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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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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 (beta -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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INTRODUCTION
METHODS
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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 lambda 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 pXbeta G-ev1, which contains the 5'-untranslated region of the Xenopus beta -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 × Delta c), where N is radiotracer uptake (in pmol/s), A is the membrane area (0.045 cm2), and Delta 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(-) (lambda 1 and lambda 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 [gamma -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 lambda 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.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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, open circle ). 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).

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 beta -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; beta -HB, beta -hydroxybutyrate; Met-glu, methyl-alpha -glucopyranoside.

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 beta -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 lambda 1 and lambda 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 (lambda 1, lambda 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.

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).

                              
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Table 1.   Exon/intron organization of the human AQP9 gene


                              
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Table 2.   Comparison of splicing site between human AQP9 and AQP3 genes

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.

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 gamma -interferon inducible binding protein IBP-1 (AAGTGA, position -763, Ref. 43) and nuclear factor NF-kappa 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
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INTRODUCTION
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DISCUSSION
REFERENCES

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., -3YYC<UNL>A</UNL>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-kappa 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/gamma -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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