 5'FL/rTSC, which was actually bound by in vitro
synthesized HFH-3. HFH-3 may be involved at least in part in the
DCT-specific expression of TSC.
Southern Blot Analysis--
To use as a probe in Southern blot
analysis of rTSC gene, an rTSC cDNA fragment
was first cloned by a PCR-based method with a rat kidney cDNA
library (rat kidney Marathon-ready cDNA,
CLONTECH) as a template using a pair of primers
(sense, 5'-CCC GGA GCC ATA ATG GCA GAG CTA CCC G-3' (from position 1 to
28 in previously reported rTSC cDNA (2)); antisense, 5'-CTT CTC CAG
CTG GAG AGC GTG AGT TCC G-3' (from position 3,070 to 3,097 in rTSC
transcript)) under the following PCR condition: 95 °C, 30 s and
70 °C, 5 min for 25 cycles. The PCR product was then subcloned into
pCR-TOPO (Invitrogen) by a TA cloning method. Sequencing was performed to confirm the complete sequence identity with the previously reported
rTSC cDNA (2). The BalI digested fragment of the rTSC cDNA (892 bp) was labeled with [ FISH--
Chromosome mapping of the rTSC gene was
carried out by FISH based on a previously reported method (17). At
first, three different DNA fragments of the rTSC gene were
amplified by PCR with three primer pairs (sense-1, 5'-CCC GGA GCC ATA
ATG GCA GAG CTA CCC G-3' (from position 1 to 28 in previously
reported rTSC cDNA (2)), and antisense-1, 5'-GCC ACA CCC ACG GCA
TTG GCA AAG GCG A-3' (from position 693 to 720 in rTSC transcript);
sense-2, 5'-CCT CCT CAT CCC TTA TCT CCT GCA CCG C-3' (from position
2,570 to 2,599 in rTSC transcript), and antisense-2, 5'-CTC GTC CTT GAG
CCG TCA TTG AGA CGG-3' (from position 2,799 to 2,826 in rTSC transcript); and sense-3, '-CCG TCT CAA TGA CGG CTT CAA GGA CGAG-3' (from position 2,799 to 2,826 in rTSC transcript), and antisense-3, 5'-CTT CTC CAG CTG GAG AGC GTG AGT TCC G-3' (from position 3,070 to
3,097 in rTSC transcript)) under the following PCR condition: 98 °C,
10 s; 68 °C, 8 min, for 30 cycles, and final extension at
72 °C for 10 min. The PCR products were labeled with digoxigenin and
used as probes in FISH.
Cloning of the 5'-Flanking Region of rTSC Gene
(5'FL/rTSC)--
The 5'FL/rTSC was cloned by a PCR-based method using
the Genome Walker Kit (CLONTECH) for rat gene
cloning. Briefly, the first PCR was carried out based on the
manufacturer's instruction with the gene-specific primer (GSP)-1,
5'-ATG GGT CAA ATG GCT GGG CTG GCT ATT G-3' (designed to hybridize with
the region from position 120 to 147 in rTSC cDNA (2)), and the
adapter primer (AP)-1, 5'-GTA ATA CGA CTC ACT ATA GGG C-3' (supplied in
the kit). Nested PCR was then performed with the GSP-2, 5'-TGC ACA GAG
CAT CGC CTG GCA TCT CTG T-3' (designed to anneal to the region from
position 31 to 58 in rTSC cDNA (2)), and the AP-2, 5'-ACT ATA GGG
CAC GCG TGG T-3', using the first PCR product as a template. The
resultant PCR product was subcloned into pCR-TOPO (Invitrogen), and
sequenced in both directions. Putative transcription factor binding
sites in 5'FL/rTSC were identified by the TRANSFAC 3.4 data base using Transcription Element Search Software (TESS) (18).
Primer Extension Method--
Primer extension was performed
using the primer extension system-AMV reverse transcriptase (Promega)
by a modification of the previously reported method (19). Total RNA was
extracted from rat renal cortex by the guanidinium
thiocyanate-cesium chloride centrifugation method (16). An
oligonucleotide (5'-ACT GCA CAG AGC ATC GCC TGG CAT CTC TGT CAC GGG TAG
C-3') designed to anneal to the region from position 21 to 60 in rTSC
cDNA (2) was labeled with IRD700 (Aloka) at the 5'-end and used as
a primer. Annealing was carried out with 1 pmol of the primer and 25 µg of total RNA at 50 °C for 8 h, and the extension
reaction was performed at 42 °C for 2 h. The extended product
was resolved on a 0.8% polyacrylamide gel. To determine the length of
the DNA product, the sequence ladder of pBluescript SK(+) (Invitrogen)
was also resolved in the same gel. The fluorescent DNA was analyzed by
automated DNA analyzer (LI-COR dNA analyzer 4200).
Rapid Amplification of the 5'-end of cDNA
(5'-RACE)--
5'-RACE was performed with rat kidney Marathon-ready
cDNA (CLONTECH). PCR was carried out according
to the manufacturer's instruction with the adapter primer, 5'-GCC ACA
CCC ACG GCA TTG GCA AAG GCG A-3' (supplied in the kit), and an
rTSC-specific primer, 5'-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3' (from
position 693 to 720 in rTSC transcript), under the following
conditions: 95 °C, 30 s; 68 °C, 2 min, for 30 cycles. The
PCR product was then subcloned into pCR-TOPO (Invitrogen) by a TA
cloning method. The 5'-end of the rTSC cDNA was determined by sequencing.
Cell Culture--
HEK293 (a human embryonic kidney epitheloid
cell line), A10 (a rat vascular smooth muscle cell line), and HepG2 (a
human hepatocyte cell line) cells were cultured in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum.
RT-PCR--
RT-PCR was performed to confirm endogenous
expression of TSC mRNA in HEK293, HepG2, or A10 cells using a
One-step RNA PCR kit (TaKaRa) according to the manufacturer's
instruction. Total RNA was extracted from these cells by RNeasy kit
(Qiagen). Endogenous hTSC mRNA in HEK293 or HepG2 cells were
detected with a pair of primers: sense, 5'-AGG CAG GCA TCG TCC TGA CCT
G-3' (from position 519 to 540 in hTSC cDNA (3)); antisense, 5'-GCA
GGG TCC TTG AGG TCA CCA G-3' (from position 1,107 to 1,128 in hTSC
transcript). rTSC mRNA in A10 cells was also detected by RT-PCR
with a pair of primers: sense, 5'-AAT GGC AAG GTC AAG TCG G-3' (from
position 608 to 626 in rTSC cDNA (2)); antisense, 5'-GAT GCG GAT
GTC ATT GAT GG-3' (from position 793 to 812 in rTSC transcript). Human mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also detected with a pair of primers: sense, 5'-CCA TGG AGA AGG CTG GGG-3'
(from position 371 to 388 in human (h) GAPDH cDNA); antisense, 5'-CAA AGT TGT CAT GGA TGA CC-3' (from position 546 to 565 of hGAPDH
transcript (20)). rGAPDH mRNA from rat renal cortex was detected by
RT-PCR with a pair of primers: sense, 5'-TCC CTC AAG ATT GTC AGC AA-3'
(from position 451 to 470 in rGAPDH cDNA (21)); antisense, 5'-AGA
TCC ACA ACG GAT ACA TT-3' (from position 739 to 758 in rGAPDH transcript).
Construction of a Human HFH-3 Expression Vector--
The protein
coding region of hHFH-3 cDNA was cloned by a PCR-based method with
human kidney Marathon-ready cDNA using a pair of primers (sense,
5'-AGT TCC CCA GCA TCG GCC AGG AGC-3' (from position 46 to 70 in hHFH-3
cDNA (12)); antisense, 5'-TCT GGC ATG TCC ACC TGG CTC AGG-3' (from
position 1,148 to 1,171 in hHFH-3 transcript)) under the following PCR
condition: 95 °C, 30 s; 68 °C, 2 min, for 30 cycles. An
expression vector of hHFH-3 (pcHFH-3) was synthesized by subcloning the
hHFH-3 cDNA into an expression vector, pcDNA1/Amp (Invitrogen).
Sequencing was performed to confirm the complete sequence identity to
the one reported previously (12).
Construction of Luciferase Reporter Gene Vectors--
To examine
the transcription activity of 5'FL/rTSC, a chimeric firefly luciferase
expression vector containing 5'FL/rTSC (rTSC/Luc) was constructed.
5'FL/rTSC was subcloned into promoter-less firefly luciferase
expression vector, PicaGene Basic Vector (Nippon Gene). Deletion
mutants of rTSC/Luc were then created with exonuclease III/Mung bean
nuclease as described previously (22, 23). A double strand DNA
containing the putative HFH-3 binding site located from position Transient Transfection and Luciferase Assay--
HEK293, A10,
and HepG2 cells were cultured in 6-well culture plates before
transfection. Transfection was performed with 2 µg/well of luciferase
expression vector as well as 0.5 µg/well control Electrophoretic Mobility Shift Assay (EMSA)--
An expression
construct to synthesize a fusion protein of the HFH-3 DNA binding
domain and glutathione S-transferase (GST) (12) was a
generous gift from Dr. Robert H. Costa (University of Illinois at
Chicago). GST-HFH-3 fusion protein was isolated from Escherichia
coli cultures and purified by glutathione-Sepharose 4B (Amersham
Pharmacia Biotech) according to the manufacturer's instruction. The
purity of GST-HFH-3 was confirmed by SDS-polyacrylamide gel
electrophoresis followed by Coomassie staining (data not shown). The
double strand DNA of HFH-3/rTSC was radiolabeled and used as a probe.
EMSA was performed with the probe and in vitro synthesized GST-HFH-3 as described previously (23, 24). A double strand DNA
containing the consensus HFH-3 binding was created by annealing with
oligonucleotides 5'-AGC TGC ACG TTC GTT GTT TAT GTA CCG AGC G-3' (sense) and 5'-TCG ACG CTC GGT ACA TAA ACA ACG AAC GTG
C-3' (antisense), in which underlining indicates the consensus HFH-3 binding sequence, and was used as a competitor. mHFH-3/rTSC
oligonucleotides (containing the mutated HFH-3 binding site) were also
used as competitor.
Generation of Transgenic Rats Bearing 5'FL/rTSC-LacZ--
The
transgene used herein consisted of the 2,110-bp rTSC promoter fused to
the LacZ gene. A fragment encoding the rTSC promoter region
(
Transgenic rats were generated using pronuclear microinjection as the
standard procedure. The microinjection was performed by YS New
Technology Laboratory (Tochigi, Japan). Briefly, the transgene fragment
was microinjected to the pronuclei of fertilized single-cell oocytes
obtained from Harlan Sprague-Dawley rats. Embryos that survived
microinjection were transferred into the oviduct of pseudo-pregnant
Wistar rats. Transgenic rats were selected with amplification of LacZ
by PCR from genomic DNA extracted from tails. The number of integrated
copies of the transgene was estimated by Southern blotting with the
transgene fragment as a probe.
Immunohistochemistry--
The transgenic and nontransgenic rats
(16-week) were anesthetized with diethyl ether, and perfused with
saline through cannulae inserted into the left ventricles. The kidneys
were removed and fixed for 24 h with 20% formaldehyde and mounted
onto paraffin blocks. Rabbit anti- Data Analysis--
p < 0.05 by Student's
t test was considered statistically significant.
Southern Blot Analysis and Chromosome Mapping of rTSC Gene--
A
single band was observed in either the BamHI or
HindIII digest of rat genomic DNA (~6.5 and ~4.2
kilobase pairs in length, respectively), suggesting that
rTSC is a single copy gene (Fig. 1). Additionally, two bands in the
EcoRI digest suggested the presence of EcoRI site
in the intron between TSC exons complementary to the probe. To
determine the chromosomal localization of rTSC gene, we next
performed FISH using three different DNA fragments of rTSC
gene as probes. After hybridization, one hundred metaphases were
analyzed by recording the number and position of fluorescent spots on
chromosomes. Twin fluorescent spots were identified in eighty three
metaphases at chromosome 19, and single fluorescent spot at the same
chromosome in twenty metaphases. A typical metaphase showing the
presence of twin fluorescent spots at chromosome 19 is shown in Fig.
2A. The position of
fluorescent spots were shown in Fig. 2B, and these spots
were clustered at 19p12-14 (p < 0.01).
Structure of 5'FL/rTSC--
A 2.1-kilobase pair fragment of
5'FL/rTSC was isolated and sequenced (DDBJ/EMBL/GenBankTM
data bases with accession number
AB024534). 5'FL/rTSC (Fig. 3) contains
several putative consensus transcription factor recognition sequences
such as: a TATA box at 42 bases upstream ( Transcription Initiation Site of rTSC Gene--
The transcription
initiation site of the rTSC gene was determined by both the
primer extension method and 5'-RACE. Total RNA extracted from rat renal
cortex was reverse transcribed using the 5'-end labeled DNA primer
(designed to be a complement to the region from position 21 to 60 of
rTSC cDNA). As shown in Fig. 4A, the longest extended
product had 67 bases in length, indicating that the transcription
initiation site of the rTSC gene is located 8 bases upstream
of the 5'-end of the cloned cDNA (2) (Fig. 4C).
Moreover, we performed 5'-RACE to confirm further the transcription initiation site. A PCR product 720 bp in length was subcloned, and the
5'-end was determined by sequencing. As shown in Fig. 4B,
the 5'-end was completely identical to the transcription initiation site determined by the primer extension method (Fig.
4C).
Transcription Activity of 5'FL/rTSC--
Luciferase reporter gene
assay was performed to examine the transcription activity of 5'FL/rTSC.
HEK293 cells (in which endogenous TSC mRNA expression was detected)
were transfected with either luciferase expression vector alone
(control) or rTSC/Luc. Luciferase expression with rTSC/Luc was
significantly higher than with control (Fig.
5A; 26.2 ± 1.8-fold
compared with control) in transfected HEK293 cells, suggesting that the
5'FL/rTSC is transcriptionally active. We next conducted the deletion
analysis of 5'FL/rTSC. As shown in Fig. 5B, transfection of
the luciferase expression vector containing the full-length (~2.1
kilobase pairs) 5'FL/rTSC (-2,093 rTSC/Luc) showed the highest
luciferase expression, and the transcription activity of 5'FL/rTSC was
dependent on the fragment length. Luciferase activity was high in cells
transfected with the rTSC/Luc containing deletion fragments from
position Tissue-specific Transcription of rTSC Gene--
Endogenous
expression of TSC mRNA was examined in HEK293, HepG2, or A10 cells
by the RT-PCR method. An expected length of PCR product (609 bp) was
detected with the total RNA from HEK293 cells (Fig.
6A), and its sequence identity
to rTSC was confirmed, whereas no PCR products for rTSC were detected
in the other cells. As a positive control, GAPDH mRNA was detected
in either HEK293, HepG2, or A10 cells by RT-PCR (Fig. 6A).
The PCR product of rTSC mRNA was also detected with the total RNA
from renal cortex by the same RT-PCR protocol. TSC was thus indicated
to be expressed in HEK293 cells but not in either HepG2 or A10 cells.
We therefore used these cell lines to examine the tissue-specific
transcription activity of 5'FL/rTSC. As shown in Fig. 6B,
the transcription activity of 5'FL/rTSC estimated by luciferase
expression was pronounced in HEK293 cells, whereas it was modest
in both HepG2 and A10 cells. It was thus suggested that the 5'FL/rTSC
is able to regulate the tissue-specific gene transcription.
Establishment of Transgenic Rats Bearing 5'FL/TSC-LacZ--
227
microinjected oocytes were transplanted into eight Wistar rats. 58 rats
were born, seven of which had the transgene. The copy number of the
integrated transgene was estimated as three to six copies by Southern
blotting. Every transgenic rat grew normally in appearance. The
transgene was inherited in the Mendelian fashion.
Expression Co-localization of LacZ with TSC--
The transgenic
rat harboring six copies of the transgene was subjected to
immunohistochemistry. In the cortical region of the transgenic rat
kidney, immunoreactive A Role of HFH-3 on Transcription Activity of 5'FL/rTSC--
To
examine the effect of HFH-3 overexpression on the transcription
activity of 5'FL/rTSC, hHFH-3 cDNA was isolated, and an expression
vector carrying the cDNA (pcHFH-3) was synthesized. In
vitro translated HFH-3 with this construct was resolved in SDS-
polyacrylamide gel electrophoresis, and we identified a new band
with the expected size corresponding to HFH-3 protein (data not shown).
As shown in Fig. 8, luciferase expression
in HepG2 cells transfected with
To evaluate the transcription responsiveness of HFH-3 to putative HFH-3
binding site, we mutated this site and examined the effect of HFH-3
overexpression on its transcription activity. In HEK293 cells
expressing HFH-3 mRNA (data not shown), transcription activity of
Functional Characterization of HFH-3 Binding Site in
5'FL/rTSC--
We next characterized the HFH-3 binding site in
5'FL/rTSC (HFH-3/rTSC) by a luciferase reporter gene assay using a
heterologous promoter. In HepG2 cells (which lacks HFH-3 mRNA
expression (data not shown)) transfected with HFH-3/rTSC-tk-Luc,
luciferase expression was increased according to the amount of
transfected pcHFH-3 (Fig. 10,
hatched column). The overexpression of HFH-3 induced
2.78 ± 0.09-fold increases in luciferase expression at the
maximum compared with control (mock transfection) (p < 0.01), whereas no increment in luciferase expression was observed in
cells transfected with mHFH-3/rTSC-tk-Luc (Fig. 10, open
column).
Direct Binding of HFH-3 to HFH-3/rTSC in EMSA--
EMSA was
performed to examine a direct binding between HFH-3 and HFH-3/rTSC. As
shown in Fig. 11, in vitro
synthesized HFH-3 protein reacted with the radiolabeled HFH-3/rTSC and
formed a protein-DNA complex (Lane 1). Using
unlabeled HFH-3/rTSC as a cold competitor, the formation of
protein-DNA complex was significantly inhibited (lane 2).
Moreover, the complex disappeared with an excess of oligonucleotides
containing the HFH-3 binding consensus sequence as a cold competitor
(lane 3). However, the protein-DNA complex was kept
unchanged with an excess of oligonucleotides of mHFH-3/rTSC as a
competitor (lane 4). These results indicate that HFH-3/rTSC
is actually bound by HFH-3 and the binding is sequence-specific.
The rTSC gene was shown to be localized to 19p12-14 by
FISH. In either human or mouse, the TSC gene has also been
mapped to chromosome 16q13 (3, 4, 25) or 8 (26), respectively. These results suggest the segmental chromosome similarity in their regions.
Genes of the Na-K-Cl cotransporter (NKCC2) (2), sodium phosphate
cotransporter (NPT2) (27), chloride channel CLC-K1 (28), and
kidney-specific cadherin (Ksp-cadherin) (29) have been shown to be
expressed specifically in the kidney. The transcription regulatory
regions of those genes have already been cloned (30-32), and the
tissue-specific transcription mechanisms have been partly examined.
Particularly, the kidney-specific CLC-K1 gene
transcription has recently been shown to depend on an interaction
between a transcription factor, MAZ, and a transcription repressor,
KKLF (33). In the present study, we analyzed the transcriptional function of the rTSC gene in terms of tissue-specific gene expression.
The 5'FL/rTSC contains two putative glucocorticoid-responsive elements
(Fig. 3). It has been reported that glucocorticoids and
mineralocorticoids stimulate the thiazide-sensitive sodium transport at
DCT, inducing an increase in the number of thiazide diuretic binding
sites (34, 35) or in TSC protein expression (36). The
glucocorticoid-responsive elements may possibly be involved in the
steroid-induced TSC expression at the gene transcription level. A
putative cAMP-responsive element was also identified, implying that TSC
transcription could be regulated by intracellular cAMP formation
by an agent such as calcitonin, which has been shown to increase the
number of renal thiazide diuretic binding sites (37). Moreover, a
couple of putative C/EBP Luciferase reporter gene analysis was performed with the chimeric
reporter expression vector containing 5'FL/rTSC (rTSC/Luc). When
rTSC/Luc was transfected into HEK293 cells (which express TSC
mRNA), marked luciferase expression was observed (Fig.
5A). Additionally, the transcription activity is dependent
upon the length of 5'FL/rTSC, indicating that the 5'FL/rTSC has a
significant transcription function. Deletion analysis also showed a
marked decrease (75%) in the transcription activity between position Tissue-specific transcription of TSC gene was focused in the
present study. As shown in Fig. 6A, transcription activity
of 5'FL/rTSC was pronounced in HEK293 cells expressing TSC mRNA, whereas it was suppressed in either HepG2 or A10 cells lacking TSC
mRNA expression (Fig. 6), suggesting that the 5'FL/rTSC plays a
crucial role in the tissue-specific TSC gene transcription. To further study the tissue-specific transcription of TSC in
kidney, we generated the transgenic rats with the 5'FL/rTSC fused
upstream of the LacZ gene as a transgene. As shown in Fig.
7, immunoreactive HFH-3 belongs to the HFH/winged helix transcription factor family (12),
and its family members are involved in tissue-specific gene expression
and cell differentiation during embryonic development (14, 15). HFH-3
mRNA is localized to DCT in kidney (12), but the target gene of
HFH-3 has not yet been identified. We hypothesized that HFH-3 might be
involved in the renal tubule-specific gene transcription of TSC,
because a putative HFH-3 binding site was identified in the 5'FL/rTSC.
We first examined the effect of HFH-3 overexpression on the
transcription activity of 5'FL/rTSC in the transfected HEK293 cells. As
shown in Fig. 7, the stimulation was significantly observed with
EMSA was performed to examine an interaction between HFH-3 and
HFH-3/rTSC. In EMSA, a protein-DNA complex was formed with the
radiolabeled HFH-3/rTSC and in vitro synthesized HFH-3. The complex formation was inhibited with an excess of the HFH-3 consensus sequence oligonucleotides but not with an excess of mHFH-3/rTSC. HFH-3
therefore can actually bind the HFH-3/rTSC and may lead to
transactivation of TSC gene. Putative target genes for
HFH-3 have been suggested to be the mineralocorticoid
receptor, Na/H exchanger NHE3,
Na/K-ATPase In conclusion, we have localized the rTSC gene to chromosome
19p12-14 and characterized its promoter region. The promoter determines the DCT-specific gene expression of rTSC, and
DCT-specific HFH-3 may be involved at least in part in renal
tubule-specific rTSC gene transcription.
580 and
141. To further examine the tissue-specific
transcription, transgenic rats harboring the 5'FL/rTSC fused upstream
of the LacZ gene were generated. Immunohistochemical
analysis clearly showed that LacZ gene expression was
co-localized to distal convoluted tubules (DCT) with TSC, indicating
that the 5'FL/rTSC regulates the renal tubule-specific TSC expression.
Because a transcription factor, HFH-3 (hepatocyte nuclear factor-3/folk
head homologue-3), had also been localized to DCT, a possible role of
the putative cis-acting element (HFH-3/rTSC,
400/
387
position) for HFH-3 binding in the tissue-specific transcription was
examined. Deletion and mutation analyses suggested that transcription
of the HFH-3/rTSC was actually responsive to HFH-3, and electrophoretic
mobility shift assay showed a direct binding of in vitro
synthesized HFH-3 to the HFH-3/rTSC. In conclusion, the
rTSC gene is localized to rat chromosome 19p12-24. The
transcription regulatory region of the TSC gene confers
DCT-specific gene expression. DCT-specific transcription factor HFH-3
may be involved in the renal tubule-specific transcription of
TSC gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP by a
random primer method (BcaBEST labeling kit, TaKaRa) and used as a
probe. Rat genomic DNA was extracted from liver as described previously
(16). Genomic DNA was then digested with restriction enzymes
BamHI, EcoRI, or HindIII. Digested DNA was then resolved on a 0.9% agarose gel and transferred to a nylon membrane (HyBond N+, Amersham Pharmacia Biotech).
Hybridization was performed in Rapid-Hyb Buffer (Amersham Pharmacia
Biotech) at 65 °C for 2 h with a radiolabeled probe (25 ng).
The membrane was then incubated with 2× SSC, 0.1% SDS at room
temperature for 20 min, 0.1× SSC, 0.1% SDS at 65 °C for 15 min,
and 0.1× SSC, 0.1% SDS at 65 °C for 15 min. The blot was exposed
to an x-ray film (Kodak) for 72 h.
400
to
387 in 5'FL/rTSC (HFH-3/rTSC) was made by annealing
oligonucleotides 5'-AGC TAC AGG CTT CCT TTT TGT TAT ATG CAT
GTC CT-3' (sense) and 5'-TCG AAG GAC ATG CAT ATA ACA AAA AGG
AAG CCT GT-3' (antisense), in which underlining indicates the
putative HFH-3 binding site (consensus sequence DBD TRT TTR YDT D (12)) located from position
400 to
387 in 5'FL/rTSC. The product was then
subcloned upstream of a thymidine kinase gene promoter in a luciferase
expression vector (HFH-3/rTSC-tk-Luc). An expression vector carrying
the mutated HFH-3 binding site (mHFH-3/rTSC-tk-Luc) was also
synthesized with oligonucleotides 5'-AGC TAC AGG CTT CCT
TGG GGT TAT ATG CAT GTC CT-3'
(sense) and 5'-TCG AAG GAC ATG CAT ATA ACC
CCA AGG AAG CCT GT-3' (antisense), in which italic
capital letters indicate mutated nucleotides and underlining indicates
the putative HFH-3 binding site. The same mutation was also introduced
by QuikChange site-directed mutagenesis kit (Stratagene) in the
580
rTSC/Luc with oligonucleotides 5'-AAG ACA GGC TTC CTT GGG
GTT ATA TGC ATG TCC-3' (sense) and 5'-GGA CAT GCA TAT
AAC CCC AAG GAA GCC TGT CTT-3'
(antisense), in which italic capital letters indicate mutated
nucleotides and underlining indicates the putative HFH-3 binding site.
The resultant mutated construct was designated
580 (mHFH-3)
rTSC/Luc.
-galactosidase
expression vector (pSV-
-galactosidase control vector, Promega) using
DOTAP liposomal transfection reagent (Roche). After the transfection,
culture medium was replaced with Dulbecco's modified Eagle's medium
containing 1% charcoal/resin-stripped fetal bovine serum.
48 h after transfection, luciferase expression was determined by
measuring its enzymatic activity using a luciferase assay system
(Promega). Transfection efficiency was normalized by
-galactosidase
expression (determined by its enzymatic activity) as reported
previously (23). To examine the effect of HFH-3 on the transcription
activity of 5'FL/rTSC, pcHFH-3 or mock DNA was co-transfected with
580 rTSC/Luc,
580 (mHFH-3) rTSC/Luc, HFH-3/rTSC-tk-Luc, or
mHFH-3/rTSC-tk-/Luc in HepG2 cells, which have been shown to lack HFH-3
expression (12).
2,110 to +45) was excised from rTSC/Luc with KpnI and subcloned to the KpnI site of pSV-
-galactosidase control
vector (Promega). The KpnI site of this plasmid was located
87-bp upstream of the LacZ coding region, and the frame for the intact
LacZ gene expression was confirmed by sequencing. The
transgene fragment excised by digestion with SalI was
purified using the QIAquick gel extraction kit (Qiagen).
-galactosidase polyclonal antibody
(Biogenesis) was used at a dilution of 1:200. Rabbit anti-TSC antibody,
a gift from Dr. Steven C. Hebert (11), was used at a dilution of 1:250. Staining was performed by the avidin-biotinylated peroxidase complex method using an RTU Vectastain universal avidin-biotin complex kit (ABC, Vector) and a VIP substrate kit (Vector). Sections were counter-stained with hematoxylin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Southern blot analysis of the rTSC
gene. Rat genomic DNA was digested with BamHI,
EcoRI, or HindIII and hybridized with the
radiolabeled BalI fragment of rTSC cDNA. A single band
is observed in the BamHI or HindIII digested
sample.
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Fig. 2.
Chromosomal localization of the
rTSC gene. One hundred myeloblasts in the
metaphase were screened by FISH with three different labeled DNA
fragments of rTSC gene. A, the typical
metaphase representing the twin spots at chromosome 19p12-14 locus in
the FISH. B, a diagram illustration of the position of
fluorescence spots. Twin spots were detected at chromosome 19 in 83 of
100 metaphases, and a single spot was also identified at the same
chromosome region in 12 metaphases. These spots are shown to be
clustered at 19p12-14 (p < 0.01).
42) of the transcription initiation site; three SRY binding sites (SRY)
at position
1,931,
1,728, and
189; five Pit-1 binding sites
(Pit-1) at positions
1,486,
1,328,
1,219,
990, and
809; a CCAAT/enhancer-binding protein (C/EBP) family
binding site at position
463 and three C/EBP
binding sites
(C/EBP
) at positions
1,461,
1,118, and
1,011; two
SP-1 binding sites (SP-1) at positions
153 and
131; two
glucocorticoid-responsive elements (GRE) at positions
1,091 and
579; cAMP-responsive element (CRE) at position
1,021; an AP-2 binding site (AP-2) at position
788; a
c-MYC binding site (c-myc) at position
141; and an HFH-3 binding site at position
393.
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Fig. 3.
Nucleic acids sequence of the 5'flanking
region of the rTSC gene. An asterisk
and position +1 indicate the transcription initiation site. An
underline indicates the protein coding sequence.
Double underlines indicate the following putative
cis-acting elements: TATA, TATA box;
SRY, SRY biding site; Pit-1, Pit-1 binding site;
C/EBP, CAAT/enhancer-binding protein (C/EBP) binding site;
SP-1, SP-1 binding site; HFH-3, HFH-3 binding
site; GRE, glucocorticoid-responsive element;
CRE, cAMP-responsive element; AP-2, AP-2 binding
site; c-MYC, c-MYC binding site. The
sequence has been deposited in the DDBJ/EMBL/GenBankTM data
bases with accession number AB024534.
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Fig. 4.
Determination of the transcription initiation
site of rTSC gene. A, a blot obtained
by the primer extension method. An asterisk indicates the
longest extended product, 67 bases in length. B, sequence of
the 5'end of rTSC cDNA obtained by the 5'RACE. The arrow
region indicates the primer sequence, and an asterisk
indicates the transcription initiation site. C, the sequence
around the transcription initiation site (asterisk) in the
rTSC gene. The arrow region is the primer
sequence used in the primer extension, and the underline
indicates the protein coding region.
2,093 to
580. However, further deletion of the promoter to
position
141 resulted in a marked decrease in luciferase
activity.
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Fig. 5.
Transcription activity of 5'FL/rTSC.
A, luciferase expression in HEK293 cells transfected with
either rTSC/Luc or a control vector (lacking the 5'FL/rTSC). The
vertical line indicates the relative luciferase activity
after normalization of transfection efficiency by -galactosidase
activity (n = 6). Bars represent the
mean ± S.E. (n = 6; *, p < 0.01). B, transcription activities of deleted fragments of
5'FL/rTSC in HEK293 cells. Deleted fragment lengths are shown on the
left, and bars on the right represent
the mean ± S.E. of relative luciferase activity
(n = 6; *, p < 0.01 compared with
580 rTSC/Luc).
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Fig. 6.
Tissue-specific transcription activity of
5'FL/rTSC. A, endogenous mRNA expression of TSC in
cultured cells, HEK293 (human renal tubular epithelial cell line),
HepG2 (human hepatocarcinoma cell line), and A10 (rat aortic smooth
muscle cell line). PCR was performed with (+) or without ( ) RT.
B, transcription activity of 5'FL/rTSC in these cells.
Bars represent the mean ± S.E. (n = 6;
*, p < 0.01 compared with the levels in HEK293
cells).
-galactosidase was observed at cortical
distal tubules (Fig. 7A),
whereas no stain was observed in the control rat (Fig.
7B). Using the sequential sections from the transgenic rat
kidney, we observed co-localization of immunoreactive
-galactosidase
(arrows, Fig. 7, C and E) and TSC
(arrowheads, panels D and F). No
immunostaining was observed in the medullar region (Fig.
7G).
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Fig. 7.
Expression of
-galactosidase in transgenic rats harboring the
5'FL/rTSC fused upstream of the LacZ gene. In the
cortical region of transgenic rat kidney, immunoreactive
-galactosidase is observed at distal tubules (A), whereas
no stains are observed in a control rat (B). In the
sequential sections from the transgenic rat, co-localization of
immunoreactive
-galactosidase (arrows, C and
E) and TSC (arrowheads, D and F) is
observed at the cortical distal tubules. In the medullar region, no
immunoreactive
-galactosidase (G) is detected.
2,093 rTSC/Luc was significantly
higher by cotransfection with pcHFH-3 than with mock (2.28 ± 0.14-fold expression compared with mock; p < 0.01).
This enhancement of transcription with pcHFH-3 was also observed in
transfection with
1,139 rTSC/Luc and
580 rTSC/Luc, whereas it was
not observed with
141 rTSC/Luc. Thus, the HFH-3-responsive element
was suggested to be present between position
580 and
141, and we
identified an element homologous to HFH-3 binding consensus sequence
(DBD TRT TTR YDT D) at position
393 (TCC TTT TTG TTA TA).
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Fig. 8.
Effect of HFH-3 overexpression on the
transcription activity of 5'FL/rTSC. HFH-3 was overexpressed with
pcHFH-3 in HepG2 cells (lacking TSC or HFH-3 mRNA expression)
transfected with deletion mutants of 5'FL/rTSC. Deleted fragment
lengths are shown on the left, and the bars on
the right represent the mean ± S.E. of relative
luciferase activity (n = 6; *, p
<0.01). Hatched or open bars represent the data
from cells transfected with pcHFH-3 (HFH-3 (+)) or with a vector
lacking HFH-3 cDNA (Control), respectively.
580 (mHFH-3) rTSC/Luc was reduced markedly (Fig. 9, open column) compared with
that of
580 rTSC/Luc (Fig. 9, hatched column).
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Fig. 9.
Loss of transcription activity by a mutation
of HFH-3/rTSC. Transcription activity of HFH-3/rTSC was examined
in HEK293 cells (expressing HFH-3 mRNA) using 580 (mHFH-3)/rTSC
in which the HFH-3/rTSC was mutated. Bars represent the
mean ± S.E. (n = 12; *, p < 0.01).
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Fig. 10.
Transactivation of HFH-3/rTSC by HFH-3.
Luciferase constructs containing the truncated wild-type HFH-3/rTSC
(HFH-3/rTSC-tk-Luc) or mutated HFH-3/rTSC (mHFH-3/rTSC-tk-Luc) fused
upstream of tk promoter was transfected into HepG2 cells (lacking TSC
expression), and HFH-3 was overexpressed by co-transfection with
pcHFH-3. The luciferase expression with HFH-3/rTSC-tk-Luc is stimulated
in proportion to the amounts of transfected pcHFH-3 DNA, whereas it is
kept unchanged in cells transfected with HFH-3/rTSC-tk-Luc.
Bars represent the mean ± S.E. (n = 10; *, p < 0.01).
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Fig. 11.
Protein-DNA interaction between HFH-3 and
HFH-3/rTSC. EMSA was performed with the in vitro
synthesized GST-HFH-3 fusion protein (GST-HFH-3) and
32P-labeled HFH-3/rTSC fragment
(32P-HFH-3-TSC) as a probe. Formation of a
protein-DNA complex is observed (lane 1). The complex
formation is significantly inhibited with an excess of unlabeled
HFH-3/rTSC fragment (T) (lane 2) as well as
oligonucleotides containing consensus HFH-3 binding site (C)
(lane 3). On the other hand, no inhibition of the complex
formation is observed with an excess of oligonucleotides containing the
mutated (m) HFH-3/rTSC (lane 4). Lane
5, radiolabeled probe alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
binding sites were identified. Because
C/EBP
is expressed in kidney and induced by interleukin-6 mediating
an inflammatory response (38), TSC gene transcription
may possibly be affected by inflammation via interleukin-6.
580 and
141 (Fig. 5B), suggesting that a major promoter
is located between these positions.
-galactosidase was present at the same tubular
region as TSC, probably at DCT in the renal cortical region, and
neither was detected in the other renal tissues. Thus, this in
vivo gene transcription model has clearly demonstrated that
DCT-specific TSC gene expression is dependent on the
5'FL/rTSC, and it is suggested that the region contains some important
elements responsible for the DCT-specific transcription.
2,093,
1,139, and
580 rTSC/Luc but not with
141 rTSC/Luc.
Consistent with this result, there is a putative HFH-3 binding site
(HFH-3/rTSC) at the
393 position (Fig. 3), 5'-TCC TTT TTG TTA T-3',
which bears a homology to the previously reported HFH-3 binding
sequence (DBD TRT TTR YDT D) (12) by 10 of 13 nucleotides
(77%). To examine the transcription activity of HFH-3/rTSC, the
HFH-3/rTSC in
580 rTSC/Luc was mutated, and its transcription
function was examined in HEK293 cells (expressing both TSC and HFH-3
mRNAs). By the mutation, we observed a marked loss of transcription
function. It is therefore suggested that the HFH-3/rTSC plays an
important role in TSC gene transcription in HEK293 cells. We
next focused on the transcriptional function of HFH-3/rTSC by examining
a functional interaction between HFH-3 and HFH-3/rTSC in HepG2 cells.
HFH-3 overexpression activated the transcription of HFH-3/rTSC-tk-Luc
in a dose-dependent manner (Fig. 8), whereas the
transcription stimulation was not observed in transfected cells with
the mHFH-3/rTSC. These results indicate that HFH-3 can stimulate the
transcription through an interaction with HFH-3/rTSC. However, the
magnitude of the transcription stimulation (~2-3-fold that of
mock) was not comparable with the transcription activity of
2,029 rTSC/Luc in HEK293 cells. Some other elements in the 5'FL/rTSC
may also be involved in the gene transcription to enhance the HFH-3 transactivation.
2 subunit, and Na/K-ATPase
2
subunit (12), although any interaction with HFH-3 has not been
proven even in these the transcription activity of the rTSC
gene genes. We have demonstrated here that HFH-3 can stimulate
through the HFH-3/rTSC via an interaction with HFH-3. This is the first
report in which a target gene for HFH-3 has been identified.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. K. Nakagawara (Nihon Gene Research Laboratories) for help with FISH; Dr. Ichiro Miyoshi for assistance in creating and breeding transgenic rats (Institute of Animal Experiments, Tohoku University Graduate School of Medicine); Yasuteru Kondo (Tohoku University School of Medicine) for laboratory assistance; Dr. R.H. Costa (Department of Biochemistry, University of Illinois at Chicago) for gift of a construct to synthesize the GST-HFH-3 fusion protein; Dr. S. C. Hebert (Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University) for supply of anti-TSC antibody; Dr. N. Takahashi (Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill) for critical reading of this manuscript; and Dr. Yoshiaki Kondo (Department of Pediatrics, Tohoku University Graduate School of Medicine) for valuable discussion.
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FOOTNOTES |
---|
* This study was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture, Japan and by the Takeda Foundation for Metabolic Disorder Research, 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB024534.
These authors were equal contributors to this study.
§ To whom correspondence should be addressed: Molecular Biology Unit, Division of Nephrology, Endocrinology, and Vascular Medicine, Dept. of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Tel.: 81-22-717-7163; Fax: 81-22-717-7168; E-mail: kazut2i@mail.cc.tohoku.ac.jp.
Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.M101614200
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ABBREVIATIONS |
---|
The abbreviations used are: TSC, thiazide-sensitive Na-Cl cotransporter; h, human; r, rat; HFH-3, hepatocyte nuclear factor-3/folk head homologue-3; DCT, distal convoluted tubule; RT-PCR, reverse transcription-polymerase chain reaction; FISH, fluorescence in situ hybridization; EMSA, electrophoretic mobility shift assay; RACE, rapid amplification of cDNA ends; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tk, thymidine kinase; Luc, luciferase; GST, glutathione S-transferase; bp, base pair(s); C/EBP, CCAAT/enhancer-binding protein.
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