Identification of a Short cis-Acting Element in the Human Vasopressin Type 2 Receptor Gene Which Confers High-Level Expression of a Reporter Gene Specifically in Collecting Duct Cells
A. Calmont,
K. Reichwald,
P. Ronco and
J. Rossert
INSERM U. 489 (A.C., P.R., J.R.) The University of Paris VI
75020 Paris, France
Institute of Molecular Biotechnology
(K.R.) 07745 Iena, Germany
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ABSTRACT
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In the kidney, water reabsorption is mainly
regulated by the binding of arginine vasopressin to vasopressin type 2
(V2) receptors. These receptors are expressed selectively in principal
cells of the collecting ducts. To identify molecular mechanisms
responsible for the cell-specific expression of the V2 receptor, we
have analyzed the proximal promoter of the corresponding gene. We
report the identification of a 33-bp enhancer [collecting duct
tissue-specific element 1 (CSE1)] that induced high levels of
expression of the luciferase reporter gene in three collecting duct
cell lines, but not in other renal cell lines. In gel shift assays,
CSE1 bound a DNA-binding protein expressed selectively in collecting
duct cell lines, and a 7-bp mutation, which abolished the activity of
CSE1 in transient transfection experiments, also abolished the binding
of this protein. Furthermore, decoy experiments performed using CSE1
showed that this sequence was involved not only in the expression of a
construct containing 4.2 kb of the V2 receptor proximal promoter, but
also in the expression of the endogenous V2 receptor gene. CSE1 appears
to act mostly by counteracting the inhibitory effects of a strong
ubiquitous repressor element that we called CIE1. Collectively, these
results identify the first functional collecting duct-specific
cis-acting element.
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INTRODUCTION
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One of the major roles of the kidney is to maintain the
homeostasis of body fluids by regulating the elimination of ions and
water. Regulation of water elimination takes place in collecting ducts,
which form the most distal part of the nephrons and which are composed
of three different cell types: intercalated cells
, intercalated
cells ß, and principal cells (reviewed in Ref. 1). Intercalated cells
are implicated in acid-base homeostasis, while principal cells, which
are by far the most abundant ones, are involved in potassium secretion,
sodium reabsorption, and water reabsorption. Water reabsorption is
mostly regulated by arginine vasopressin (AVP), a hormone produced in
the pituitary gland. AVP binds to vasopressin type 2 (V2) receptors,
resulting in an increase of adenylate cyclase activity and promoting
the cAMP-mediated incorporation of water channels into the luminal
surface of principal cells (reviewed in Ref. 2). The importance of V2
receptors in the regulation of urine concentration is illustrated by
the consequences of mutations in the coding sequence of the
corresponding gene. Such mutations are responsible for nephrogenic
diabetes insipidus, a disease that is characterized by an inability to
concentrate urine, leading to polyuria and polydipsia, and which can be
responsible for repeated episodes of dehydration in early infancy, and
thus for mental retardation (reviewed in Ref. 2).
The V2 receptor is a polypeptide of 371 amino acids, with a
structure typical of G protein-coupled receptors, which contain seven
transmembrane domains (3, 4). It is encoded by a gene located on the X
chromosome, which is formed by three exons separated by two short
introns (5). In different mammalian species, Northern blot analyses
have shown that the expression of the V2 receptor gene is restricted to
the kidney (3, 4). In situ hybridization experiments and
immunohistochemical studies have confirmed this restricted pattern of
expression. They have shown that, in the kidney, the V2 receptor gene
is expressed predominantly by principal cells of the collecting ducts.
It is also expressed at lower levels by some cells of the thick
ascending limb of the loop of Henle, while it is not expressed by other
renal cells (6, 7, 8). Furthermore, during embryonic development, in
situ hybridization studies performed in rat have shown that
expression of the V2 receptor gene begins around day 16 post
conception, i.e. at a time corresponding to the formation of
the permanent collecting ducts, and that its expression is restricted
to collecting ducts (7). The V2 receptor gene can thus be
considered as a good model for studying collecting duct-specific
transcription mechanisms.
Two rabbit collecting duct cell lines have been generated in our
laboratory (9, 10), providing us with very useful tools to study the
expression of the V2 receptor gene. These cells have been obtained by
infecting primary cultures of renal cortical cells with either
wild-type SV40 strain (RC.SV3A2 cells) or with a
temperature-sensitive mutant strain (RC.SVtsA58 cells) (9, 10).
RC.SVtsA58 cells, which are the most extensively characterized, have
typical features of principal cells of the collecting ducts. They
express the V2 receptor and they are responsive to AVP, which induces a
dose-dependent increase in the levels of cAMP; they are responsive to
bradykinin, to atrial natriuretic peptide, and to prostacyclin
E2, they take up sodium via an
amiloride-sensitive sodium channel, and they express the cell adhesion
molecule L1 (10, 11, 12). RC.SV3 cells have also characteristic features of
principal cells of the collecting ducts. They respond to AVP, which
increases the production of cAMP, and they express the cell adhesion
molecule L1 (9, 11).
To delineate molecular mechanisms responsible for the cell-specific
expression of the human V2 receptor gene, we performed transfection
experiments and DNA-binding assays, using different segments of the V2
receptor proximal promoter. We report the identification of a 33-bp
enhancing sequence that strongly increases promoter activity
selectively in collecting duct cell lines and which binds a nuclear
protein present only in these cell lines. Furthermore, in decoy
experiments, this element down-regulates the expression of the
endogenous V2 receptor gene. To give rise to a tissue-specific
expression, this short tissue-specific sequence appears to act
mostly by counteracting the effects of a strong repressor element.
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RESULTS
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Identification of a Segment of the V2 Receptor Promoter That Is
Active Selectively in Collecting Duct Cell Lines
Our first goal was to delineate a segment of the V2 receptor
proximal promoter able to drive reporter gene activity in collecting
duct cell lines, but not in other cell lines. To do so, we performed
transient transfection experiments using segments of the V2 receptor
proximal promoter of increasing length, cloned upstream of the
luciferase reporter gene. The 3'-end of all these promoter segments
extended up to a BamHI site located 230 bp upstream of the
translation initiation codon, which was known to be downstream of the
transcription start site (3, 5, 13). These constructs were transfected
in four different rabbit cell lines derived either from collecting
ducts (RC.SVtsA58 cells and RC.SV3A2 cells), or from other segments of
the nephron (RC.SV1 cells and RC.SV2 cells), and in Caco 2 intestinal
cells. The V2 receptor gene was known to be expressed in RC.SVtsA58
cells, and in RV.SV3A2 cells but not in RC.SV1 cells, in RC.SV2 cells,
and in Caco 2 cells (9, 10, 11, 12).
We first cloned a 1.0-kb fragment of the V2 receptor proximal promoter
upstream of the luciferase reporter gene (pluc 1.0) (Fig. 1A
). The transgene was active at high
levels in all four rabbit cell lines (Fig. 1B
), which suggested that
this 1.0-kb fragment contains a strong ubiquitous enhancer, but no
cell-specific element. A 2.0-kb segment of the V2 receptor proximal
promoter was then cloned upstream of the luciferase gene (pluc 2.0)
(Fig. 1A
). pluc 2.0 was active at very low levels in all four renal
cell lines, as well as in Caco 2 cells (Fig. 1B
). It was at least
35-fold less active than pluc 1.0 in all four renal cell lines (Fig. 1B
), and its expression level was similar to a construct containing
only 47 bp of the mouse ß-globin minimal promoter (pluc 47G) (data
not shown). These results showed that the segment of the V2 receptor
promoter extending from -2.0 kb to -1.0 kb contains a very strong
negative regulatory element, which was able to inhibit the activity of
pluc 1.0 in all renal cell lines. We called this element CIE1 (for
collecting duct inhibitory element 1).

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Figure 1. Functional Analysis of Different Segments of the V2
Receptor Proximal Promoter
A, Schematic representation of the constructs used for transfection
experiments. B, Results of transient transfection experiments performed
using the constructs depicted in panel A. These constructs were
transfected in two collecting duct cell lines (RC.SVtsA58 cells and
RC.SV3A2 cells), in two epithelial cell lines derived from other
segments of the nephron (RC.SV1 cells and RC.SV2 cells), and in an
epithelial intestinal cell line (Caco 2 cells).
pSV-ß-galactosidase control vector was used to correct for
transfection efficiency. Values represent the mean ±
SD. Experiments were repeated at least three times in
triplicate.
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A construct harboring a segment of the V2 receptor proximal promoter
extending up to -2.2 kb (pluc 2.2) had the same activity as pluc 2.0
in all cell lines (data not shown). Similarly, when a 4.0-kb fragment
of V2 receptor proximal promoter was cloned upstream of the luciferase
gene (pluc 4.0), it was active at very low levels in all cell lines
(Fig. 1
). On the contrary, a transgene containing a 4.2-kb segment of
the V2 receptor proximal promoter cloned upstream of the luciferase
gene (pluc 4.2) was much more active in collecting duct cell lines than
in other renal cell lines or in Caco 2 cells (Fig. 1
). Furthermore,
pluc 4.2 was only 4.3 and 3.5 times more active than pluc 2.0 in RC.SV1
cells and in RC.SV2 cells, respectively, but it was 25 and 30 times
more active in RC.SVtsA58 cells and in RC.SV3A2 cells, respectively.
pluc 4.2 was also active at high levels when it was stably transfected
in RC.SV3A2 cells (data not shown). These results showed that a 4.2-kb
segment of the V2 receptor proximal promoter was able to induce a
cell-specific expression of the reporter gene in transient transfection
experiments and suggested that the cis-acting element
responsible for this cell-specific expression lies between -4.2 kb and
-4.0 kb. They also suggested that this tissue-specific
cis-acting element acts mostly by counteracting the
inhibitory effects of CIE1.
Mapping of the Transcription Start Site of the V2 Receptor Gene
Before studying in detail the promoter fragment delineated above,
the transcription start site of the human V2 receptor gene was mapped.
To obtain cDNA sequences corresponding to the full-length 5'-end of the
V2 receptor mRNA, 5'-RACE (rapid amplification of cDNA ends)
experiments were performed using total RNA from RC.SV3A2 cells
stably transfected with pluc 4.2, and primers specific of the
luciferase gene. After two rounds of PCR, a single band of about 550 bp
could be detected by electrophoresis of the PCR products (Fig. 2A
). This band was cloned in pGEM-T, and
all of the 36 different clones tested contained an insert of the same
size. Four of these clones were sequenced, and all of them contained an
identical sequence, corresponding to a transcriptional start site
located 422 bp upstream of the translation initiation codon (Fig. 2B
).
Analysis of the sequence located immediately upstream of the
transcription start site in the V2 receptor gene (GenBank accession
number U52111) confirmed that the V2 receptor promoter is a TATA-less
promoter (data not shown).

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Figure 2. Identification of the Transcriptional Start Site of
the V2 Receptor Gene
A, Agarose gel electrophoresis of the PCR products obtained in a
5'-RACE assay. Total RNA from RC.SV3A2 cells stably transfected with
pluc 4.2 was used as a template to synthesize first strand cDNA, in the
presence of a primer specific of the luciferase gene. After labeling
this first strand cDNA using dCTP and the terminal deoxynucleotidyl
transferase, two rounds of PCRs were performed using primers specific
of the 5'-tail and of the luciferase gene. The nested PCR products were
electrophoresed in an agarose gel and stained with ethidium bromide.
Lane 1, 100-bp ladder (Life Technologies, Inc.); lane 2, a
single PCR product of approximately 550 bp was obtained. B, Complete
sequence of the human V2 receptor mRNA, and of the corresponding
protein. It was deduced from the sequence of the PCR product shown in
panel A, and from sequences already available (GenBank accession number
NM 000054).
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Identification of a Short Cell-Specific cis-Acting
Element within the V2 Receptor Promoter
To delineate more precisely the cell-specific element located
between -4.2 kb and -4.0 kb in the V2 receptor promoter, we looked
for a minimal sequence that would be able to increase the activity of
the luciferase reporter gene specifically in RC.SVtsA58 cells and in
RC.SV3A2 cells, when cloned upstream of the 2.2 kb promoter segment in
pluc 2.2. The activity of these constructs was compared with the
activity of pluc 2.0, since it was almost inactive in all four renal
cell lines (Fig. 1
). First, a 148-bp sequence extending from -4,255 bp
to -4,107 bp was cloned upstream of the V2 receptor proximal promoter
in pluc 2.2 (pluc 2.4) (Fig. 3A
). pluc
2.4 was 4 times more active than pluc 2.0 in RC.SV1 cells and
in RC.SV2 cells, but it was 37 and 28 times more active than this
latter construct in RC.SVtsA58 cells and in RC.SV3A2 cells,
respectively (Fig. 3B
), which indicated that the -4,255-bp to
-4,107-bp segment contains a cis-acting element that could
enhance the expression of the reporter gene specifically in collecting
duct cell lines. Computer-based analysis of this 148-bp sequence showed
that its 3'-end contains a potential binding site for winged-helix
transcription factors and another one for MADS-box transcription
factors related to MEF-2, while its 5'-end contains mostly binding
sites for ubiquitous transcriptions factors such as Sp1 and AP-1 (14).
Winged-helix proteins and MADS-box transcription factors being involved
in cell-type-specific gene expression and in cell differentiation, we
focused our attention on the most 3'-part of the 148-bp segment. A
33-bp sequence located between -4,140 bp and -4,107 bp was cloned
upstream of the V2 receptor proximal promoter in pluc 2.2 (pluc 2.24)
(Fig. 3A
), and this construct was used in transfection experiments.
pluc 2.24 had the same activity as pluc 2.4 in RC.SVtsA58 cells and in
RC.SV3A2 cells (Fig. 3B
), confirming that the enhancer element is
located within this 33-bp segment. It is of note that pluc 2.24 was
about 2 times more active than pluc 2.4 in control cell lines (Fig. 3B
). A 7-bp mutation was then introduced in the 33-bp sequence, and the
mutated construct was cloned upstream of the V2 receptor proximal
promoter in pluc 2.2 (pluc 2.24 m7) (Fig. 3A
). This mutation was
located within the potential binding sites for winged-helix proteins
and for MADS-box proteins (Fig. 3C
). The mutated construct was almost
as active as pluc 2.24 in RC.SV1 cells and RC.SV2 cells (Fig. 3B
). On
the contrary, in the two collecting duct cell lines, its activity was
dramatically decreased when compared with pluc 2.24, and it was similar
to the one observed in control cell lines (Fig. 3B
). These results
strongly suggested that the 33-bp sequence located between -4,140 bp
and -4,107 bp contains a tissue-specific element that we called CSE1
(for collecting duct tissue-specific element 1).

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Figure 3. Identification of a cis-Acting
Element Active Selectively in Collecting Duct Cell Lines
A, Schematic representation of the different constructs used for
transfection experiments. B, Results of transient transfection
experiments performed using the constructs depicted in panel A. Each
construct was transfected in two collecting duct cell lines (RC.SVtsA58
cells and RC.SV3A2 cells), and in two epithelial cell lines derived
from other segments of the nephron (RC.SV1 cells and RC.SV2 cells).
pSV-ß-galactosidase control vector was used to correct for
transfection efficiency. Results are expressed as a ratio between the
activity of the tested construct and the activity of pluc 2.0. C,
Sequence of the 33-bp segment that is located between -4,140 bp and
-4,107 bp, and which was used to construct pluc 2.24 (wild-type CSE1).
This sequence contains a potential binding site for winged-helix
proteins, and another one for MADS-box proteins related to MEF-2.
Sequence of a segment containing a 7-bp mutation (light-face
letters) and used to construct pluc 2.24 m7 (mutated CSE1).
Values represent the mean ± SD. Experiments were
repeated at least three times in triplicate.
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To confirm that CSE1 is a positive regulatory element within the
context of 4.2 kb of the V2 receptor proximal promoter, decoy
experiments were performed, using a plasmid containing eight copies of
CSE1 (pBS-8M). pBluescript KS containing no insert (pBKS) was used as a
negative control, and pBS-8M or pBKS was transiently transfected in
RC.SV3A2 cells that had been stably transfected with pluc 4.2. pBS-8M
reduced the activity of pluc 4.2 by more than 50%, when compared with
pBKS (Fig. 4A
). The same approach was
then used to determine whether CSE1 plays a role in the regulation of
the expression of the endogenous V2 receptor gene. We used RC.SVtsA58
cells, since these cells express the V2 receptor and respond to AVP at
a restrictive temperature (39.5 C) but not at a permissive temperature
(33 C) (10); these phenotypical modifications were due to modification
of mRNA stability (R. Piedagnel, unpublished results). Different
amounts of pBS-8M or of pBKS were transiently transfected in RC.SVtsA58
at 33 C, and, after 3 h, the cells were switched at 39.5 C to
induce the expression of V2 receptors at the cell surface. After 2
days, the production of cAMP in response to AVP was assessed.
Transfection with increasing amounts of pBS-8M induced a reduction of
the production of cAMP and for the highest amounts of pBS-8M, this
reduction was as high as 18%, when compared with pBKS (Fig. 4B
).

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Figure 4. Study of the Role of CSE1, Using Decoy Experiments
A, RC.SV3A2 collecting duct cells stably transfected with pluc 4.2 were
transiently transfected either with a decoy plasmid containing eight
copies of CSE1 (pBS-8M) or with a plasmid containing no insert (pBKS).
Transfections were performed using either 2.5 pg or 5 pg of plasmid per
cell. The luciferase activity was arbitrarily defined as 100% when
RC.SV3A2 cells stably transfected with pluc 4.2 were not cotransfected.
B, RC.SVtsA58 cells were transfected with either a decoy plasmid
containing eight copies of CSE1 (pBS-8M) or a plasmid containing no
insert (pBKS). Transfections were performed using 1.5 pg, 2.5 pg, 3.0
pg, 3.5 pg, 5 pg, 6.0 pg of plasmid per cell. The cAMP production was
arbitrary defined at 100% when RC.SVtsA58 were not transfected. Values
represent the mean ± SD. Experiments were repeated at
least three times in triplicate.
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CSE1 Can Act as a Cell-Specific Enhancer
Transfection experiments performed using pluc 1.0, pluc 2.0, pluc
2.2, pluc 4.0, and pluc 4.2 suggested that CSE1 acts mostly by
relieving the inhibitory effect of CIE1 (Fig. 1
). To test the ability
of CSE1 to directly enhance the activity of the V2 receptor proximal
promoter, transfection experiments were performed using a construct
containing CSE1 but not CIE1. CSE1 was cloned upstream of a 1.0-kb
segment of the V2 receptor proximal promoter (pluc 1.0+), and the
activity of pluc 1.0+ was compared with the activity of a construct
containing only a 1.0-kb segment of the V2 receptor proximal promoter
(pluc 1.0) (Fig. 5A
). pluc 1.0+ was about
3 times more active than pluc 1.0 in the two collecting duct cell lines
(RC.SV3A2 cells and RC.SVtsA58 cells), but its activity was identical
to the activity of pluc 1.0 in the two other renal cell lines (RC.SV1
cells and RC.SV2 cells) (Fig. 5B
). The luciferase activities observed
when the four cell lines were transfected with pluc 1.0+ were very
similar to the ones detected when these cell lines were transfected
with a construct containing CSE1 cloned upstream of a 736-bp segment of
the V2 receptor proximal promoter (pluc 0.7+). They were only slightly
higher than the luciferase activities observed when the cell lines were
transfected with a construct containing CSE1 cloned upstream of a
393-bp segment of the V2 receptor proximal promoter (pluc 0.4+) (Fig. 5
). Conversely, when CSE1 was cloned upstream of a segment of the V2
receptor promoter extending from -10 bp to +192 bp (pluc 0.05+), it
was almost inactive in all cell lines (data not shown).

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Figure 5. Study of the Enhancing Activity of CSE1
A, Schematic representation of the different constructs used for
transfection experiments. B, Transient transfection experiments were
carried out in two collecting duct cell lines (RC.SVtsA58 cells and
RC.SV3A2 cells) and in two epithelial cell lines derived from other
segments of the nephron (RC.SV1 cells and RC.SV2 cells), using the
constructs depicted in panel A. pSV-ß-galactosidase control
vector was used to correct for transfection efficiency. Results are
expressed as a ratio between the activity of the tested construct and
the activity of pluc 1.0. Values represent the mean ±
SD. Experiments were repeated at least three times in
triplicate.
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Identification of the Proteins Binding to CSE1
To identify the DNA-binding proteins binding to CSE1, gel shift
experiments were performed using a double-stranded oligonucleotide
extending from -4,140 bp to -4,107 bp as a probe, and nuclear
extracts obtained from all four renal cell lines and from NIH/3T3
cells. When RC.SVtsA58 cells or RC.SV3A2 cells were used as a source of
nuclear extracts, four DNA-protein complexes (complexes C1C4) were
observed, and all these complexes were competed by a 50- to 100-fold
molar excess of cold oligonucleotide (Fig. 6A
). In contrast, when gel shift
experiments were performed using nuclear extracts from RC.SV1 cells,
RC.SV2 cells, or NIH/3T3 cells, a different pattern of DNA-protein
complexes was observed. Specifically, complex C2 was not seen with any
of these three nuclear extracts, and complex C3 was seen only when
nuclear extracts from RC.SV2 cells were used (Fig. 6B
). When a
double-stranded oligonucleotide containing a 7-bp mutation identical to
the one used to construct pluc 2.55 m7 (Fig. 3
) was employed as cold
competitor, it did not modify the pattern of DNA-protein complexes
observed with nuclear extracts from RC.SVtsA58 cells (Fig. 6C
) or from
RV.SV3A2 cells (data not shown). In particular, the formation of the
complex C2 was not inhibited. When this mutated oligonucleotide was
used as a probe, complexes C1, C2, and C3 could not be detected with
nuclear extracts from RC.SVtsA58 cells (Fig. 6C
), or from RV.SV3A2
cells (data not shown). Hence, our DNA-binding assays suggest that a
protein specific for principal cells of the collecting ducts binds to
CSE1 (corresponding to complex C2), and not to a mutated sequence
that has no cell-specific activity in transfection experiments.

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Figure 6. Electrophoretic Mobility Shift Analysis of the
Proteins Binding to the 33-bp Sequence Extending from -4,140 bp to
-4,107 bp (wt CSE1)
A, Lane 1 and lane 4 represent the different DNA-protein complexes
observed using nuclear extracts from RC.SVtsA58 cells and from RC.SV3A2
cells. In lanes 2, 3, 5, and 6, competition assays were performed using
a 50- and 100-fold molar excess of unlabeled oligonucleotide. B,
DNA-protein complexes observed using nuclear extracts from five
different cell lines. Complex C2 is only seen with nuclear extracts
derived from collecting duct cell lines (lanes 1 and 2). C, Lane 1
represents the different DNA-protein complexes observed using nuclear
extracts from RC.SVtsA58 cells and wt CSE1 as a probe. In lanes 2 and
3, competition assays were performed using a 50- to 100-fold molar
excess of unlabeled oligonucleotide harboring a 7-bp mutation (m CSE1).
Lane 4 represents the different DNA-protein complexes observed using
nuclear extracts from RC.SVtsA58 cells and m CSE1 as a probe.
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Transfection of a Human Collecting Duct Cell Line
Results described above were obtained using rabbit renal cell
lines. To test the relevance of our results in human collecting duct
cells, we performed transfection experiments using a human collecting
duct cell line (HCD.A8 cell line). The results obtained using this cell
line were essentially the same as the ones obtained using the two
rabbit collecting duct cell lines (RC.SVtsA58 cells and RC.SV3A2
cells). In particular, pluc 2.0, pluc 4.0 and pluc 2.24m7 were
expressed at very low levels, while pluc 1.0, pluc 4.2, and pluc 2.24
were expressed at much higher levels (Fig. 7
). The expression level of pluc 1.0+ was
also 6 times higher than the expression level of pluc 1.0, confirming
the enhancing activity of CSE1 in human collecting duct cells (data not
shown).

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Figure 7. Functional Analysis of Different Segments of the V2
Receptor Proximal Promoter in a Human Collecting Duct Cell Line
A, Schematic representation of the different constructs used for
transfection experiments. B, Results of transient transfection
experiments performed using the constructs depicted in panel A. Each
construct was transfected in HCD.A8 cells, and pSV-ß-galactosidase
control vector was used to correct for transfection efficiency. Values
represent the mean ± SD. Experiments were repeated at
least three times in triplicate.
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DISCUSSION
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In kidney, V2 receptors are expressed selectively in
differentiated principal cells of the collecting ducts (6, 7, 8), and the
regulation of water reabsorption is mostly due to the binding of AVP to
these receptors. With the long-term goal of identifying DNA-binding
proteins responsible for the cell-specific expression of the human V2
receptor gene, we have analyzed regulatory sequences of the
corresponding proximal promoter.
The coding sequence of the human V2 receptor gene has been extensively
studied, since mutations that lead to the synthesis of an abnormal
protein are responsible for X-linked nephrogenic diabetes insipidus
(reviewed in Ref. 2), but the V2 receptor promoter has not been studied
in detail. In particular, the transcription start site of this gene has
not been precisely mapped, although sequencing of cDNAs obtained from
cells transfected with the human V2 receptor gene or from malignant
cells have shown that it is located at least 235 bp upstream of the
translation initiation codon (3, 5, 13). By performing 5'-RACE
experiments with cells stably transfected with a construct containing
more than 4 kb of the human V2 receptor proximal promoter, we have
shown that the transcription start site is located 422 bp upstream of
the translation initiation codon. Previous studies performed with the
rat gene have shown that, in this species, the transcription start site
is also located 422 bp upstream of the translation initiation codon
(15). Using this transcription start site, the deduced length of the
human V2 receptor mRNA is 1,976 bp, which corresponds precisely to the
size of the mRNA determined by Northern blot analyses in different
species, including human (3).
By transfecting constructs containing different segments of the V2
receptor proximal promoter in four rabbit renal cell lines, in a human
collecting duct cell line and in a human intestinal cell line, we have
identified a 33-bp sequence located between -4,140 bp and -4,107 bp,
which was able to induce high-level expression of the reporter gene
specifically in three different collecting duct cell lines. When this
sequence, named CSE1, was cloned upstream of a 2.2-kb segment of the V2
receptor proximal promoter, which had almost no activity by itself, it
induced high levels of expression of the reporter gene in three
collecting duct cell lines (RV.SVtsA58 cells, RC.SV3A2 cells, and
HCD.A8 cells), but not in cell lines derived from other segments of the
nephron (RC.SV1 cells and RC.SV2 cells). A 7-bp mutation in CSE1
abolished the cell-specific activation of the reporter gene, the levels
of expression of the mutated construct being similar in collecting duct
cell lines and in control cell lines. Furthermore, when RC.SV3A2 cells
were stably transfected with a construct containing 4.2 kb of the V2
receptor proximal promoter cloned upstream of the luciferase gene (pluc
4.2), transient transfection of these cells with a decoy plasmid
containing eight copies of CSE1 induced a 2-fold reduction in the
levels of expression of the reporter gene. Similarly, transfection of
RC.SVtsA58 cells with the decoy plasmid induced a significant decrease
in the production of cAMP in response to AVP and thus very probably
inhibited the expression of the V2 receptor gene. These results were
supported by gel shift assays, which showed that CSE1 bound a
DNA-binding protein present in cell lines derived from collecting ducts
(RV.SVtsA58 cells and RC.SV3A2 cells) but not in cell lines derived
from other segments of the nephron (RC.SV1 cells and RC.SV2 cells) or
in fibroblastic cells (NIH/3T3 cells). The 7-bp mutation that abolished
the enhancing activity of CSE1 in transfection experiments also
abolished the binding of this cell-specific DNA-binding protein in gel
shift assays.
The cell-specific DNA-binding protein binding to CSE1 has not yet been
identified, but a computer-based analysis of CSE1 showed that it
contains a potential binding site for MADS-box transcription factors
related to MEF-2 and another one for winged-helix proteins, in addition
to a consensus binding site for TBP (TATA binding protein) and
to a weaker consensus binding site for the ubiquitously expressed
transcription factor Oct-1 (14). DNA-binding proteins containing a
MADS-box have been involved in cell differentiation and, for example,
knock-out experiments have shown that MEF-2 plays an important role in
muscle development (reviewed in Ref. 16), but no MEF-2-related protein
is known to be present in the developing kidney. Furthermore, in gel
shift experiments, MEF-2 and MEF-2-related proteins such as MEF-2C give
rise to very low-mobility complexes (17). Thus, it seems somehow
unlikely that the cell-specific protein binding to CSE1 belongs to the
MADS-box family. Forkhead/winged-helix proteins have also been
implicated in cell fate determination and in cell-specific gene
expression, and at least nine winged-helix transcription factors have
been shown to be expressed in the developing kidney (18, 19, 20, 21, 22, 23, 24, 25, 26).
Nevertheless, almost all of these transcription factors are expressed
in the metanephric mesenchyme, and not in the ureteric bud or in its
derivatives (18, 19, 20, 21, 22, 23, 24). Among forkhead transcription factors that are
expressed in the ureteric bud, HNF-3 is expressed in the urothelium of
the renal pelvis but not in collecting ducts (25), and similarly HFH-4
is expressed in the ureteric bud before its differentiation in
collecting ducts but not in collecting duct cells (26). It is therefore
possible that the cell-specific protein that binds to CSE1 is a yet
unknown DNA-binding protein, and that it belongs to the winged-helix
family of transcription factors. This hypothesis is supported by the
recent identification of a cDNA encoding a winged-helix protein that is
expressed selectively in collecting ducts cells and which starts to be
expressed at a time corresponding to the expression of the V2 receptor
(A. Calmont, unpublished results).
In transient transfection experiments, a construct containing
2,061 bp of the V2 receptor proximal promoter was at least 35-fold less
active in all renal cell lines than a construct containing only 1,004
bp of the same promoter (compare pluc 2.0 with pluc 1.0), which
suggests that a suppressor cis-acting element, named CIE1,
is located between -2.0 kb and -1.0 kb. CIE1 may be important to
silence transcription of the V2 receptor gene in cells that do not
express the V2 receptor, in the same way as repressor elements present
in the L1 adhesion molecule gene, in the Fgf4 gene, or in the GATA-1
gene (27, 28, 29). For each of these three genes, deletion of a repressor
cis-acting element induced a ubiquitous expression of the
reporter gene, in transgenic animals (27, 28, 29). In this model, CSE1
would act mostly by counteracting the inhibitory effects of CIE1. This
hypothesis is supported by the results of transient transfection
experiments performed using constructs containing or not containing
CIE1. In collecting duct cell lines, a construct containing CSE1 cloned
upstream of CIE1 was at least 30 times more active than a construct
that did not contain CSE1 (compare pluc 2.24 with pluc 2.0).
Conversely, a construct containing CSE1 cloned upstream of a 1-kb
segment of the V2 receptor proximal promoter, and which did not include
CIE1, was only about 3 times more active than a construct containing
only the 1-kb promoter segment (compare pluc 1.0+ and pluc 1.0). Thus,
our data suggest that CSE1 is a rather weak enhancer, but that it can
very efficiently antagonize the inhibitory effects of CIE1 (Fig. 8
).

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Figure 8. Model for the Action of the Cell-Specific Protein
Binding to CSE1
A, Summary of the results of transfection experiments perfomed using
constructs containing various combinations of CSE1, of CIE1, and of the
V2 receptor minimal promoter (m.p.) (see Results for
details). B, Schematic representation of the interactions between
proteins binding to CSE1, to CIE1, and to the V2 receptor minimal
promoter (m.p.). In this model, collecting duct cells contain a
cell-specific protein able to bind to CSE1 and to inhibit the repressor
effect of CIE1. This DNA-binding protein has also a weak enhancer
activity on the V2 receptor minimal promoter (see text for details).
|
|
In conclusion, we have identified a 33-bp segment of the V2 receptor
promoter, named CSE1, which induced high levels of expression of the
luciferase reporter gene specifically in collecting duct cell lines,
and which bound a DNA-binding protein expressed selectively in these
cell lines. CSE1 appears to act mostly by relieving the inhibitory
effects of an ubiquitous repressor element named CIE1. CSE1 is the
first functional collecting duct-specific cis-acting element
identified so far.
 |
MATERIALS AND METHODS
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Cell Lines
Four rabbit cell lines derived from renal cortex and obtained in
our laboratory were used in transfection experiments. The RC.SVtsA58
cell line has been obtained by infection of isolated renal cortical
cells with the temperature-sensitive SV40 mutant tsA58, and its
characteristics are those of principal cells of the collecting ducts
(10, 11, 12). Run on experiments having shown that the V2 receptor gene is
transcribed at similar levels at permissive temperature (33 C) and at
restrictive temperature (39.5 C) (R. Piedagnel, unpublished results),
all transfection experiments were performed at 33 C. The RC.SV1 cell
line, the RC.SV2 cell line, and the RC.SV3 cell line have been obtained
by infecting isolated renal cortical cells with wild-type SV40, and
they were cultured at 37 C. The RC.SV3 cell line, and its subclone
RC.SV3A2 cells, have functional characteristics of principal cells of
the collecting ducts (9). RC.SV1 cells and RC.SV2 cells have
characteristics of proximal tubular cells, and of cells of the thick
ascending limb of the Henles loop, respectively (9). All four rabbit
cell lines were grown in 50% DMEM (Life Technologies, Inc., Gaithersburg, MD), 50% HAM (Life Technologies, Inc.) supplemented with 2 mM glutamine, 5 mg/liter
insulin, 50 nM dexamethasone, 5 mg/liter transferrin, 30
nM selenium, and 20 mM HEPES (MD medium).
HCD.A8 cells, which are a subclone of the human HCD cell line (30),
were provided by M. Géniteau-Legendre. The HCD cell line has been
obtained by transfection of isolated human renal cortical cells with a
recombinant plasmid DNA harboring a complete but replication-defective
SV40 genome (30). It has characteristics of principal cells of the
collecting ducts, since it expresses the cell adhesion molecule L1 and
responds to AVP by an increased production of cAMP (11, 30). These
cells were grown in MD medium supplemented with 2% FCS (Life Technologies, Inc.).
NIH/3T3 mouse fibroblasts were obtained from ATCC
(Manassas, VA), and cultured in DMEM supplemented with 10% FCS
(Life Technologies, Inc.).
The Caco 2 cell line, which is derived from a human colon carcinoma,
was given to us by G. Trugnan. It was grown in DMEM supplemented with
20% FCS (Life Technologies, Inc.) and 1% (vol/vol)
nonessential amino acids.
DNA Constructions
For transfection experiments, different inserts were cloned in
the pluc 5 promoterless luciferase expression vector, which was
obtained by modifying the polycloning site in pluc 4 (31). In this
vector, the firefly luciferase gene is cloned upstream of an SV40
splice site and polyadenylation site and downstream of a
polyadenylation cassette that prevents read-through transcription. Most
inserts were derived from a cosmid named QC7C1, which contains an
insert extending from about 19 kb upstream of the first exon of the V2
receptor to about 18 kb downstream of this gene (GenBank accession
number U52111). In some constructs, a 47-bp fragment of the mouse
ß-globin proximal promoter was used as an heterologous minimal
promoter, as in other studies (32).
To describe the promoter segments of the V2 receptor gene that we used,
we numbered the nucleotides according to the transcription start site
defined by RACE experiments (cf. Results). Nucleotide +1 is
thus located 422 bp upstream of the translation initiation codon. pluc
2.0 contains a BamHI-BamHI segment of the V2
receptor gene extending from -2,061 bp to +192 bp, cloned upstream of
the luciferase reporter gene in pluc 5. pluc 4.2 contains a -4,255-bp
to +192-bp segment of the V2 receptor gene, cloned in pluc 5. It was
generated by cloning a BglII-SacII segment
upstream of a -1,004-bp to +192-bp element derived from pluc 2.0. pluc
4.0 contains a -4,046-bp to +192-bp segment of the V2 receptor gene.
It was derived from pluc 4.2 by deleting a
BglII-HindIII fragment. pluc 2.4 was derived from
pluc 4.2 by deleting an XbaI-NcoI fragment
extending from -4,107 bp to -2,205 bp. pluc 2.24 was derived from
pluc 2.0 by cloning a double-stranded oligonucleotide corresponding to
the sequence extending from -4,140 bp to -4,107 bp
(TACTCTATGATTCTATTTATATAAAATTCTAGA) upstream of the NcoI
site located at -2,205 bp. pluc 2.24m7 is identical to pluc 2.24
except that we used a double- stranded oligonucleotide containing a
7-bp mutation (TACTCTATGATTCCCCGGGGATAAAATTCTAGA, mutated
nucleotides being underlined). pluc 0.05+ was derived from
pluc 2.24 by deleting a NcoI-BglII fragment
extending from 2,205 bp to -10 bp. pluc 1.0+ was derived from pluc
2.24 by deleting a NcoI-SacII fragment extending
from -2,205 bp to -1,004 bp. pluc 1.0, which contains a segment
located between -1,004 bp and +192 bp, was derived from pluc 2.24 by
deleting a BglII-SacII fragment. pluc 0.7+ was
derived from pluc 2.24 by deleting a NcoI-BstXI
fragment extending from -2,205 bp to -736 bp. pluc 0.4+ was derived
from pluc 2.24 by deleting a NcoI-ApaI fragment
extending from -2,205 bp to -393 bp.
For transfection experiments, in addition to the constructs described
above, we used: pluc 47G, which contains 47 bp of the mouse ß-globin
proximal promoter cloned in pluc 5; pSV-ß-galactosidase control
vector (Promega Corp., Madison, WI), which contains the
SV40 early promoter and enhancer cloned upstream of the lacZ
gene; pSV Neo (Promega Corp.), which contains the SV40
early promoter and enhancer cloned upstream of the neomycin resistance
gene; and pBS-8M, which contains a 33-bp sequence extending from
-4,140 bp to -4,107 bp multimerized eight times in pBluescript KS
(Stratagene, La Jolla, CA). Multimerization was made
possible by introducing a BamHI site at one end of the
sequence and a BglII site at the other end.
Transient Transfection Experiments
The day before transfection, cells were plated in six-well 25-mm
dishes (Nunc, Kamstrup, Denmark) at a density of 2 x
105 cells per well. Transfections were carried
out with plasmid DNA coated with the polycationic lipid lipofectamine
(Life Technologies, Inc.) according to the manufacturers
instructions. Briefly, 900 ng of a luciferase construct and 100 ng of
pSV-ß-galactosidase control vector were mixed with 6 µl of
lipofectamine. Cells were incubated with this mixture in 1 ml
serum-free medium for 24 h. They were then washed twice in PBS
(150 mM NaCl, 10 mM sodium sulfate, pH 7.8) and
incubated for 48 h in a medium containing twice the normal
concentration of FCS. Cells were then washed twice with cold PBS,
harvested on ice by scraping them in 0.2 ml potassium phosphate,
0.1 M, pH 7.8, and lysed by three freezing-thawing
cycles.
Luciferase activity was assayed using a luminometer (EG&G Berthold, Bad
Wilbad, Germany). Briefly, 50 µl of cell lysates were incubated with
28 ng of D-luciferin (Fermentas AB, Vilnius, Lithuania) in
100 mM potassium phosphate, pH 7.8, 5 mM ATP,
15 mM MgSO4, and 1 mM
dithiothreitol, and light units were counted for 5 sec (31).
ß-Galactosidase activity was used to correct for transfection
efficiency. It was measured by a colorimetric assay using resorufin
ß-D-galactopyranoside (Sigma, St. Louis, MO)
as a substrate, as previously described (33). Briefly, 20 µl of cell
lysates were incubated in reaction buffer (50 mM Tris-HCl,
pH 7.5, 10 mM MgCl2, 100
mM NaCl, 75 µg resorufin
ß-D-galactopyranoside in dimethylsulfoxide), at 30 C.
When a significant change in color was seen, ß-galactosidase
activities were measured at 572 nm.
All transfection experiments were done in triplicate and repeated at
least three times. Results are expressed as mean ±
SD.
Stable Transfection Experiments
Stable transfections were carried out as previously described
for transient transfections with the following modifications. The day
before transfection, cells were plated in 90-mm dishes (Nunc) at a
density of 106 cells per dish. Linearized pluc
4.4 (4.5 µg) and 0.5 µg of linearized pSV neo were mixed with 30
µl of lipofectamine. Transfected cells were selected in MD medium
containing 100 µg/ml of G418 (Sigma). Cells transfected
in the same manner but without pSV Neo and cultured in the same
selecting medium died after 1 week of culture.
Determination of the Transcriptional Start Site
The transcriptional start site of the human V2 receptor gene was
determined by using a 5'-RACE system (Life Technologies, Inc.) and RC.SV3A2 cells stably transfected with pluc 4.2. These
cells harbor a transgene containing a segment of the V2 receptor gene
extending from -4,255 bp to +192 bp, cloned upstream of the luciferase
reporter gene. Briefly, total RNA was prepared according to Chomczynski
and Sacchi (34), and used as a template to synthesize first-strand cDNA
by reverse transcription in the presence of a luciferase-specific
antisense primer (GSPluc1, 5'-AACACTACGGTAGGCTGCGAAATG-3'). The 5'-end
of this first-strand cDNA was labeled using dCTP and the terminal
deoxynucleotidyl transferase. The tailed DNA was then used to perform
the first PCR using a sense DNA primer complementary to the 5'-tail and
a second luciferase-specific antisense primer (GSPluc2,
5'-GCAACTCCGATAAATAACGCGCCC-3'). Samples were amplified for 45 cycles
under the following conditions: denaturation for 60 sec at 94 C,
annealing for 45 sec at 55 C, and extension for 90 sec at 72 C.
Aliquots of the first PCR reaction were used as template in a second
PCR. This nested PCR was performed using a nested sense DNA primer
complementary to the 5'-tail, paired with a third luciferase-specific
antisense primer (GSPluc3, 5'-CATAGCTTCTGCCAACCGAACGGA-3'). Samples
were amplified for 45 cycles under the following conditions:
denaturation for 60 sec at 94 C, annealing for 45 sec at 55 C, and
extension for 90 sec at 72 C. The PCR products were electrophoresed
through a 1% agarose gel, excised, cloned into the pGEM-T-easy vector
(Promega Corp.), and sequenced.
Decoy Experiments and Determination of cAMP Production
In a first set of experiments, RV.SV3A2 cells stably transfected
with pluc 4.2 were transiently transfected with various amounts of
either the decoy construct (pBS-8M) or pBluescript KS.
pSV-ß-galactosidase (0.1 µg) was cotransfected to assess
transfection efficiency. Luciferase and ß-galactosidase activities
were assayed as described above.
In a second set of experiments, RC.SVtsA58 cells, cultured in six-well
dishes at 33 C, were transfected with various amounts of either the
decoy construct (pBS-8M) or pBluescript KS. After 3 h, the cells
were switched to 39.5 C, to allow the expression of V2 receptors at the
cell surface. After 36 h, determination of cAMP production in
response to AVP was performed as previously described (9). Briefly,
cells were first incubated for 10 mn at 37 C in MD medium containing
0.1 mM 3-isobutyl 1-methyl xanthine (IBMX,
Sigma). Thereafter, they were incubated for 7 mn at 37 C
in the same medium supplemented or not with 10-7
M AVP (Sigma) or 10-5
M forskolin (Sigma). The reaction was stopped
by rapid removal of the medium, immediately followed by addition of 1
ml of a solution containing 95% ice-cold ethanol and 5% formic acid.
After 45 min of incubation at 4 C, the supernatant was
recovered, evaporated, and resuspended in 50 mM sodium
acetate, pH 6.2, and used in a RIA. cAMP production was calculated as
picomoles per µg of protein. To measure the protein content of each
cell culture, 1 ml of 1M NaOH was added to each well. After
overnight incubation at 4 C, supernatants were removed and protein
content was measured according to Bradford (35).
Gel Retardation Assays
Nuclear extracts were prepared from 80% confluent cells as
previously described (36), except that the buffer used to extract
nuclear proteins contained 0.55 M NaCl. Fifteen femtomoles
of a double-stranded oligonucleotide probe labeled with the Klenow
fragment of Escherichia coli DNA polymerase I and 30 µCi
[
-32P] dCTP were incubated for 30 min at
room temperature in the presence of 58 mg of nuclear proteins, 300 ng
of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech,
Björkgatan, Sweden), and 1 µg of salmon sperm DNA (Roche Molecular Biochemicals, Indianapolis, IN), in a solution
containing 20 mM HEPES, pH 7.9, 25
mM NaCl, 1 mM EDTA, pH 8.0,
0.5% Nonidet P-40, and 8% glycerol. The reaction mixture was then
fractionated by electrophoresis on a 4% polyacrylamide gel.
Competition experiments were performed in the presence of a 50- to
100-fold molar excess of competitor.
 |
ACKNOWLEDGMENTS
|
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We thank B. de Crombrugghe and G. Trugnan for the generous gift
of the mouse ß-globin minimal promoter and of the Caco 2 cell line,
respectively. We thank M. Géniteau-Legendre for providing us
with HCD.A8 cells. We thank J. Chambaz, C. Terraz, and R.
Piedagnel for carefully reading the manuscript.
 |
FOOTNOTES
|
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Address requests for reprints to: Jérome Rossert, M.D., Ph.D., INSERM U. 489, Tenon Hospital, 4 rue de la Chine, 75020 Paris, France. E-mail: jerome.rossert{at}tnn.ap-hop-paris.fr
This work was supported by Grant 9871 from the Association pour la
Recherche sur le Cancer (to J. R.), by INSERM, and by The
University of Paris VI. A. C. is a recipient of a fellowship
from the Ministère de lEducation Nationale.
Received for publication February 14, 2000.
Revision received June 22, 2000.
Accepted for publication July 3, 2000.
 |
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