(Received for publication, May 28, 1996, and in revised form, August 12, 1996)
From the Laboratoire de Reproduction et Développement, CNRS, URA 1940, Université Blaise Pascal-Clermont-Ferrand II, 63177 Aubiere Cédex, France
Aldose reductase (AR; EC 1.1.1.21) is an
oxidoreductase that catalyzes the NADPH-dependent
conversion of glucose to sorbitol, the first step of the polyol
pathway. AR is of great interest due to its implication in the etiology
of diabetic complications. In renal medullary cells, AR also plays an
osmoregulatory role by accumulating sorbitol to maintain the
intracellular osmotic balance during antidiuresis. We have previously
cloned the AR cDNA from mouse kidney, and we report here the
isolation of the mouse AR gene promoter. Transient transfection of
chloramphenicol acetyltransferase reporter constructs containing
various 5-flanking regions of the mouse AR gene in CV1 cells led to
the identification of a sequence spanning base pairs
1053 to
1040,
required for an enhancer activity in hypertonic compared with isotonic
cell culture conditions. This sequence is similar to the
tonicity-responsive element first characterized in the
betaine-
-aminobutyric acid transporter promoter.
The osmotic balance between intracellular and extracellular compartments of cells is critical for the maintenance of cellular homeostasis. Exposure to anisotonic media initiates a response that counteracts volume perturbations by complex mechanisms involving changes in the intracellular concentrations of active organic solutes (osmolytes) such as sorbitol, inositol, betaine, myo-inositol, and glycerophosphorylcholine (1-5). Among the organic osmolytes, sorbitol has received special attention since it is a beneficial factor during antidiuresis and yet appears to be detrimental in diabetes (6, 7). For example, by accumulating sorbitol, renal medullary cells maintain both their volume and their intracellular medium unperturbed under hyperosmotic stress. In target tissues of diabetes such as kidney, nerve, and eye, sorbitol accumulation exerts a hyperosmotic effect that contributes to some complications of diabetes mellitus (see Ref. 8 for review).
Sorbitol is formed by the reduction of glucose by the enzyme aldose reductase (AR1; EC 1.1.1.21). AR is a ubiquitous "housekeeping" enzyme probably functional in all cells (9, 10). An osmoregulatory role of AR has been suggested by studies in cell lines derived from renal inner medulla showing that an increase in the osmolality of the medium is associated with increases in cellular sorbitol levels, AR activity, and AR gene expression (3, 4, 11). Induction of AR by hypertonic media was demonstrated also in kidney mesangial cells (12), glomerular endothelial cells (13), Chinese hamster ovary cells (12), lens epithelial cells (14), and human embryonic epithelial cells (15). The molecular mechanism of transcriptional regulation of the AR gene in response to hypertonicity is still unknown.
Nucleotide and deduced amino acid sequences for mouse AR (mAR) have
been recently reported (16, 17). We report here the isolation and
sequence of the 5-flanking region of the mAR gene and its functional
characterization.
Genomic DNA was isolated from mouse Balb/c
liver for PCR amplification of mAR gene intron-2 (Taq
polymerase, Perkin-Elmer). Exon boundaries were delimited on the AR
cDNA sequence (17) by homology to the rat AR sequence (18). The
upstream primer used for PCR amplification matched the mAR gene exon-2
end, with the last four nucleotides identical to the beginning of rat
AR gene intron-2. The sequence of this primer was
5-CTCTTCATTGTCAGCAAGGTAC-3
. The downstream primer matched exon-3
(positions 287-304 on the mAR cDNA sequence). Its sequence was
5
-TTCACCATGCTCTTGTCA-3
. PCR was performed with 5% formamide. The
640-bp amplified DNA fragment was cloned in the pGEM-T vector (Promega)
and sequenced using the T7 sequencing kit (Pharmacia Biotech Inc.)
according to the manufacturer's instructions.
A second PCR was performed from this clone to amplify intron-2 without
exon sequences. For this PCR, the primers were 5-TGTGAGGATGCTGGGGCC-3
and 5
-AGGCAGCAAAGGCACAAG-3
. This amplified DNA fragment was used to
screen a genomic library obtained by partial Sau3A1
digestion of Balb/c tail DNA and insertion in BamHI sites of
the
EMBL12 vector. A single positive clone of 13.8 kb (
AR 1-2)
was further characterized.
A
restriction map of the AR 1-2 clone was obtained by digestion with
16 different enzymes from Boehringer Mannheim. Digestion products were
electrophoresed on a 0.8% agarose gel and transferred to nylon filters
(Hybond-N, Amersham Corp.). Hybridization of filters with the cDNA
probe led to the orientation of the clone and to the localization of
the 5
-end of the gene. Digestion of the
AR 1-2 clone with
SalI/XhoI released a 9-kb and a 4.8-kb fragment,
which were subcloned separately in the pGEM7Zf(
) vector from Promega
(see Fig. 2). An EcoRI fragment of 1.9 kb was selected from
the 9-kb clone, subcloned into the pGEM7Zf(
) vector, and fully
sequenced (see Fig. 2). Sequence data were analyzed using BISANCE
programs at CITI 2 (Paris) (19). The transcription start site was
positioned by homology to the rat AR gene (18).
Genomic Southern Analysis
Mouse Balb/c liver genomic DNA
was digested with 13 different enzymes (listed in the Fig. 1 legend)
and subjected to Southern blot analysis. The filter was first
hybridized with the 32P-labeled intron-2-specific probe
described above in 3 × SSC, 0.2% polyvinylpyrrolidone
(Mr 40,000), 0.2% Ficoll 400, 5% polyethylene glycol, 1% glycine, 0.1% SDS, and 10 µg/ml denatured salmon sperm DNA at 62 °C for 24 h. Washes were performed at 60 °C. After
autoradiography, the filter was rehybridized with the
32P-labeled mAR cDNA (17) under the same conditions as
described for the intron-2 probe.
Construction of CAT Fusion Plasmids
All constructs were
obtained by cloning PCR-amplified fragments from the 9-kb clone in the
promoterless basic plasmid pBLCAT3 (20). PCR products were isolated
from agarose gels, digested with PstI/XbaI, and
directionally inserted in pBLCAT3 in front of the CAT coding sequence.
In the longest construct (p1586CAT), the distal and proximal primers
used to amplify the genomic DNA fragment extending from positions
1586 to +35 in the mAR promoter were
5
-CGCCTGCAGGCTACGTAGTGCATTTCCCTAGC-3
and
5
-CGCTCTAGACGCTGCACGTTACAAACCCGG-3
, respectively. Shorter constructs
were PCR-generated always using the same proximal primer. The distal
primers used to obtain p1067CAT, p1067mCAT, p998CAT, and p142CAT were
5
-AGACTGCAGCGACTGGAAAATCACCAGAATGGG-3
, 5
-AGACTGCAGCGACTGTCCCCTCACCAGAATGGG-3
, 5
-CAGCTGCAGTTGTCCCTGTTG-3
, and 5
-TATCTGCAGAGCTTTCCGTCTG-3
, respectively. Recombinant plasmids were purified from clear bacterial lysates on cesium chloride gradients
and verified by sequencing.
CAT fusion plasmids were tested by lipofection in monkey kidney CV1 cells using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (Boehringer, Mannheim, Germany) according to the manufacturer's instructions. Before transfection, cells were grown in basal medium (Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 4 µg/ml insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum). Five micrograms of CAT constructs were transfected when cells were 60-80% confluent in 60-mm plastic dishes (Falcon, Oxnard, CA). After 14 h, the medium containing the liposome-DNA complex was removed and replaced with either fresh isotonic medium (basal medium, 330 mosm/kg of H2O) or a medium made hypertonic (500 mosm/kg of H2O) by the addition of NaCl. Cells were harvested 24 h after transfection. Each transfection included 5 µg of the different mAR promoter constructs and 2.5 µg of pSV2CAT plasmid (21), the expression of which was similar under all culture conditions.
CAT AssaysCAT activity of cell extracts was assayed according to the method of Neumann et al. (22). Protein concentration was determined by the Bradford method (Bio-Rad). Samples were incubated in 100 µl of 0.25 M Tris (pH 7.8), 0.4 µM acetyl coenzyme A, and 0.2 µCi of [14C]chloramphenicol for 1 h at 37 °C. After thin layer chromatography and autoradiography, acetylated and non-acetylated forms were cut out and quantified by scintillation counting. CAT activity corresponds to the ratio of acetylated form radioactivity to total radioactivity (both forms). To compare basal promoter activity of mAR-CAT constructs, CAT activity was measured as described above in samples containing 50 µg of proteins. Variations in transfection efficiency were determined by repeated measurements of the CAT activity of the p1586CAT construct in 10 different assays. The average percentage of conversion was 41.73 ± 7.31%. The standard deviation was low enough to consider that transfection efficiency was constant for one construct in each transfection. To study the response to hypertonicity, the protein quantity in CAT assays was reduced to 25 µg for p1586CAT, p1067CAT, and p1067mCAT in order to obtain percentages of conversion in the range of 5-75% under both isotonic and hypertonic conditions (in this range, neither [14C]chloramphenicol nor acetyl coenzyme A is limiting for the enzymatic reaction). Average inductions (hypertonic/isotonic CAT activity ratios) and standard deviations were calculated from at least four independent transfections.
Northern Blot AnalysisNorthern blot analysis of total RNAs isolated from CV1 cells (grown under the same conditions as described for the transfected cells) was performed according to the method previously described (17).
A first
screening of a mouse genomic DNA library with the mAR cDNA (17)
revealed the presence of many AR pseudogenes. This is why an
intron-specific probe was required to isolate the functional mAR gene.
By comparison with the rat AR gene (18), intron-2 was observed to be
the 5-shortest intron of the gene (600 bp in the rat sequence), so it
was selected to target the 5
-end of the gene with its promoter and
upstream regulation sequences. mAR gene intron-2 was amplified by PCR
(see "Experimental Procedures"). The probe derived from this
intron-2 sequence was used to hybridize a Southern blot of mouse
genomic DNA digested with enzymes that cut infrequently in the genome.
Fig. 1A shows a single hybridizing band in
each digest in contrast to the complex pattern observed when the same
filter was hybridized with the mAR cDNA probe (Fig. 1B).
The intron-2-specific probe was further used to screen a
EMBL12
genomic DNA library. A single positive clone (
AR 1-2; insert size of
13.8 kb) was isolated. According to the restriction map and to the
cDNA hybridization pattern (data not shown),
AR 1-2 was supposed
to span the 5
-end of the gene (up to intron-2) and ~9 kb of
5
-flanking region. Digestion of
AR 1-2 with
SalI/XhoI released a 9-kb and a 4.8-kb fragment,
which were subcloned in the pGEM7Zf(
) vector (Fig. 2).
The 1.9-kb EcoRI fragment released from the 9-kb clone was
fully sequenced. It contains 141 bp of intron-1, exon-1 (104 bp), and
1.7 kb of 5
-flanking sequences including the promoter.
This fragment shows 85 and 49% sequence identity to the rat (18) and
rabbit (23) AR gene promoters, respectively. When multialignment was
carried out in the 650-bp equivalent sequenced region of the human AR
promoter (24), the sequence identity of the mAR promoter to rat,
rabbit, and human sequences was 74, 54, and 52%, respectively. The
putative transcription start site was defined by homology to the rat
sequence. The A of the methionine initiator codon corresponds to
nucleotide +39. Exon-1 is identical to that published for the mAR
cDNA (17). Only the first three nucleotides of the 5-untranslated
region were missing in the cDNA sequence. One TATA box and two
CCAAT boxes are located at positions
30,
66, and
99, respectively
(Fig. 2).
Numerous potential regulatory sites for binding of ubiquitous NF1, Sp1,
and C/EBP transcription factors and components of the signaling network
(NF-B, AP1, AP2, PEA3, Ets-1, c-Myc, and the cAMP response element
(25)) are present in the mAR promoter. Several potential
cis-acting steroid response sequences corresponding just to
the right half-site consensus sequence of the estrogen response element
and the androgen/glucocorticoid/progesterone response element were
identified in the 5
-flanking region of the mAR gene. The most
important feature is the presence, 1053 bp upstream of the putative
transcription start site, of a sequence similar to the
tonicity-responsive element (TonE), described by Takenaka et
al. (26), in the promoter of the canine betaine transporter (BGT1)
gene. This sequence, located near an AP1 site, was shown to be
essential for the osmotic regulation of the BGT1 gene. A likely
sequence arrangement was observed in the 5
-region of the mAR gene
(TonE-like at position
1053 and an AP1 site at position
1014; Fig.
2). This sequence organization is well conserved among the different AR
genes (Fig. 2B). This region was further investigated to
study the response of the mAR gene to hypertonicity.
The response of the
mAR gene to hypertonicity was studied by transient transfections of
reporter gene constructs in CV1 cells. As previously shown (27, 28),
NaCl added to make the medium hypertonic is a potent inhibitor of cell
proliferation. After 24 h of exposure to hypertonic medium
(H; 500 mosm/kg of H2O), CV1 cells appeared to
be less confluent (Fig. 3B) than in isotonic medium (I; 330 mosm/kg of H2O) (Fig.
3A), but no cell death was observed. The presence of
endogenous AR mRNA in these cells was tested under isotonic and
hypertonic conditions (Fig. 3C). Northern analysis of total
RNAs hybridized with the coding region of mAR cDNA revealed a
single band of ~1.5 kb. After 24 h of exposure of CV1 cells to
hypertonic medium, the relative abundance of AR mRNAs already
increased 2.6-fold compared with cells maintained in isotonic
medium.
To test the ability of mAR sequences to direct hypertonicity-induced
stimulation, constructs containing 5-flanking sequences from the mAR
gene linked to the indicator CAT gene were transfected in the CV1
cells. All the constructs transfected in CV1 cells exposed to isotonic
medium supported detectable levels of expression of the CAT gene in the
range of 9.98-43.89%, indicating that this is indeed a functional
promoter (Fig. 4). Deletion of the
1586/
998 region
results in a reduction of the basal promoter activity of p998CAT and
p142CAT. It is interesting to note that the AP1 site is absent from
these two constructs. The p1586CAT and p1067CAT constructs showed a
hypertonicity-dependent enhancement of transcriptional activity. In cells exposed to hypertonic medium, CAT activity increased
~3-fold over the level of basal activity measured under isotonic
conditions (Fig. 4). This effect seems to be promoter-specific since,
under the same conditions, the activity of the pSV2CAT plasmid control is not stimulated. To identify the sequences involved in the response to hypertonicity, subfragments were analyzed. As shown
in Fig. 4, deletions up to nucleotides
998 and
142 resulted in a
loss of the transcriptional activation by hypertonic stress. The
hypertonic/isotonic CAT activity ratios of p998CAT and p142CAT are
significantly different from those of p1586CAT and p1067CAT
(p < 0.05), whereas they are not significantly
different from that of pSV2CAT. The TonE-like sequence at
position
1053 is the most likely candidate to be involved in the
response to hypertonicity. To determine the role of this
5
-TGGAAAATCACCAG-3
sequence present in the p1067CAT and p1586CAT
constructs, it was mutated to 5
-TGTCCCCTCACCAG-3
in p1067mCAT. No
enhancer activity was observed with this construct. The
hypertonic/isotonic ratio calculated for p1067mCAT was significantly
different from those of p1586CAT and p1067CAT (p < 0.05), but was similar to that reported for pSV2CAT. These
results, showing that if the TonE-like sequence is mutated or deleted,
the response to hypertonicity is lost, strongly suggest that this motif
might function as a hypertonicity-responsive element.
The isolation and sequence of the 5-flanking region of the mAR
gene led us to underline its transcriptional regulation by hypertonicity and to identify a tonicity-responsive element in a small
region spanning base pairs
1053 to
1040. The complex pattern
obtained when a mouse genomic DNA Southern blot was hybridized with the
mAR cDNA compared with the single band observed with the
intron-2-specific probe confirms previous results obtained for human
(29) and rat (18) genomes concerning the existence of AR
pseudogenes.
Two half-palindromic sites for binding of the
androgen/glucocorticoid/progesterone receptor (5-TGTTCT-3
) are found
in the mAR sequence, but such half-sites are not known to be functional for other genes in the literature. Until now, we had no evidence concerning hormonal regulation of the mAR gene. AR mRNA levels in
seminal vesicle, vas deferens, epididymis, and kidney are not altered
in adult castrated mice (17). Moreover, there is no sequence similarity
between the mAR and mouse vas deferens protein promoters (30). Mouse
vas deferens protein, a member of the aldoketoreductase superfamily
sharing 69% identity with mAR (17), is highly expressed in the vas
deferens under androgenic control (31-33).
In contrast, regulation by hypertonicity is now well established for
the rabbit AR gene (3, 23, 34). We report here the same regulation for
the mAR gene in transfected CV1 cells and precisely localize one of the
osmotic response elements proposed by Ferraris et al. (23).
We demonstrated that the region spanning nucleotides 1067 to
998
upstream of the transcription start site and containing a TonE sequence
similar to that described in the BGT1 promoter (26) is necessary for
the response to hypertonicity. When the TonE-like sequence is mutated
(p1067mCAT) or when the
1067/
998 region is deleted (p998CAT), the
response to hypertonicity is lost. In the BGT1 TonE, if the middle
sequence GAAA or the 3
-end sequence GTCCA is mutated, there is no
longer enhancer activity under hypertonic conditions, whereas mutation
in the 5
-end of the TonE does not alter its enhancer activity.
Moreover, complex
involved in the response is not competed by
oligonucleotides mutated in the middle or in the 3
-end sequence of the
TonE in contrast to oligonucleotides mutated in the 5
-end, suggesting that this complex results from the binding of transcription factors on
the middle and the 3
-end of the TonE. Mutation in the middle of the
mouse TonE-like sequence (p1067mCAT) leads to the loss of response to
hypertonicity. The 3
-end of the mouse TonE-like sequence, which was
originally an imperfect palindrome compared with that of the BGT1 gene,
with insertion of an additional nucleotide after the four adenines
seems nevertheless to be able to confer response to hypertonicity. The
response obtained with the p1586CAT construct is quite modest
(~3-fold), but is in good agreement with the increase in endogenous
AR mRNA (2.6-fold). However, it cannot be excluded that other
sequences could be involved in the response to hypertonicity.
Deletion of the TonE-like sequence and the downstream AP1 site (p998CAT and p142CAT) resulted also in a reduction of the basal promoter activity. This last observation suggests that the AP1 site, which is absent from these two constructs, is necessary for the basal promoter activity. Moreover, in the BGT1 gene, a complex was formed when the DNA fragment containing the TonE and the AP1 site was incubated with both isotonic and hypertonic cell extracts. This complex was competed by an excess of AP1 sequence. So, in the BGT1 gene, the AP1 site is occupied even under isotonic conditions and probably participates in the basal promoter activity.
Stimulation of the mitogen-activated protein kinase cascade is required in Madin-Darby canine kidney epithelial cells to adapt to hyperosmolality (35). Although several transcription factors such as ATF-2 (which binds to the cAMP response element), c-Myc, p62TCF/Elk-1 (which belongs to the ets gene family), and AP1 (which corresponds to homodimer or heterodimer of c-Jun) have been identified as substrates for mitogen-activated protein kinase (36), none of them has been clearly implicated in the mechanism of regulation by hyperosmolality. Nevertheless, it would be of great interest to test if such cis-acting sequences present in the mAR promoter are functionally active.
We thank J. M. Garnier (Laboratoire de Génétique Moléculaire des Eucaryotes, INSERM U184, Strasbourg, France), who kindly provided the mouse genomic DNA library used in this study.