Functional and molecular characterization of the human neutral
solute channel aquaporin-9
Hiroyasu
Tsukaguchi1,
Stanislawa
Weremowicz2,
Cynthia C.
Morton2, and
Matthias
A.
Hediger2,3
1 Membrane Biology Program and
Renal Division, Department of Medicine, and
2 Departments of Pathology and
Obstetrics, Gynecology and Reproductive Biology, Brigham and
Women's Hospital and Harvard Medical School, and
3 Department of Biological
Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, Massachusetts 02115
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ABSTRACT |
In metabolically active cells, the coordinated transport of
water and solutes is important for maintaining osmotic homeostasis. We
recently identified a broad selective-neutral solute channel, AQP9,
from rat liver that allows the passage of a wide variety of water and
neutral solutes (H. Tsukaguchi, C. Shayakul, U. V. Berger, B. Mackenzie, S. Devidas, W. B. Guggino, A. N. van Hoek, and M. A. Hediger. J. Biol. Chem. 273:
24737-24743, 1998). A human homolog (hAQP9) with 76% amino acid
sequence identity to rat AQP9 (rAQP9) was described, but its
permeability was found to be restricted to water and urea (K. Ishibashi, M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki.
Biochem. Biophys. Res. Commun. 244: 268-274, 1998). Here we report a reevaluation of the functional characteristics of hAQP9, its tissue distribution, the structure of its
gene, and its chromosomal localization. When expressed in
Xenopus oocytes, hAQP9 allowed passage
of a wide variety of noncharged solutes, including carbamides, polyols,
purines, and pyrimidines in a phloretin- and mercurial-sensitive
manner. These functional characteristics are similar to those of rAQP9.
Based on Northern blot analysis, both rat and human AQP9 are abundantly expressed in liver, whereas, in contrast to rAQP9, hAQP9 is also expressed in peripheral leukocytes and in tissues that accumulate leukocytes, such as lung, spleen, and bone marrow. The human AQP9 gene
is composed of 6 exons and 5 introns distributed over approximately ~25 kb. The gene organization is strikingly similar to that reported for human AQP3 and AQP7, suggesting their evolution from a common ancestral gene. The promoter region contains putative tonicity and
glucocorticoid-responsive elements, suggesting that AQP9 may be
regulated by osmolality and catabolism. Fluorescence in situ hybridization assigned its locus to chromosome 15 q22.1-22.2. Our
data show that hAQP9 serves as a promiscuous solute channel expressed
in both liver and peripheral leukocytes, where it is ideally suited to
transport of metabolites and/or nutrients into and out of these cells
water channel; urea transport; chromosomal localization; gene
organization; osmoregulation
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INTRODUCTION |
SOLUTES SUCH AS IONS, nutrients, metabolites, and
neurotransmitters are known to cross cell membranes via a variety of
transporters (1, 21, 47). The liver plays a central role in the
production and elimination of biological compounds, and hepatocytes
must have specific solute transport mechanisms to allow uptake and exit
of large amounts of solutes with minimal osmotic perturbation (11).
Urea is mostly produced in the liver as a major end product of nitrogen
metabolism and is secreted into the urine via the kidney. We initially
hypothesized that a specialized urea transporter might be responsible
for urea secretion in hepatocytes. Our laboratory previously isolated
three members of the urea transporter (UT) family from rat kidney,
named UT1, UT2, and UT3 (41). None of these transcripts were found to
be expressed in liver, suggesting that hepatocytes have a structurally
distinct urea transporter.
To elucidate the molecular basis of this putative urea transporter in
liver, we employed expression cloning with
Xenopus oocytes and isolated a
295-amino acid residue protein, aquaporin-9 (AQP9), which belongs to
the major intrinsic protein (MIP) family (1, 21, 47). Rat AQP9 (rAQP9)
displays moderate homology to mammalian AQP3 (9, 15, 29) and AQP7 (13)
(46-48% amino acid sequence identity) as well as to the bacterial
glycerol facilitator (GlpF) (37% identity) (Ref. 7a). Surprisingly,
the substrate selectivity of rAQP9 was found to be strikingly different
from that of the other aquaporins (1, 21, 47). Rat AQP9 allows
permeation of a wide variety of structurally unrelated solutes
including carbamides, polyols, purines, and pyrimidines, as well as
water, in a phloretin- and mercury-sensitive manner. The protonated
forms of monocarboxylates and ketone bodies (
-hydroxybutyrate) were also found to be permeable, whereas amino acids, cyclic sugars, Na+,
K+,
Cl
, and deprotonated
monocarboxylates and ketone bodies were excluded. These data revealed
that rAQP9 behaves as a broad selectivity neutral solute channel that
represents a new branch class of the MIP protein family.
A long-standing puzzle in transport physiology has been whether water
and solutes share a common pathway when they cross cell membranes. The
classic concept was that these processes are of distinct biophysical
nature. Earlier studies with red blood cells (2) and kidney medulla
(20) suggested that there must be a separate transport pathway for
water and urea. Consistent with the concept that urea transporters are
highly selective carriers for urea, we and others found that the urea
transporters UT1, UT2, and UT3 transport urea and urea analogs in a
phloretin-sensitive manner but are not permeable to water and glycerol
(30, 41). In contrast, our functional analysis of rat AQP9 (42)
revealed that it forms a common pathway for water and solutes.
A broad selectivity water/neutral solute channel may have profound
pharmacological and clinical implications. Since the purine and
pyrimidine analogs, e.g., 5-fluorouracil, which readily permeate AQP9,
are commonly used as chemotherapeutic agents, AQP9 may influence the
chemosensitivity and resistance in cancer tissues. We observed significant upregulation of AQP9 mRNA in the liver of
streptozotocin-induced diabetic
rats,1
suggesting that AQP9 is particularly important for cells that must
adapt to osmotic stress in metabolically active states. Regulation of
gene expression by hypertonicity has been extensively studied for
several kidney osmolyte transporters and glucose-catalyzing enzymes
(6). The observations suggested that the AQP9 gene may be
controlled by similar osmoregulatory mechanism.
In this study, we report the functional characteristics of the human
isoform of AQP9 (hAQP9). This analysis is of particular importance,
since Ishibashi et al. (14) reported that the permeability of hAQP9 is
restricted to urea and water. Furthermore, to study the regulatory
mechanisms of AQP9 expression and to search for possible linkage to
human diseases, we isolated the hAQP9 gene, including its 5'
flanking region, and mapped it to the human chromosomes.
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METHODS |
Cloning of hAQP9 cDNA. The cDNA probe
(420 bp) was generated by PCR from a human liver cDNA using
oligonucleotide primers according to the sequence of rat AQP9
(accession no. AF016406, nucleotide 396-816). A human liver cDNA
library was constructed in a
ZAPII phage vector (Stratagene, La
Jolla, CA) by using the Superscript Choice System (GIBCO-BRL).
Approximately 4 × 105 clones
were screened with 32P-labeled PCR
probe under high-stringency conditions that included washing with
0.1× SSC and 0.1% SDS, at 65°C. A positive clone was
selected and subcloned into pBluescript II SK(
) (Stratagene).
In vitro translation. cRNA was
translated in vitro using a rabbit reticulocyte lysate system in the
absence or presence of canine microsomal membrane (Promega, Madison,
WI) according to the manufacturer's protocol. The products were
analyzed on a 10% SDS-PAGE gel.
Northern analysis. A multiple tissue
Northern blot (Clontech, Palo Alto, CA) was hybridized in 50%
formamide with 32P-labeled
full-length hAQP9 cDNA probe at 42°C, and washed with 0.1% SDS and
0.1× SSC, at 65°C. Autoradiography was performed at
80°C for 5 days.
Oocyte expression and radiotracer uptake
assay. Human AQP9 cDNA was digested by
Not I and
BamH I and blunted by 3' fill-in and then blunt-end ligated into the
Bgl II site of the high level expression vector pX
G-ev1, which contains the 5'-untranslated region of the Xenopus
-globulin
cDNA (a gift of Dr. Peter Agre; Ref. 38). AQP9-cRNA was synthesized
after linearization with Sma I, using T3 RNA polymerase, and 25 ng of cRNA was injected into collagenase-treated oocytes. After
incubation at 18°C for 2-3 days, radiotracer studies were
performed as described (42). Briefly, oocytes were incubated for 90 s
with Barth's solution [88 mM NaCl, 1 mM KCl, 0.33 mM
Ca(NO3)2,
0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM
NaHCO3, and 10 mM HEPES, pH
7.4] including 1 mM unlabeled compound and 1-2 µCi/ml of
radiolabeled compound. Uptake was terminated by adding ice-cold
Barth's solution with 1 mM unlabeled compound, and oocytes were
solubilized in 200 µl 10% SDS. The diffusive solute permeability
coefficient (Ps,
cm/s) was determined from the relation
Ps = N/(A ×
c), where
N is radiotracer uptake (in pmol/s),
A is the membrane area (0.045 cm2), and
c is the concentration difference
of the solute (in pmol/cm3).
Isolation of genomic clones.
Approximately 5 × 105 clones
from a human lambda phage genomic library (Lambda FIX II, Stratagene) were screened by using the
32P-labeled full-length hAQP9
cDNA. Two positive clones were isolated, and the inserts were subcloned
into the Not I site of pBluescript SK(
) (
1 and
2-2). To obtain further 5' upstream
sequence, a human P1 artificial chromosome (PAC) library (Genome
Systems, St. Louis, MO) was screened with a 382-bp cDNA probe
corresponding to the 5' end of the hAQP9 cDNA (nucleotides
209 to +173). The isolated PAC clone (PAC
19-h12) was subjected to restriction
enzyme and Southern blot analysis. A 5.5-kb
EcoR I fragment from PAC 19-h12, spanning the translation
initiation site as well as the 5' flanking region, was subcloned
into pBluescript SK(
) (pBS H12). pBS
H12 construct was used for sequence
analysis to determine the gene promoter sequence and for primer
extension analysis. Sequence was determined on both strands and
analyzed by the GCG analysis package (version 8.1; Genetic Computer
Group, Madison, WI). The size of introns was determined by PCR using
two primers placed in consecutive exons.
Primer extension. An antisense
oligonucleotide was designed
(5'-GCCGTTCCAATTAGAGGCTGTGGC-3', nucleotides
144 to
167) and end-labeled with
[
-32P]ATP (6,000 Ci/mmol, New England Nuclear, Boston, MA) by T4 polynucleotide kinase.
Poly(A)+ RNA from human liver (2 µg) was incubated with 0.1 pmol of the primer in a reaction buffer
containing 50 mM Tris · HCl, pH 8.3, 50 mM KCl, 10 mM
MgCl2, 10 mM dithiothreitol, 1 mM
of each dNTP, and 0.5 mM spermidine at 58°C for 20 min. The annealed samples were reverse-transcribed by using avian
myeloblastosis virus (AMV) reverse transcriptase (Promega) in the same
buffer containing 6.25 mM sodium pyrophosphate. After 30-min incubation
at 42°C, the samples were analyzed on an 8% acrylamide gel in
Tris/borate/EDTA buffer.
Rapid amplification of 5' cDNA
ends. 5'-cDNA ends were amplified by PCR from
human liver Marathon-Ready cDNA (Clontech) using two gene-specific
antisense primers: AS1, 5'-TAAGCTGCTCTTCAAGACCAGTC-3' (nucleotides +44 to +66); AS2,
5-ATGACCCCTCCAAAACGTCCTCG-3' (nucleotides +151 to +173), in
combination with adaptor primers 1 and 2 (AP1 and AP2, Clontech).
Following the first round of PCR with primer sets AP1 and AS2, the
products were subjected to a second round PCR using primer sets AP2 and
AS1. PCR products were analyzed by agarose gel electrophoresis,
transferred, and hybridized with a hAQP9 cDNA probe corresponding to
the 5' end (nucleotide positions
209 to +173).
Hybridization was performed at 42°C in 50% formamide, and filters
washed with 0.1% SDS and 0.1× SSC, at 65°C.
Fluorescence in situ hybridization.
The human genomic clone
2-2 (0.5 µg) was labeled with
digoxigenin-11-dUTP, coprecipitated with 10 µg Cot-1 DNA (GIBCO-BRL),
and resuspended in 1× Tris-EDTA buffer at 100 µg/ml (48).
Hybridization of metaphase chromosome preparations from peripheral
blood lymphocytes from normal males was performed with the probe at 15 µg/ml in Hybrisol VI as described (34). The probe was detected using
the Oncor kit (Oncor, Gaithersburg, MD) according to the
manufacturer's protocol. Twenty-two metaphase chromosomes were
counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)
and were assessed for probe localization with a Zeiss Axiophot
microscope. Images were captured and photographed using the CytoVision
Imaging System (Applied Imaging, Pittsburgh, PA).
Radiation hybrid mapping. The Stanford
G3 radiation hybrid panel (Research Genetics, Huntsville, AL) was
screened by PCR assay with a set of primers designed on the 3'
untranslated region of hAQP9 cDNA (sense,
5'-TGATATTCTCCAAACCTAG-3', nucleotides 1413-1431; and
antisense, 5'-GAAAGTGCCTTGTGAATTGTTAAGC-3', nucleotides
1856-1880). The data were processed at the Stanford
Human Genome Center RH server (rhserver{at}shgc.stanford.edu) for
calculation of a two-point maximum likelihood analysis to framework markers.
 |
RESULTS |
cDNA cloning and tissue distribution of
hAQP9. The human AQP9 cDNA we isolated consists of
2,890 nucleotides and its sequence is identical to that previously
reported (GenBank accession no. AB008775, Ref. 14) with the exception
that our clone has an additional 14 nucleotides at the 5' end.
The sequence encodes a 295-amino acid residue protein with a predicted
molecular mass of 31 kDa (Fig.
1A).
Human AQP9 cDNA has a potential N-linked glycosylation site at Asn142, and in vitro translation
generated a single band of 28 kDa that increased in size by adding the
microsome membrane, suggesting that this site is indeed glycosylated
(Fig. 1B). Human AQP9 also has a
potential protein kinase C (PKC) phosphorylation site at Ser11, but there are no protein
kinase A (PKA) sites. Hydropathy analysis showed that, in analogy to
rat AQP9 (rAQP9), hAQP9 has six putative transmembrane domains (Fig.
1C). The amino acid sequence of
hAQP9 is 76% identical to that of rAQP9 (42) and has moderate identity with human AQP3 (47%) (9, 15, 29), human AQP7 (41%) (17), and the
bacterial glycerol facilitator GlpF (39%) (7a), whereas it displays
only weak identity with AQP1 (28%) (1, 21, 47). High-stringency
Northern analysis (Fig. 2) gave a strong
signal of 2.9 kb in liver, in analogy to rAQP9 (42). In contrast to rAQP9, hAQP9 is also abundantly expressed in peripheral leukocytes as
well as the tissues accumulating leukocytes, such as the lung, spleen,
and bone marrow (22, 42). hAQP9 mRNA was not detected in testis and
brain, whereas rAQP9 is abundantly expressed in these tissues (42). We
did not detect hAQP9 mRNA in cancer cell lines of nonepithelial origin
such as leukemia, lymphoma, and melanoma, or of epithelial origin
(colon and lung carcinoma).

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Fig. 1.
Sequence analysis of human aquaporin-9 (hAQP9) cDNA.
A: amino acid sequence alignment of
hAQP9 cDNA. Sequences from human AQP1 (GenBank accession no. P29975),
rat and human AQP3 (L35108 and AB001325, respectively), rat AQP7
(AB000507), and rat AQP9 (AF016406) were aligned using the PILEUP
program (Genetics Computer Group). Putative membrane-spanning regions
are underscored and numbered 1-6.
Conserved residues are indicated by gray shading. Asn-Pro-Ala (NPA)
consensus motifs are in boxes. Note potential sites for
N-linked glycosylation
(Asn142, *), protein kinase C
phosphorylation (Ser11, ), and
casein kinase II phosphorylation site
(Thr26, ).
B: autoradiograph of SDS gel showing
in vitro translation products; , absence; +, presence of canine
pancreatic microsome membrane. C:
structural model of AQP9. Topology was determined according to the
Kyte-Doolittle algorithm. Numbering of putative membrane-spanning
domains corresponds to that of
A.
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Fig. 2.
Distribution of hAQP9 mRNA in human tissues. Northern blots with 2 µg
of poly(A)+ RNA of various human
tissues were hybridized with a full-length hAQP9 cDNA probe. mRNA
sources of cancer cell lines are: promyelocytic leukemia (HL-60,
lymphoblastic), HeLa cell (S3), chronic myelogenous leukemia (K-562),
lymphoblastic leukemia (MOLT-4, T-lymphoblastic), Burkitt lymphoma
(Raji, B-lymphoblastic), colorectal adenocarcinoma (SW480), lung
carcinoma (A549), and melanoma (G361).
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Functional properties of hAQP9. When
expressed in oocytes, hAQP9 increased the urea permeability coefficient
(Ps) from 1.5 ± 0.2 × 10
6 cm/s
(water-injected) to 30.2 ± 1.7 × 10
6 cm/s (Fig.
3A). The
increase in Ps
was similar to that observed for the urea transporters (UT2 and UT3)
and rAQP9 (Ps = 25-45 × 10
6
cm/s) (41, 42). Human AQP9 induced permeabilities of a variety of
structurally unrelated solutes including polyols (glycerol, mannitol,
sorbitol), purines (adenine), pyrimidines (uracil and the
chemotherapeutic agent 5-fluorouracil), and urea analogs (thiourea) (Fig. 3, A and
B), and the
Ps values ranged
from 20 to 30 × 10
6
cm/s. The broad permselectivity contrasts the functional
characteristics of hAQP9 previously reported by Ishibashi et al. (14),
showing that hAQP9 is only slightly permeable to urea
(Ps = 5.5 × 10
6 cm/s) and impermeable
to glycerol. Consistent with the characteristics of rAQP9, radiotracer
uptake studies revealed that hAQP9 is impermeable to cyclic sugars
(D-glucose,
D-mannose,
myo-inositol), the nucleoside uridine,
and amino acids (glutamine, glycine) (data not shown). Also consistent
with rAQP9 properties, we found a three- to fourfold higher
permeability for the monocarboxylates lactate and
-hydroxybutyrate at reduced pH (pH 5.5) compared with physiological pH (data not shown).
As alluded to in our previous study, the pH sensitivity indicates that
monocarboxylates permeate AQP9 in their protonated, neutral form.
Likewise, the low permeabilities of the purine analogs xanthine and
uric acid suggests that these compounds permeate hAQP9 in their
protonated form.

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Fig. 3.
Functional properties of hAQP9 expressed in
Xenopus oocytes.
A: solute permeability in oocytes
expressing hAQP9. Uptake of 1 mM
14C- or
3H-labeled carbamides, polyols,
purines, pyrimidines, nucleosides, and monocarboxylates was measured
over 90 s in oocytes injected with water (control) or hAQP9 cRNA.
Measurements were performed at 22°C, except for lactate uptake,
which was performed at 4°C to minimize the contribution from
endogenous lactate transport. Data are means ± SE from
6-8 oocytes. B: molecular
structures of substrates. C:
inhibition by phloretin. Percent inhibition of solute permeability (1 mM) was examined by 10-min incubation with phloretin at 0.1 mM (solid
bars) or 0.5 mM (gray bars). D:
inhibition by HgCl2. Percent
inhibition was obtained by 5-min incubation with 0.3 mM
HgCl2. * No significant
inhibition. 5-FU, 5-fluorouracil; -HB, -hydroxybutyrate; Met-glu,
methyl- -glucopyranoside.
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When oocytes expressing hAQP9 were transferred from regular Barth's
solution (200 mosmol/kgH2O) to
diluted Barth's solution (70 mosmol/kgH2O), we observed osmotic
lysis of the oocytes within 2-3 min following exposure to
hypotonicity. This indicates that hAQP9 is permeable to water (data not
shown), a property that was investigated in detail for rAQP9 (42).
Phloretin (0.1 mM) and HgCl2 (0.3 mM) effectively inhibited the permeability of mannitol, sorbitol,
adenine, 5-fluorouracil, and
-hydroxybutyrate (70-90%) (Fig.
3, C and
D). The urea and glycerol
permeabilities were inhibited only 20-50% by 0.1 mM phloretin,
and glycerol uptake was inhibited only ~50% by 0.3 mM
HgCl2. The urea permeability was
not significantly affected by 0.3 mM
HgCl2. Overall, these inhibitor
sensitivities were very similar to those reported for rAQP9 (42).
Thus our data indicate that, despite the report of Sasaki and
colleagues (14, 22), the functional properties of human and rat AQP9 are almost identical (42).
Genomic cloning and exon-intron
organization. To determine the genomic structure of the
human AQP9 gene, we screened a human bacteriophage genomic library
using the full-length hAQP9 cDNA as a probe. We isolated two
overlapping clones, called
1 and
2-2, each containing ~15
kb (Fig. 4). Southern analysis revealed that the two clones span an ~20-kb genomic region that
includes the coding region of the last two-thirds of the carboxy
terminus of hAQP9, but they do not contain the 5' flanking region
of the hAQP9 gene. This suggests that the size of this gene is much
larger than that of other aquaporins that are 3-11 kb long (12,
24, 28, 32, 44). To isolate the missing upstream sequence, we screened
a human PAC genomic library, using a cDNA probe specific to the
5' end (382 bp) of hAQP9 cDNA. We isolated a PAC clone (PAC
19-h12), which contains both the
5'-flanking region and the 5' coding sequence.

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Fig. 4.
Structure of the human AQP9 gene. Exon and intron organization were
determined by comparing the sequences between hAQP9 cDNA and three
genomic clones. Two overlapping lambda phage clones ( 1, 2-2)
and a PAC clone (PAC 19-h12) are
shown at the bottom. Translational and
nontranslational regions of hAQP9 cDNA are shown by solid black and
gray boxes, respectively. Exons and introns of the human AQP9 gene are
indicated by numbered boxes and solid lines, respectively.
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Sequence comparison of AQP9 cDNA with the three genomic clones revealed
that the hAQP9 gene is composed of six exons and five introns (Fig. 4).
The position and sizes of exons and introns and the sequence of
exon-intron junctions are summarized in Table 1. The sizes of exons 1-6 are 398, 127, 138, 119, 218, and 1,958 bp, respectively. Exon 6 has a stop codon
and a polyadenylation signal (AATAAA). All splice sites
are consistent with the consensus sequence for splice-acceptor and
-donor sites (GT-AG rule) (33). Notably, the overall gene structure of
hAQP9 is analogous to that of hAQP3 and hAQP7 with regard to the number
of exons and introns, the sites of the junctions, and the codon phases
(12, 17) (Table 2). However, introns for
hAQP9, ranging from 1.5 kb (intron 3) to more than 10 kb (intron 1),
were larger than those of AQP3 and AQP7 (90 to 3,500 bp) (12, 17). The
hAQP9 gene appears to be evolutionarily closely related to hAQP3 and
hAQP7, a water channel that is permeable to urea and glycerol (9, 13,
15, 29). The gene architecture of AQP9, AQP3, and AQP7 remarkably contrasts those genes encoding AQP channels that are selective for
water, including AQP1, AQP2, AQP4, and AQP5. These genes are composed
only of four exons located at identical sites with a clear preference
of class 0 introns (24, 28, 32, 44).
Transcription initiation site. To
determine the transcription initiation site (TS), we used the primer
extension assay and 5' rapid amplification of 5' cDNA ends
(5'-RACE) analysis. Human liver
poly(A)+ mRNA was used as a
template for primer extension using a primer corresponding to
nucleotides
167 to
144 of the noncoding region of hAQP9
cDNA. We detected the most intense band at nucleotide position
286 (TS1, 286 bp upstream of the AUG translation
initiation site position) and another band with less intensity at
nucleotide
211 (TS2, Fig.
5A). TS1 and TS2 are located 58 bp
upstream and 17 bp downstream, respectively, within the 5' end of
the cloned AQP9 cDNA. To confirm the transcription initiation sites, we
performed 5'-RACE and subsequent Southern analysis. For
5'-RACE analysis, we used human liver Marathon-Ready cDNA
(Clontech), which has 5' adaptors attached containing primer
sites AP1 and AP2 (Fig. 5B). The
5' ends of hAQP9 cDNA were then amplified using 5' AP1 or
AP2 primers and 3' AQP9-specific primers (AS1 or AS2, see Fig. 5B) and analyzed by Southern
blotting. Whereas in the cloned hAQP9 cDNA the distance between the AS1
primer site and its 5' end is ~300 bp, the two 5'-RACE
products that were generated gave a strong band at ~350 bp (5'
sequence of TS1 product) and a weaker band ~270 bp (5' sequence
of TS2 product), again consistent with the existence of two
transcriptional start sites, TS1 and TS2. We conclude that nucleotide
286 relative to the AUG translation start site is the major
transcriptional start site and therefore corresponds to the beginning
of exon 1.

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Fig. 5.
Mapping of the transcription initiation site.
A: primer extension. An antisense
oligonucleotide, ASPE (nucleotide positions 144 to 167 of
the human AQP9 cDNA), was end-labeled and annealed to 2 µg of the
human liver poly(A)+ RNA. The
annealed mixture was further extended by avian myeloblastosis virus
reverse transcriptase, and the reaction products (PE) were analyzed on
an 8% polyacrylamide gel. Sequencing products of a plasmid pBS
H12, generated from the antisense
primer, were run on the gel as a size marker. TS1 and TS2 indicate the
positions of the extension products that represent the major and minor
transcriptional start sites. Asterisks indicate the nucleotide position
of the initiation sites (the numbers at either end of the sequences
represent the nucleotide positions relative to the translation start
site). B: diagram of 5'-RACE
cDNA products. Coding and 5'-untranslated regions of the hAQP9
cDNA are shown by the solid box and the bold line, respectively.
Positions of the two transcriptional start sites (TS1 and TS2)
determined by primer extension analysis are indicated. Human AQP9
cDNA-specific primers are AS1 and AS2, and two overlapping adaptor
primers are AP1 and AP2. A 295-bp PCR product is expected from the
5' end of the cloned cDNA (accession no. AF016495) when primer
set AP2-AS1 is used. C: Southern blot
analysis of 5'-RACE products. The PCR products from the primer
pair AP1-AS2 were further amplified with the nested sets of primers
AS1-AP2. The resulting PCR products (AS1-AP2) were electrophoresed,
transferred to nitrocellulose filters, and probed with
32P-labeled hAQP9 cDNA (nucleotide
209 to +173). The sizes of the upper and lower bands are
consistent with the positions of the TS1 and TS2 sites shown in
A and
B.
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Analysis of the 5' flanking
region. We analyzed ~1 kb upstream of the AUG
translation initiation codon (Fig. 6) by
sequencing a pBS H12 plasmid. The
5' flanking region did not contain a typical TATA box sequence
(TATAWA, Ref. 4) nor a CCAAT box (7) at the transcriptional start site.
The human AQP5 gene is reported to lack a TATA box (24), whereas most
other human aquaporin genes (AQP0, AQP1, AQP2, AQP3, and
AQP4) contain a TATA box element (12, 28, 32, 37, 44). Thus AQP9
provides the second example of a "TATA-less" promoter
in the aquaporin gene family. The gene promoter also did
not contain GC-rich elements (G+C content was ~35% at positions from
900 to
400) nor Sp-1 binding sites, although these motifs
were reported to contribute to transcription regulation in TATA-less
gene promoters (8, 39).

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Fig. 6.
Nucleotide sequence of the 5' flanking region. First nucleotide
of the translation start site is numbered +1. Initiation codon (ATG) is
set in bold and boxed, and the amino acid sequence is shown according
to the single letter convention (at
bottom). Transcriptional start sites
(TS1 and TS2 in Fig. 5), as determined by primer extension assay, are
indicated by arrows. An Asterisk indicates position of 5'
terminus of the cloned cDNA. Consensus recognition sequences for
transcriptional factors were analyzed by using data bases of TFD SITES
and Transfac SITES (10, 46), and putative regulatory elements are
indicated by boxes. Sequence was deposited as GenBank accession no.
AF102870. See Analysis of the 5' flanking
region for complete description of motif
abbreviations.
|
|
When searching for regulatory cis
elements, we identified several putative transcription factor
recognition sites. The 5' flanking sequence contained AP-1
(activator protein-1, TGANTMA, position
580; Ref. 25) and GATA-1
(WGATAMS, position
602; Ref. 43) sites. Potential binding sites
were also found for the transcription enhancer factor TEF-1 (TGGAATGTT,
position
849), the sis-inducible factor SIF (CCCGTC, position
371), and the histone H4 transcriptional factor H4TF (GATTTC,
position
238). The sequence TGTCCT at position
723
corresponds to a binding motif for the glucocorticoid
receptor (GR) (19). We furthermore found two TonE
tonicity response enhancer consensus motifs (TGGAANNNYNY; Refs. 6, 40)
at position
847 and
886.
Transcription factor recognition sites may be involved in
tissue-specific expression. We identified a CCAAT/enhancer-binding protein (C/EBP) site (TKNNGYAAK, position
766; Ref. 18) and a
hepatocyte nuclear factor (HNF-1) site (ATTAATCATTACC, in reversed orientation at position
273, distal to TS1; Ref. 36), both of
which are known to be involved in liver-specific expression. There are
also putative
-interferon inducible binding protein IBP-1 (AAGTGA,
position
763, Ref. 43) and nuclear factor NF-
B (GGGRNTYYC,
position
238, Ref. 26) sites that may be associated with
lymphocyte-specific expression.
Localization of hAQP9 on the human
chromosomes. We analyzed the chromosomal localization
by fluorescence in situ hybridization (FISH), using a 15-kb genomic
probe (Fig. 7). The map position was
determined by visual inspection of the fluorescent hybridization signals on DAPI-stained metaphase chromosomes. In all of the 22 metaphase preparations, a hybridization signal was found on the long
arm of chromosome 15 in bands q22.1-22.2. In 21 metaphase spreads,
both copies of chromosome 15 were labeled and, in one metaphase spread,
the signal was detected on a single chromosome 15. We next performed a
radiation hybrid mapping, using the Stanford G3 radiation panel. PCR
amplification with primers specific to the 3'-untranslated region
of hAQP9 cDNA indicated that it is most likely linked to the
polymorphic locus D15S117 (marker AFM098yg1) with a centiray distance
(cR10000) of 10.95 (lod score = 13.05). The physical position of D15S117 correlated with
the cytogenetic localization of the band q22.1-q22.2 (3), confirming
that the hAQP9 gene exists at this locus. This locus is 10 centimorgans proximal to the centromeric marker of Bardet-Biedl syndrome (chromosome 15q22.3-q23) (5), an autosomal recessive disorder that is characterized by retinal degeneration, polydactyly, obesity, and hypogenitalism (OMIM
209900). No other genes of aquaporins and transporters were found in
the vicinity of 15q22.1-22.2.

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Fig. 7.
Chromosomal localization of the human AQP9 gene.
A: ideogram of human chromosome 15. hAQP9 gene was mapped at 15q22.1-22.2 (arrow).
B: photograph of human metaphase
chromosomes counterstained with 4,6,-diamidino-2-phenylindole
dihydrochloride (DAPI). The two chromosomes 15 are indicated by
numbers; arrows point the site of hybridization of the hAQP9 probe on
both chromosomes 15 in bands q22.1-22.2.
|
|
 |
DISCUSSION |
An important goal of this study was the reevaluation of the functional
characteristics of human AQP9. Ishibashi et al. (14) reported that the
permeability of human AQP9 is restricted to urea and water, a finding
which would be in contrast to the broad permselectivity of its rat
homolog (42). In addition, the same group recently reported that rat
AQP9L (22), of which amino acid sequence is 99% identical to rat AQP9
we reported (42), is permeable only to urea and glycerol as well as to
water. The discrepancy between our findings and those of Sasaki and
colleagues (14, 22) is not clear. Our data establish that
the permselectivity of human AQP9 is very similar to that of rat AQP9.
Northern analysis showed that human AQP9 mRNA is expressed in
leukocytes and in tissues that accumulate white blood cells, including
spleen, bone marrow, and lung. Notably, in rat, AQP9 mRNA is not
expressed in peripheral leukocytes. The distinct pattern of tissue
distribution in rat and human may be due to different requirements of metabolites among the two species. The AQP9 expression may also be developmentally regulated and may change according to the
metabolic state of tissues.
We assume that cell types of leukocytes expressing hAQP9 are
neutrophils and/or B-lymphocytes, because
1) no AQP9 mRNA was detected in
thymus (~100% of the cells are T-lymphocytes) and in T-lymphoblastic
cell lines (MOLT-4), 2) moderate
AQP9 expression was detected in spleen and lymph node (most of the
cells are B-lymphocytes), and 3) the
most abundant expression was found in peripheral leukocytes, of which
cells are usually a mixture of ~80% neutrophil, 15% T-lymphocytes, and 5% B-lymphocytes.
As a first step toward understanding the regulatory mechanisms of
hAQP9, we isolated and characterized its promoter. Interestingly, there
are no typical TATA or CCAAT box elements in the hAQP9 promoter. The
A-T rich region found at position
320 to
340 may serve as a TATA box. Alternatively, the Inr initiator element was reported to
participate in the transcription of genes which do not have TATA or
CAAT boxes (27, 45). Inr elements typically consist of an A (adenine)
at the transcriptional start site and flanking polypyrimidine clusters,
i.e.,
3Y
YYYY+5
(27, 45). The putative hAQP9 transcriptional start sites (Figs. 5 and
6) have an adenine at the initiation position, but the flanking sequence does not have typical polypyrimidine motifs as reported previously. These findings were not unexpected for AQP, since the AQP5
gene also lacks TATA box and Inr elements (24).
With regard to tissue-specific expression of AQP9, Northern analysis
(Fig. 2) showed that hAQP9 is expressed mainly in liver and leukocytes.
The 5' flanking region of the hAQP9 gene contains several motifs
that may serve as recognition sites for tissue-specific factors such as
the hepatocyte-specific regulatory factors HNF-1 and C/EBP (18,
36) and the lymphocyte-specific transactivating factors IBP-1 and
NF-
B (26, 43).
Since we observed that AQP9 mRNA is upregulated in the liver of
streptozotocin-treated diabetic rats (see footnote 1), we were further
interested in whether AQP9 expression is controlled by osmoregulation.
In the diabetic liver, hepatocytes are exposed to hypertonic conditions
because of the accelerated glyconeogenesis and ketosis,
which may trigger AQP9 upregulation through osmoregulation. Since AQP9
confers a broadly selective aqueous pore, upregulation of
AQP9 may facilitate secretion of metabolites such as ketone bodies and
protect cells from damage caused by unwanted volume changes. Numerous
studies with kidney medullary cells (6) suggested that regulation of
osmolyte transport by external hypertonicity is important for these
cells to adapt to osmotic stress (6). Recently, an osmotic response
element has been identified in several gene promoters responsible for
the osmoregulation of the sodium-dependent myo-inositol transporter (SMIT), the
betaine/
-amino-n-butyric acid
transporter (BGT), and the aldose reductase. We found two tonicity
response enhancer (TonE) consensus motifs in the hAQP9 promoter, which
reside in close proximity, only 18 bp apart, 600 bp upstream of the
transcription initiation site (Fig. 6). A glucocorticoid-responsive element motif at position
723 may also affect AQP9 gene
expression. AQP9 may be regulated by the catabolic hormones such as
glucagon and glucocorticoid, because these hormones promote the
breakdown and oxidation of stored body fuels, thereby increasing the
requirement for transport of metabolites.
The results from the genomic analysis of AQP9 provide information on
how MIP family members with diverse permselectivities may have emerged.
Based on functional analysis, we propose that the MIP family can be
divided into three classes: class I, which includes water-selective
channels such as AQP1, AQP2, AQP4, and AQP5; class II,
which has limited permselectivity for certain neutral solutes, e.g.,
AQP3 and AQP7; and class III which has broad permselectivity, e.g.,
AQP9 from rat and human. Class II and class III channels are also known
as aquaglyceroporins (1). The division into three distinct classes is
consistent with the structures of the human AQP genes: in general,
class II and III genes have six exons, whereas class I genes have four
exons. Recently, the human AQP8 gene has been shown to have six exons
but to belong to the class I water-selective channels (23), raising the
possibility that it may represent another phylogenic branch.
Identification of other AQP members will help to define more detailed
characteristics of such a subclass. Furthermore, genes within a given
class often cluster on specific chromosomes. For example, the class I
genes AQP2 and AQP5 cluster on a single band of chromosome 12 (12q13) (47), and the class II genes AQP3 and AQP7 cluster within a single band
on chromosome 9 (9p13) (16, 17). As shown in Fig. 8, the class III AQP9 gene is located on
chromosome 15 (15q22.1-22.2), and it is conceivable that
additional AQP9-like genes cluster in this region. Given the close
homology (~40% amino acid sequence identity) of class II and III but
not class I genes with the water-impermeable bacterial glycerol
facilitator GlpF (7a), we propose that class I and class II/III genes
evolved from a common ancestral gene ("primitive pore") and that
the II/III prototypes further evolved to form subtypes with specialized
permselectivities (Fig. 8).

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Fig. 8.
Hypothetical model of the evolution of the major intrinsic protein
(MIP) family. A: on the basis of
functional and genomic analysis, we propose that the MIP family can be
divided into 3 classes, class I, II and III, which may have evolved
from a common "primitive pore".
B: summary of permselectivity,
chromosome localization, and amino acid identities of MIP family
members. Percent identities are based on comparison with the hAQP9
amino acid sequence. All identities refer to comparison of human
sequences. Human chromosomes with AQP cluster are shown in bold. ND,
not determined.
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants RO1-DK-46289 (to M. A. Hediger).
H. Tsukaguchi was supported by a Research Fellowship of the National
Kidney Foundation.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
1
Tsukaguchi and Hediger, unpublished data. cDNA
and genomic sequences were deposited in GenBank accession nos. AF016495 and AF102870.
Address for reprint requests and other correspondence: M. A. Hediger,
Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115 (E-mail: mhediger{at}rics.bwh.harvard.edu).
Received 1 April 1999; accepted in final form 4 June 1999.
 |
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