(Received for publication, May 15, 1995)
From the
Human aquaporin 3 (AQP3) gene was isolated, and its structural
organization was characterized. The gene appeared to exist as a single
copy in the human genome and to comprise six exons distributing over 7
kilobases. The sizes of the exons are 171, 127, 138, 119, 218, and 1035
base pairs, and those of introns are approximately 3530, 300, 350, 330,
and 90 base pairs, respectively. The initiation site of transcription
was identified to locate 64 base pairs upstream of the first ATG codon
by primer extension analysis and ribonuclease protection assay. The
5`-flanking region has a TATA box, two Sp1 sequences, and some
consensus sequences including AP2 sites. With luciferase assay, the
5`-flanking region was demonstrated to have a promoter activity, which
is up-regulated 4-fold by phorbol ester. These findings about the
genomic clone of human AQP3 will contribute to elucidate the molecular
mechanism of transcriptional regulation of AQP3. Water transport in highly water-permeable membranes is
facilitated by some specialized pathways, which are called aquaporins
(AQP) To elucidate the molecular mechanism of transcriptional regulation
of aquaporins, the isolation of aquaporin gene is needed. Previously,
human AQP1 gene (9) and human AQP2 gene (10) were
isolated, and their structural organization was characterized. Human
genes of AQP1 and AQP2 consist of four exons like human MIP26
gene(11) , and the sites of exon-intron boundary in these three
genes exist at the identical points. However, no information about the
structural organization of AQP3 gene is available. The structure of
AQP3 is interesting in the viewpoint of evolution of the aquaporin
family, since AQP3 is most homologous to Escherichia coli glycerol facilitator (GlpF, 42%) and its homology to other AQPs is
low (less than 35%)(5) . In the present study, we isolate
human AQP3 gene and characterize its entire structural organization. We
also describe the 5`-flanking region of human AQP3 gene and evaluate
its promoter activity.
Figure 1:
Schematic
representation of human AQP3 gene. The gene loci are demonstrated by rectangles (exons 1-6). Blackrectangles in human AQP3 gene and hatchedboxes in human AQP3 cDNA indicate the coding
region.
Figure 2:
Southern blot analysis of human AQP3 gene.
Human genomic DNA (10 µg) digested with XbaI, BamHI, BglII, and SacI was separated by
electrophoresis and transferred to nylon membrane. The membrane was
hybridized with a labeled probe for human AQP3 cDNA (1.4
kb).
Figure 3:
Structure of the 5`-flanking region of
human AQP3 gene. The first nucleotide of the ATG codon (underlined) is designated +1. The initiation site of
transcription determined by primer extension analysis and ribonuclease
protection assay is indicated by an arrow. The TATA box is boxed. Sp1, AP2, ATF, and SIF sites are underlined.
The initiation site of
transcription was determined by primer extension analysis and
ribonuclease protection assay with RNA from human A549 cells. In primer
extension analysis, major bands were present at -64 (site of T, Fig. 4). Moreover, a protected band was also found at -64
in the ribonuclease protection assay (Fig. 5). These findings
indicate that the initiation site of transcription is at -64,
which is 31 bases downstream of TATA box.
Figure 4:
Primer extension analysis for mapping the
initiation site of transcription. A synthetic primer (+10 to
-30) was end-labeled and hybridized to RNA from human A549 cells.
The hybridized primer was extended with reverse transcriptase, and the
synthesized product was separated by electrophoresis. The sequence
ladder was generated by sequencing the 5`-flanking region with a
synthetic primer (+10 to -14). Lane1, 10
µg of total RNA; lane2, 20 µg of total RNA; lane3, 50 µg of total RNA; lane4, 10 µg of poly(A)
Figure 5:
Ribonuclease protection assay for mapping
the initiation site of transcription. The labeled antisense RNA probe
covering the SalI-SalI region (340 bp) was
synthesized by T7 RNA polymerase and hybridized with 10 µg of
poly(A)
A search for consensus
sequences (13) revealed three AP2 sites (CCCMNSSS) at
-37, -198, and -293, one possible ATF (CRE) site
(TGACGT) at -299, and one SIF site (CCCGTC) at -264.
Figure 6:
Luciferase activity of the 5`-flanking
region of human AQP3. The reporting vector containing the 5`-flanking
region (pGL2-5`) and that without the 5`-flanking region
(pGL2-Basic) were transfected into human A549 cells. TPA
(10
In the present study, we have isolated human AQP3 gene, which
exists as a single copy in human genome, and characterized the entire
structural organization. We have also demonstrated that the 5`-flanking
region of human AQP3 gene has a promoter activity, which is
up-regulated by phorbol ester. Previously five cDNA clones of AQP
(AQP1-AQP5) have been identified, and only two genomic clones of
AQP (AQP1 and AQP2) have been characterized. Human AQP1 gene and human
AQP2 gene consist of four exons like human MIP26 gene with the
identical points of exon-intron boundary(10) . On the other
hand, human AQP3 gene consists of six exons, and its points of
exon-intron boundary are different from other AQP genes. The
phylogenetic comparison analysis between AQP3 and other MIP family
proteins reveals the separate clustering of AQP3 and GlpF from other
aquaporins, which suggests that AQP3 is most related to GlpF and
developed in a different branch from other aquaporins(5) . The
different structural organization of AQP3 from AQP1 and AQP2 is
consistent with the above suggestions. Although AQP3 is expressed in
several tissues including kidney, gastrointestinal tract, and lung,
AQP3 is expressed exclusively in principal cells of the collecting duct
in the kidney like AQP2. To elucidate the mechanism of cell-specific
expression, analysis of the promoter region of AQP3 is necessary. The
5`-flanking region demonstrated in this study has a typical TATA box,
the initiation site of transcription at 31 bp downstream of the TATA
box, and several consensus sequences, which indicates that this region
contains the promoter sequence of AQP3. Moreover, the 5`-flanking
region was shown to have a promoter activity demonstrated by luciferase
assay. Further examination of the 5`-flanking region of these two genes
(AQP2 and AQP3) will reveal a clue to solve the mechanism of collecting
duct-specific gene expression. In human AQP2 gene, the promoter
region has a ATF (CRE) site, an AP1 site, and an AP2 site, and its
promoter activity is up-regulated by cAMP but not TPA. The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
D50486[GenBank Link].
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(1) . These proteins belong to an
ancient channel family, the major intrinsic protein (MIP) family (2) . AQP1 (AQP-CHIP) is the first recognized aquaporin
purified from red cells and renal proximal tubules and is widely
expressed throughout the body(3) . Two other clones, AQP2 (4) and AQP3(5) , were reported from our laboratory.
AQP2 (AQP-CD) is selectively expressed in kidney, and inside kidney it
is localized only at the apical membrane of collecting duct cell. AQP3
is expressed in several tissues including kidney, colon, small
intestine, lung, liver, pancreas, spleen, and prostate. Localization of
AQP3 in the kidney is also restricted to collecting duct cells, but in
contrast to AQP2, it distributes on the basolateral membrane. AQP3 also
facilitates the transport of nonionic small solutes such as urea and
glycerol, while the previously cloned aquaporins are permeable only to
water. AQP4 is a mercury-insensitive water channel, which is most
abundantly expressed in brain(6, 7) . AQP5, which has
recently been cloned from rat salivary gland, is implicated in the
generation of saliva, tears, and pulmonary secretions (8) .
Isolation and Characterization of Human AQP3
Gene
To isolate human AQP3 gene, about 6 10
phage clones from the human placental genomic library
(Stratagene) were screened with human AQP3 cDNA (1.4 kb) labeled with
[
P]dCTP (3000 Ci/mmol, Amersham Corp.) by random
priming(12) . Positive clones were purified and characterized
by restriction endonuclease mapping and Southern blot analysis. DNA
fragments from positive clones were subcloned into pBluescript II
SK(+) (Stratagene) and sequenced using a fluorescence DNA
sequencer (373A, Applied Biosystems).
Genomic Southern Blot Analysis
Ten µg of human
genomic DNA extracted from leukocytes were digested with restriction
endonuclease (XbaI, BamHI, BglII, and SacI), electrophoresed on a 0.7% agarose gel, and transferred
to a nylon membrane. After prehybridization for 4 h, the membrane was
hybridized with human AQP3 cDNA (1.4 kb) labeled with
[P]dCTP in 50% formamide, 5
saline/sodium/phosphate/EDTA, 5
Denhardt's, 1.6% SDS, and
50 µg/ml salmon sperm DNA for 18 h at 42 °C. The membrane was
washed in 0.1
SSC and 0.5% SDS for 60 min at 68 °C and was
exposed to a film for 3 days at -70 °C.
Primer Extension Analysis
A synthetic
oligonucleotide primer corresponding to nucleotide +10 to
-30 in the human AQP3 cDNA was end-labeled with
[-
P]ATP (6000 Ci/mmol, Amersham Corp.).
Human lung epithelial (A549) cells (JCRB, Japan), which demonstrated
mRNA expression of AQP3 in our preliminary study, were grown in minimal
essential medium supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin. RNA extracted from A549
cells was hybridized with the end-labeled primer (5
10
cpm) in 250 mM KCl, 10 mM Tris-Cl (pH 8.0), and
1 mM EDTA at 55 °C for 1 h. Primer extension was carried
out with 100 units/ml Moloney murine leukemia virus reverse
transcriptase (Superscript II, Life Technologies, Inc.) in 75 mM KCl, 10 mM MgCl
, 20 mM Tris-Cl (pH
8.0), 0.25 mM EDTA, 10 mM dithiothreitol, 0.25 mM dNTP, and 100 µg/ml actinomycin D. The synthesized product was
electrophoresed on an 8% denaturing polyacrylamide gel. The gel was
dried under vacuum at 80 °C for 40 min and was exposed to a film
for several days at -70 °C.
Ribonuclease Protection Assay
To prepare RNA probe
for ribonuclease protection assay, SalI-SalI DNA
fragment (340 bp) containing the 5`-flanking region of human AQP3 gene
was subcloned into pBluescript II SK(+). This clone (designated
Sal#2) was digested with HindIII to make a linearized template
for antisense RNA probe. Labeled RNA probe was synthesized by T7 RNA
polymerase (Stratagene) with [P]CTP (800
Ci/mmol, Amersham Corp.). RNA extracted from A549 cells was hybridized
with the labeled RNA probe in 80% formamide, 40 mM PIPES (pH
6.4), 400 mM sodium acetate, and 1 mM EDTA at 42
°C for 18 h and treated with 0.5 unit/ml RNase A and 10 units/ml T1
at 37 °C for 30 min. After treatment, the protected fragments were
precipitated and electrophoresed on an 8% denaturing polyacrylamide
gel. The gel was dried under vacuum at 80 °C for 40 min and was
exposed to a film for several days at -70 °C.
Construction and Transfection of the Reporting
Vector
To measure promoter activity of the 5`-flanking region by
luciferase assay, a reporting vector for transcription was constructed.
From the Sal#2 clone, the SacI-XhoI DNA fragment (420
bp) containing the 5`-flanking region was subcloned into pGL2-Basic
vector (Promega). The constructed vector (pGL2-5`) and pGL2-Basic
were purified before transfection into A549 cells. A549 cells were
grown in minimal essential medium supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin on 15-cm
dishes. About 5 10
cells growing in exponential
phase were harvested by 0.25% trypsin and resuspended in K-PBS buffer
(30.8 mM NaCl, 120.7 mM KCl, 1.46 mM KH
PO
, 8.1 mM Na
HPO
, 10 mM MgCl
).
Transfection was performed by electroporation under 400 V at 960
microfarads in a 0.4-mm width cuvette. To evaluate the efficiency of
transfection, pSV-
-galactosidase vector (pSV
Gal) was
transfected together with pGL2-5` or pGL2-Basic. Transfected
cells were seeded on 9-cm dishes in the full medium for 48 h. To
examine the effect of phorbol ester and cAMP on the promoter activity
of the 5`-flanking region, tetradecanoylphorbol acetate (TPA) and
chlorophenylthio-cAMP (cpt-cAMP) were added to the medium 24 h after
transfection. After washing with calcium- and magnesium-free
phosphate-buffered saline twice, the cells were incubated in 900 µl
of cell lysis buffer (Promega) at room temperature for 15 min. The cell
lysate was kept at -70 °C for luciferase assay or
-galactosidase assay.
Luciferase Assay
After 20 µl of the cell
lysate was mixed with 100 µl of luciferase assay reagent (Promega)
briefly at room temperature, the light intensity of the sample was
immediately counted in a scintillation counter for 1 min. Luciferase
concentration was estimated by calculating the square root of measured
counts/min minus background counts/min. To measure background
counts/min, we prepared the cell lysate of non-transfected cells.
After 15 µl of the
cell lysate was mixed with 15 µl of -Galactosidase Assay
-galactosidase assay
buffer (Promega), the sample was incubated at 37 °C for 30 min and
mixed with 50 µl of 1 M sodium carbonate. The absorbance
of the sample was measured at 420 nm.
Isolation and Structural Organization of Human AQP3
Gene
By screening the human placental genomic library with human
AQP3 cDNA, we isolated two clones. The digestion of one genomic clone
(designated #42) with SalI and BglII released three
fragments (0.3, 5.5, and 1.2 kb). These fragments were subcloned and
sequenced, which demonstrated the structural organization of human AQP3
gene. Human AQP3 gene consists of six exons and five introns (Fig. 1). The sizes of exons are 171, 127, 138, 119, 218, and
1035 bp, and those of introns are approximately 3530, 300, 350, 330,
and 90 bp, respectively. All splice sites conform to the GT-AG rule (Table 1). The sixth exon has a stop codon and a polyadenylation
signal.
Southern Blot Analysis of Human AQP3 Gene
To
determine whether human AQP3 gene exists as a single copy or multiple
copies, we performed genomic Southern blot analysis. On this blot only
one or two distinct hybridization signals were detected (Fig. 2), which suggests that a single AQP3 gene locus exists.
Moreover, the pattern of hybridization on this blot was consistent with
the known restriction site in the #42 genomic clone (Fig. 1).
The 5`-Flanking Sequence of Human AQP3 Gene
The 5`
end-SalI fragment in #42 genomic clone was subcloned and
sequenced (Fig. 3). The TATAAA sequence (TATA box) is present at
-95. No CCAAT sequence is observed. Two Sp1 sequences (KRGGCKRRK)
are found at -180 and -186.
RNA.
RNA from human A549 cells. After RNase A and
T1 treatment, the protected fragments were precipitated and separated
by electrophoresis (lane1). The sequence ladder was
generated by sequencing the 5`-flanking region with a synthetic primer
(+10 to -14).
Promoter Activity of the 5`-Flanking Region of Human AQP3
Gene
To verify whether the 5`-flanking region of human AQP3 gene (Fig. 3) has promoter activity, we measured luciferase activity
of transfected A549 cells. Luciferase activity was corrected by
-galactosidase activity in each experiment. As shown in Fig. 6, pGL2-5` showed about 5 times greater luciferase
activity than pGL2-Basic, which indicates that the 5`-flanking region
has a promoter activity. In addition, we also examined the effect of
TPA and cpt-cAMP on promoter activity of the 5`-flanking region. TPA
(10
M) up-regulated luciferase activity
about 4-fold in pGL2-5`, whereas cpt-cAMP (200 µM)
had no effect (Fig. 6).
M) or cpt-cAMP (200 µM)
was added to the medium 24 h after transfection, and luciferase
activity was measured 48 h after
transfection.
(
)In human AQP3 gene, the promoter region has a
possible ATF site (the last eighth base (C) is different from G/A) and
three AP2 sites, and its promoter activity is up-regulated by TPA but
not cAMP. These findings suggest that the transcriptional regulation of
AQP3 is different from that of AQP2. In the rat experimental models,
expression of AQP2 is highly up-regulated by dehydration for several
days(14) . This up-regulation may be mediated by an increased
level of systemic vasopressin in dehydrated state. Vasopressin, acting
on vasopressin V2 receptor, increases the intracellular cAMP
concentration, which in turn stimulates the transcription of AQP2 gene.
AQP3 is also up-regulated by dehydration, although its extent of
up-regulation is less than that of AQP2.
(
)It is
possible that this up-regulation of AQP3 is similarly mediated by
vasopressin, since principal cells of the collecting duct have
vasopressin V1 receptors beside V2 receptors, and V1 receptor is known
to be coupled to the Ca
/protein kinase C system.
Activation of the Ca
/protein kinase C system will
stimulate the transcription of AQP3 as demonstrated in this study (Fig. 6). The possibility that two aquaporins located in the
same cells use different signaling systems is noteworthy, and further
studies are needed to evaluate this speculation. Up-regulation of these
two aquaporins, namely AQP2 located at the apical membrane and AQP3 at
the basolateral membrane, would be helpful for increased water
reabsorption in the collecting duct in a dehydrated state.
We thank Drs. N. Tojo, M. Furuno, and Y. Matsumura for
their assistance and helpful discussion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.