©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of Human Aquaporin 3 Gene (*)

(Received for publication, May 15, 1995)

Naohiko Inase Kiyohide Fushimi Kenichi Ishibashi Shinichi Uchida Masahiko Ichioka Sei Sasaki (§) Fumiaki Marumo

From the Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Water transport in highly water-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP)()(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) .

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.


MATERIALS AND METHODS

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 KHPO, 8.1 mM NaHPO, 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 (pSVGal) 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.

-Galactosidase Assay

After 15 µl of the cell lysate was mixed with 15 µl of -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.


RESULTS

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.


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.





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).


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).



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.


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) RNA.




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) 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).



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.

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 (10M) up-regulated luciferase activity about 4-fold in pGL2-5`, whereas cpt-cAMP (200 µM) had no effect (Fig. 6).


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 (10M) or cpt-cAMP (200 µM) was added to the medium 24 h after transfection, and luciferase activity was measured 48 h after transfection.




DISCUSSION

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.()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.


FOOTNOTES

*
This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture and from the Salt Science Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) D50486[GenBank Link].

§
To whom correspondence and reprint requests should be addressed. Tel.: 81-3-3813-6111 (ext. 3221); Fax: 81-3-3818-7177.

The abbreviations used are: AQP, aquaporin; MIP, major intrinsic protein; kb, kilobase(s); bp, base pair(s); PIPES, piperazine-N,N`-bis(2-ethanesulfonic acid); TPA, tetradecanoylphorbol acetate; GlpF, glycerol facilitator; cpt-cAMP, chlorophenylthio-cAMP; ATF, acting transcription factor; CRE, cyclic AMP response element; SIF, sis-inducible factor.

Y. Matsumura, S. Uchida, M. Furuno, Y. Takeuchi, F. Marumo, and S. Sasaki, unpublished observation.

Y. Takeuchi and S. Sasaki, unpublished observation.


ACKNOWLEDGEMENTS

We thank Drs. N. Tojo, M. Furuno, and Y. Matsumura for their assistance and helpful discussion.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.