(Received for publication, February 8, 1996)
From the
The activity of the apical membrane
Na/H
exchanger NHE3 isoform of renal
or intestinal epithelial cells is chronically regulated by a wide
variety of stimuli, including acidosis, cAMP, glucocorticoids, and
thyroid hormone. To understand the molecular mechanisms responsible for
long term regulation of this cation transporter, we have isolated and
determined the structure of this gene from a rat genomic library. The Nhe3 gene spans >40 kilobases and contains 17 exons that
are flanked by typical splice donor and acceptor sequences at the
exon-intron boundaries. The transcription initiation site was mapped by
S1 nuclease protection analyses of mRNA from rat kidney and intestine.
Multiple start sites were clustered between nucleotides -100 and
-96 relative to the translation initiation codon. An atypical
TATA-box and CCAAT-box are centered 30 and 147 nucleotides,
respectively, upstream of the predominant transcription initiation
site. Sequence analysis of approximately 1.4 kilobases of the
5`-flanking promoter region also revealed the presence of other
putative cis-acting elements recognized by various
transcription factors (e.g. AP-1, AP-2, C/EBP, NF-I,
OCT-1/OTF-1, PEA3, Sp1, glucocorticoid, and thyroid hormone receptors),
some of which may participate in the chronic regulation of this gene.
The glucocorticoid responsiveness of the Nhe3 gene was
assessed by fusing its 5` regulatory region to the firefly luciferase
reporter gene and then by measuring the expression of the chimeric gene
in transiently transfected renal epithelial OK and LLC-PK
cells. Glucocorticoid treatment significantly increased the luciferase
activity of the chimeric gene in both cell lines, thereby indicating
that glucocorticoid regulation of Nhe3 is mediated primarily
by a transcriptional mechanism.
The Na/H
exchanger (NHE) (
)is an integral membrane protein present in all mammalian
cells. Multiple isoforms (NHE1 to NHE5) have been identified by
molecular cloning
techniques(1, 2, 3, 4, 5, 6, 7) .
They range in size between
81 and 93 kDa and appear to exist in
the membrane as homodimers, at least in the cases of NHE1 and
NHE3(8) . These isoforms exhibit differences in their patterns
of tissue expression, biochemical and pharmacological characteristics,
and physiological functions (reviewed in (9) and (10) ).
In addition, Na/H
exchanger activity is influenced by a wide variety of molecular
signals (e.g. neurotransmitters, growth factors, peptide
hormones, phorbol esters, cAMP, chemotactic factors, lectins, osmotic
shrinkage, acidosis, glucocorticoids, and thyroid hormone) that act
either rapidly (seconds to minutes) or following a considerable latent
period (hours to days) before the effects on the rate of transport are
manifested (reviewed in (11) and (12) ). Recent
studies have begun to identify the stimuli and signaling pathways that
acutely modulate the individual NHE isoforms (13, 14, 15, 16, 17, 18, 19, 20) .
The mechanisms responsible for these alterations, although not fully
resolved, appear to involve both phosphorylation-dependent and
-independent processes.
Considerably less is known about the
molecular mechanisms involved in the chronic regulation of the NHE
isoforms, although recent investigations are beginning to provide some
insight. Prolonged exposure of cultured renal epithelial cells to
acidic medium, which serves as a paradigm for studying physiological
adaptations to chronic metabolic and respiratory acidosis, elevates the
activities and/or mRNA abundances of NHE1 (21, 22) and
NHE3(22, 23) , but inhibits those of
NHE2(24) . Interestingly, acid-mediated stimulation of NHE1 in
renal MCT cells is associated with activation of protein kinase C and
the transcription factor AP-1(25) , whereas acid induction of
NHE3 in renal OKP cells is linked to the c-src family of
non-receptor protein-tyrosine kinases(26) . Aside from
acidosis, chronic incubation of renal IMCD cells in hyperosmotic media
also stimulates Na/H
exchanger
activity, which is associated with increased NHE2 and reduced NHE1 mRNA
abundances (24) . Last, long-term administration of
glucocorticoids to rabbits or sheep selectively elevates NHE3 activity
and mRNA in renal proximal convoluted tubules (27, 28) and in ileum(29) , but has no effect
on NHE1 or NHE2. At present, the underlying mechanisms for acid,
hyperosmolarity, and glucocorticoid regulation of specific isoform mRNA
abundances have yet to be resolved in detail, but are likely to involve
altered rates of gene transcription and/or mRNA stability. Further
progress in this area will require the isolation and characterization
of the NHE genes and their associated cis-acting DNA
regulatory elements.
Chromosomal mapping analyses have revealed that
the members of the Na/H
exchanger
gene family are dispersed in the mammalian
genome(4, 30, 31, 32) . To date, the
complete gene encoding human NHE1 has been characterized (33) and shown to bind several transcription factors in its
5`-flanking region(34) , although their functional importance
was not established. However, more recent studies have shown that
binding of the transcription factor AP-2 to the mouse Nhe1 gene promoter increases gene transcription(35) . No
information is available regarding the other isoforms.
In order to understand the molecular mechanisms involved in the tissue-specific, developmental, and hormonal regulation of the NHE isoforms, we have characterized the genomic organization of the rat Nhe3 gene. Furthermore, we demonstrate that the transcriptional activity of the 5`-flanking promoter region is significantly elevated in response to glucocorticoid stimulation of transiently transfected renal cell lines.
Screening of both genomic libraries involved filter hybridization
with rat NHE3 cDNA probes radiolabeled with
[-
P]dCTP to a specific activity of
0.5-1.0
10
cpm/µg of DNA using the random
primer oligonucleotide labeling kit (Pharmacia). The filters were
prehybridized at 65 °C for 3-5 h in 6
SSC, 5
Denhardt's solution, 0.1% SDS, and 100 mg of denatured salmon
sperm DNA/ml (see (36) for composition of SSC and
Denhardt's solution). The filters were then hybridized in the
same solution with denatured cDNA probe for 48 h at 65 °C. The
replica filters were washed twice for 30 min each at 22 °C in 2
SSC, 0.1% SDS, and then once for 60 min at 65 °C in 2
SSC, 0.1% SDS. The filters were examined by autoradiography for
positive hybridization signals, and the corresponding phage colonies
were purified by 3 successive screenings using standard procedures.
Rat genomic DNA from the purified positive phage clones was mapped
by cleavage with different restriction endonucleases and Southern blot
analysis(37) . Briefly, the digested DNA was fractionated by
electrophoresis on 1% agarose gels, denatured, and transferred to nylon
membranes by capillary transfer. These membranes were successively
hybridized to [-
P]ATP end-labeled,
synthetic oligonucleotides corresponding to various regions of the NHE3
cDNA.
Following plating overnight,
subconfluent monolayers of OK and LLC-PK cells were washed
twice with phosphate-buffered saline and then changed to standard
-minimum essential medium supplemented with 10%
``charcoal-stripped'' fetal bovine serum at least 2 h before
transfection. Cells were transfected using the calcium phosphate-DNA
coprecipitation technique of Chen and Okayama(40) . The
functional activity of the chimeric gene was assessed by cotransfection
of 20 µg of plasmid DNA into cells. The composition of the DNA
included either the promoterless pXP1 vector (negative control) or the
pRSV-Luc vector containing the constitutively active Rous sarcoma virus
(RSV) promoter linked to luciferase (positive control) or the
pNhe3-1380 vector, plus pRSV110 (a vector containing the RSV promoter
linked to the
-galactosidase gene) and pHG1 (expression plasmid
containing the SV40 promoter and human glucocorticoid receptor;
generously provided by Dr. John White, McGill University) in a weight
ratio of 3:1:1, respectively. The activity of the
-galactosidase
gene served as an internal control to monitor for transfection
efficiency. The plasmid containing the human glucocorticoid receptor
was transfected to ensure that a sufficient concentration of receptor
was available for binding to the overexpressed Nhe3-1380 gene.
After a 20-h DNA precipitation period, the cells were washed and then
cultured in fresh medium in the absence or presence of 100 nM dexamethasone. The cells were harvested 72 h later by washing
twice with phosphate-buffered saline, followed by the addition of
Triton glycylglycine lysis buffer and then scraping the cells off the
dish with a rubber policeman. Cell extracts were diluted in 25 mM glycylglycine (pH 7.8) and assayed for luciferase and
-galactosidase activities using protocols and reagents provided by
Promega. Luciferase activity was measured using a BioOrbit 1250
luminometer.
Figure 1:
Genomic organization of the rat
Na/H
exchanger Nhe3 gene. A, overlapping genomic inserts from recombinant
phage
clones that encode the rat Nhe3 gene are aligned relative to
the exon-intron organization of the gene (shown in B). The
overlap regions between
3B and
6-2 and between
11-2 and
12-1 were determined by DNA sequencing. An overlap between
6-2 and
11-2 was not identified, presumably due to the
presence of a large intron. B, the relative locations of the Nhe3 exons within the gene, depicted by gray bars,
and their sizes (in base pairs) are illustrated. C, the
positions of the boundaries between exons in the NHE3 protein are
indicated by the arrows. The hatched boxes represent
the proposed membrane-spanning segments.
The sizes of the introns were determined either by DNA
sequencing or were estimated by agarose gel electrophoresis of
PCR-generated DNA fragments using oligonucleotide primers complementary
to the flanking exons. They generally varied in length from 0.1 to
1.2 kilobases, excluding the first intron which is estimated to be
>25 kilobases. This estimate is based on the sizes of the
6-2
and
11-2 phage clones which did not overlap. As shown in Table 1, the exon-intron boundaries conform to typical splice
donor AG/GT(A/G)AGT and acceptor (T/C)
N(C/T)AG/G (n > 11) consensus sequences(41) . Each splice donor site
begins with an invariant GT dinucleotide, whereas each splice acceptor
site ends with an invariant AG dinucleotide and is preceded by a
polypyrimidine tract.
It has generally been observed that members of
gene families share similar genomic organizations, such as the
Na,K
-ATPase
isoforms(42, 43) . This also applies to the NHE gene
family members that have been characterized to date. A comparison of
the exons within the protein coding sequences of rat NHE3 and human
NHE1 (33) is illustrated in Fig. 2. With the exception
of exons 2 and 3 in human NHE1, which are both split by an apparent
intron in rat Nhe3, the exon-intron boundaries occur in
exactly the same positions within the proposed N-terminal
transmembranous region and the first
50 amino acids of the
C-terminal cytoplasmic region. These regions share the highest degree
of amino acid identity among the isoforms. However, little similarity
in exon organization exists in the remainder of the C-terminal
cytoplasmic regions of the isoforms which also share minimal sequence
identity and are believed to encode the diverse regulatory elements of
the transporters.
Figure 2:
Comparison of the exon arrangement of the
Na/H
exchanger NHE1 and NHE3 isoforms
from human and rat, respectively. The deduced amino acid sequences of
human NHE1 and rat NHE3 were aligned using the CLUSTAL W alignment
program(83) . The locations of the exon boundaries for rat NHE3
(see Table 1) and human NHE1 (33) are illustrated by arrows.
To resolve this difficulty, S1 nuclease protection studies were performed using total RNA isolated from adult rat intestine and kidney (Fig. 3). The S1 probe consisted of a 416-nucleotide single-stranded DNA fragment that extended from position -412 to +4 relative to the translation start site. A cluster of apparent transcription initiation sites located within a 5-bp region were observed in total RNA isolated from adult rat intestine and kidney, with a major site at -97 (T nucleotide) and two minor sites at -100 (A nucleotide) and -96 (G nucleotide). Since T is the most predominant start site in rat Nhe3, it is designated as +1 and all 5` elements have been numbered relative to this site.
Figure 3:
Determination of the transcription
initiation site of the rat Na/H
exchanger Nhe3 gene by S1 nuclease protection analysis.
A single-stranded
P-labeled DNA fragment beginning 4
nucleotides 3` to the translation start site and extending 416
nucleotides in the 5` direction was hybridized to 50 µg of total
RNA from rat kidney or intestine. S1 nuclease protection analysis was
performed as described under ``Experimental Procedures.'' The
protected fragments were analyzed on a 6% denaturing polyacrylamide
sequencing gel. Lane 1, 50 µg of total RNA from rat
kidney; lanes 2-5, sequencing reaction using the same
primer that was used in generating the S1 probe; lane 6, 50
µg of total RNA from rat intestine. The nucleotide sequences
flanking the transcription initiation sites (indicated by asterisks) is illustrated on the right of the
figure.
The nucleotide sequence of the 5` end of the Nhe3 gene is shown in Fig. 4. The center of an atypical TATA sequence, ATTAAA, is located 30 bp upstream of the major initiation site and potentially binds the multiprotein complex, TFIID (reviewed in (44) ). As well, although a classical CCAAT consensus sequence capable of recognizing NF-Y/CP1 (45, 46) is not observed between -200 and -50 bp of the transcription start site, an atypical sequence, CCAAG, that resembles the core binding motif is located 147 bp 5` of the major transcription initiation site. However, a classical TATA-box and CCAAT-box are positioned further upstream at -428 and -501, respectively, but are considered too far from the promoter region to be functional. This premise is based on our inability to obtain a primer extension product following hybridization of specific oligonucleotides in this region to total RNA from adult rat intestine and kidney (data not shown).
Figure 4:
Nucleotide sequence of the 5`-flanking
region of the rat Na/H
exchanger Nhe3 gene. The sequence of the 5`-flanking region (1.38
kilobases) of the rat Nhe3 gene is shown (GenBank
accession number U49386). A SalI site used in generating
the 416-nucleotide S1 nuclease protection probe is indicated.
Transcription initiation sites are indicated by large (major
site) and small (minor sites) arrows. The DNA
sequence was scanned for elements that share homology to known
transcription factor binding sites using the computer program SIGNAL
SCAN(84) . An apparent TATA-box and sequences exhibiting
similarity to transcription factor binding sites (AP-1, AP-2, C/EBP,
NF-I, NF-Y, OCT-1/OTF-1, PEA3, Sp1) or hormone receptor response
elements (glucocorticoid, GR; and triiodothyronine, T3R) are underlined. Numbers to the left of
the figure refer to nucleotide positions relative to the major
transcription initiation site.
The promoter region also contains several potential GC-box motifs or Sp1-binding sites that resemble the core hexanucleotide motif, GGGCGG (47) , and are located at positions -588, -350, -139, -72, and -54 (reverse orientation) upstream of the apparent cap signal. Indeed, the first 200 bp of the promoter region is highly (G + C)-rich (67%), a characteristic that is commonly observed in the immediate 5`-flanking region of housekeeping genes.
The 5`-flanking region also contains a
number of other potential cis-acting elements recognized by
known transcription factors that may play a role in the basal or
chronic regulation of the Nhe3 gene (see
``Discussion''), including four half-sites for the
glucocorticoid responsive element (GRE) (TGTTCT; positions -1273,
-1235, -479, and -459)(48) , six half-sites
for the thyroid hormone responsive element (AGGTCA; positions
-1020, -1014, -1007, -234, -225,
-215)(48) , one PEA3 site (AGGAAGT; position -1309) (49) , four AP-1 sites (TGA(C/G)T(C/A)A; positions -1178,
-1130, -1065, and -907)(50, 51) ,
two C/EBP sites (ATTGCGCAAT; positions -979 and
-948)(52) , one OCT-1/OTF-1 site (ATGCAAAT; position
-628) (53) , four AP-2 sites (CCCC(A/G)(G/C)(G/C)C;
positions -412, -295, -48, and
-4)(54) , and one NF-I site (TTGGC(N)GCCAA;
position -271)(46, 55) .
Figure 5:
Glucocorticoid induction of a chimeric rat Nhe3 promoter-firefly luciferase reporter gene in transiently
transfected renal OK and LLC-PK cell lines. A,
schematic illustration of the promoterless firefly luciferase gene in
the parent vector pXP1 and a chimeric Nhe3 gene (pNhe3-1380)
containing 1380 bp (relative to the transcription start site) of the
5`-flanking region of Nhe3 (BamHI-KpnI
fragment) fused to the luciferase reporter gene. Each recombinant
plasmid was transiently cotransfected into renal OK (B) or
LLC-PK
(C) in the presence of pRSV110 which
expresses the
-galactosidase gene (to normalize for transfection
efficiency) and pHG1 which expresses the human glucocorticoid receptor
(to provide a sufficient concentration of receptor for binding to the
overexpressed Nhe3-1380 gene) in a weight ratio of 3:1:1,
respectively. The cells were incubated in the absence (gray shaded
box) or presence (black box) of 100 nM dexamethasone for 72 h. Cell extracts were then prepared for
measurements of luciferase and
-galactosidase activities. For
comparative purposes, the corrected values obtained for luciferase
activity in unstimulated cells transfected with pNhe3-1380 were
normalized to a value of 1 and all other activities were adjusted
accordingly. The data represent fold stimulation (mean ± S.D.)
from two experiments, each performed in
quadruplicate.
The promoterless expression plasmid, pXP1,
showed negligible luciferase activity when transfected into both cell
lines, whereas pNhe3-1380 exhibited a 69- and 7-fold increase in basal
luciferase activity in OK and LLC-PK cells, respectively (Fig. 5, B and C). This indicated that the
inserted 5`-flanking region of the Nhe3 gene contains a
functional promoter. However, the basal activity of pNhe3-1380 in OK
and LLC-PK
cells is considerably less (691- and 16-fold,
respectively) than that obtained with the expression plasmid pRSV-Luc
that contains the more powerful Rous sarcoma virus promoter linked to
the luciferase gene (data not shown). The weak promoter activity of the Nhe3 gene could account, at least in part, for the generally
low abundance of NHE3 mRNA in rat tissues(2) .
The
glucocorticoid responsiveness of the pNhe3-1380 was tested by
incubating the transfected cells in the absence or presence of 100
nM dexamethasone for 72 h. This treatment substantially
elevated luciferase activity in OK and LLC-PK cells by 15-
and 6.5-fold, respectively (Fig. 5, B and C).
In contrast, transfected cells containing the promoterless pXP1 plasmid
did not show a significant change in luciferase activity.
We have isolated and characterized the structural
organization of the rat Na/H
exchanger Nhe3 gene and its 5`-flanking region from a
phage genomic library. This gene, which has previously been
mapped to rat chromosome 1 (32) and human chromosome
5p15.1(31) , spans >40 kb in length and contains at least 17
exons. The organization of the first 9 exons encoding the entire
proposed N-terminal transmembranous region (exons 1 to 7;
453
amino acids) and the first
50 amino acids of the adjacent
C-terminal cytoplasmic region (exons 8 and 9) is conserved for the most
part with that of the human NHE1 gene, the only other NHE gene to be
characterized to date. The only exceptions are exons 2 and 3 of rat Nhe3 which are continuous in human NHE1 (forming exon 2) and
exons 4 and 5 of rat Nhe3 which are also uninterrupted in
human NHE1 (forming exon 3). The N-terminal transmembranous segments
share high amino acid identity (
55-95%) among the NHE
isoforms and are believed to comprise the structural domains necessary
for ion translocation. In contrast, the organization of exons 10 to 17
of the Nhe3 gene is dissimilar to the exon arrangement of the
comparable region (exons 8 to 12) of human NHE1. This C-terminal region
in both isoforms, which is proposed to reside on the cytoplasmic side
of the plasma membrane, shares minimal amino acid identity
(25-35%) and contains numerous putative regulatory motifs that
respond in a distinctive manner to a variety of
stimuli(14, 16, 17, 18, 60) .
To begin addressing questions concerning the regulation of Nhe3 gene expression at the transcriptional level, the transcription initiation sites were determined. In addition, the 5`-flanking sequence was examined for potential promoter elements that are fixed close to the start of transcription (i.e. TATA-box and cap signal) and enhancer motifs (e.g. CCAAT-box, Sp1, AP-1, AP-2, C/EBP, OCT-1/OTF-1, and NF-I binding sites) and hormone receptor responsive elements (e.g. triiodothyronine and glucocorticoid receptor binding sites) that are less restricted in both position and orientation. Only those potential sites that may be of physiological relevance to Nhe3 are discussed below.
Multiple
transcription initiation sites were identified for the Nhe3 gene using S1 nuclease protection analyses from rat kidney and
intestine, the two major tissues where the gene is expressed. Although
three initiation sites were found to cluster between nucleotides
-100 and -96 relative to the translation ATG start codon,
the predominant site used in both tissues occurred at position
-97 (thymidine) with minor sites located at -100 (adenine)
and -96 (guanine). The cap signal CCTG closely
matches the consensus cap signal NCA
(G/C/T)(61) .
Although the site of initiation is frequently an A preceded by an
invariant C nucleotide, the use of T (but never C or G) is observed in
a minority of cases. However, this motif is only present in
approximately 60% of all promoters, suggesting that this sequence is
not essential for promoter function(61) . Multiple start sites
scattered around the promoter region have also been reported for
several genes (62, 63) that lack a canonical TATA
sequence. The presence of an atypical TATA-box (discussed below) in rat Nhe3 may also account for the multiple start sites.
Consequently, multiple transcription initiation sites may be a
characteristic of genes that lack a TATA-box, or a well defined TATA
motif, in the promoter region.
The promoter/enhancer region of the
rat Nhe3 gene contains an atypical TATA-box and CCAAT-box that
are centered 30 and 147 nucleotides, respectively, upstream of the
major transcription initiation start site. The core and flanking
residues of the atypical TATA-box, GATTAAAGG, differ from
the extended canonical sequence (G/C)TAT
AAA(A/T)(G/A) by
having an A in the -2-position, a T in the -1-position, and
a G in the +4 position. Nonetheless, a recent analysis of the
promoter region of
400 genes has revealed that A, T, and G can be
present in these positions, respectively, but at low frequencies (4%,
9%, and 11%, respectively)(61) . The atypical CCAAT-box,
AACCA
AGTAG, is centered at position -147 and exhibits
similarity (8/10 match) to the extended CCAAT consensus sequence
(A/G)(G/A)CCA
AT(C/G)(A/G)G(45, 46, 61) .
This pentanucleotide core sequence is recognized by the transcription
factor NF-Y/CP1 and is typically located between 50 and 200 nucleotides
upstream of the transcription start site. The atypical CCAAT-box in Nhe3 differs from the canonical sequence by substitution of G
and T in the +2 and +3 positions, respectively. The presence
of G and T in these positions is rare, being observed in only 9% and
2%, respectively, of genes examined(61) . However, since 6 out
of 7 nucleotides that were identified to contact the NF-Y protein
(based on methylation interference analysis) (45) are conserved
in the proposed CCAAT-box, it is reasonable to suspect that the
observed motif may be transcriptionally relevant. However, functional
studies are required to verify this possibility.
In addition, other
CCAAT-like elements are known to exist, such as the palindromic
consensus sequence TTGGC(N)GCCAA that is recognized by
nuclear factor I (NF-I/CTF)(46, 55) . One potential
NF-I binding site in the Nhe3 promoter region is located
between nucleotides -271 and -257 and maintains all 6
essential contact points in the NF-I binding site that, based on
methylation interference analysis, seem to contact the NF-I
protein(46) . A third class of nuclear transcription factors
that is able to bind to CCAAT-box and related enhancer motifs belongs
to the CCAAT-box/enhancer binding protein (C/EBP) gene
family(52, 64) . C/EBP DNA-binding proteins recognize
the optimal dyad-symmetrical sequence ATTGCGCAAT and regulate the
expression of genes during cellular differentiation (52, 64) and in response to
cAMP(65, 66) . Two potential C/EBP elements that match
at least 7 out of 10 nucleotides of the optimal motif are located in
the Nhe3 gene at positions -979 and -948.
The promoter region also contains five potential binding sites for Sp1 that are positioned at nucleotides -588 (TTGGCGGGAG), -350 (AAGGCGGGAG), -139 (GGGGCGTGAG), -72 (GGGGCGGGAA), and -54 (GCCCCGCCCT; reverse orientation) upstream of the apparent cap signal. These sites closely match the consensus decanucleotide sequence for Sp1, (G/T)GGGCGG(G/A)(G/A)(C/T), which contains an optimized core hexanucleotide sequence GGGCGG and 5`- and 3`-flanking nucleotides that, although degenerate, can significantly influence Sp1 binding(47, 61) . Sp1 sites, which can function in either orientation, are often found within 200 nucleotides upstream of the start of transcription and can act in conjunction with NF-I to increase the rate of transcription (47) . It is also noted that the sequence of the promoter region is (G + C)-rich (67% from nucleotides -1 to -201) which is frequently found in the 5` promoter region of housekeeping genes. Thus, it appears that the Nhe3 promoter region is a mosaic of potential elements that are characteristic of both cell-specific and housekeeping genes.
Since the mRNA and/or activity of Na/H
exchangers in renal or intestinal epithelial cells is elevated
following prolonged exposure to various stimuli, including phorbol
esters(67) , cAMP(68) , acidic
medium(22, 23, 25, 26, 69, 70) ,
triiodothyronine(71) , and
glucocorticoids(27, 28, 29, 56) , we
examined the 5`-flanking region of the Nhe3 gene for potential cis-acting DNA response elements that may be involved in some
of these responses.
The trans-acting factor AP-1 is a heterodimer composed of the c-jun and c-fos proto-oncogenes that influences basal transcription and is also required for induction of transcription by phorbol esters(50, 51) . The 5`-flanking region of the Nhe3 gene contains four potential AP-1 sequences; three match 6 out of 7 nucleotides of the AP-1 consensus sequence, TGA(G/C)T(C/A)A and the other exhibits a 5/7 match. In addition to the AP-1 site, other phorbol ester responsive elements have been identified (reviewed in (72) ). Insertion of an oligonucleotide containing the PEA3 consensus sequence, AGGAAGT, upstream of a heterologous promoter can confer responsiveness to phorbol esters(49) . Furthermore, PEA3 can act synergistically with AP-1 to achieve maximal induction of transcription by phorbol esters(73) . A PEA3 site (in the reverse orientation) that precisely matches the consensus sequence and is in close proximity to the AP-1 sites is located at nucleotide -1309 of the Nhe3 gene. Last, the regulatory region of this gene contains four potential sites at positions -412, -295, -48, and -4 that share homology with AP-2 elements, CCCC(A/G)(G/C)(G/C)C, that act as basal transcription enhancers but are also responsive to both phorbol esters and cAMP(54, 74) . The sequence of individual sites, however, can vary considerably from the canonical binding motif. The responsiveness of AP-2 sites, as well as C/EBP sites(66) , to cAMP is of particular relevance since cAMP chronically up-regulates NHE3 activity in renal OK cells(68) . Moreover, a consensus site for the classical cAMP-responsive binding protein CREB, TGACGT(A/C)A(74) , was not identified in the 1.4-kb 5`-flanking region, although potential CREB-binding sites may be present further upstream.
Triiodothyronine, estrogen, retinoids, and vitamin D bind to specific intracellular receptors that are part of a large family of ligand-dependent transcription factors. Interestingly, the DNA recognition site for each of these receptors, which may bind as monomers, homodimers, or heterodimers, is a variant of the optimal hexanucleotide half-site AGGTCA (reviewed in (48) ). However, the specificity of hormone receptor binding to the cognate response element and the magnitude of transcriptional activity is highly dependent upon the number, orientation, and spacing between half-sites(75, 76, 77) . For example, direct repeats separated by 3, 4, and 5 bp are selectively responsive to vitamin D, triiodothyronine, and retinoic acid, respectively(76) . Two clusters of AGGTCA-like elements (each containing 3 core binding motifs) are present in the rat Nhe3 gene at positions -1025 to -1002 and -239 to -210. Each cluster contains two direct repeats in one orientation and a single half-site in the reverse orientation. The selective hormone responsiveness, if any, awaits further characterization.
The
glucocorticoid hormone receptor complex binds to an inverted
palindromic sequence AGAACA(N)TGTTCT that is distinct from
the triiodothyronine/estrogen/retinoid/vitamin D core binding motif (48) . Within this consensus glucocorticoid responsive element
(GRE), the hexanucleotide core TGTTCT is generally highly conserved,
whereas the leftmost hexanucleotide sequence (AGAACA) can be quite
variable. The TGTTCT half-site is also recognized by mineralocorticoid,
androgen, and progesterone receptors(48) . Similar to the core
binding motif for the thyroid hormone class of receptors, hormone
selectivity for the GRE is influenced by the number, spacing,
orientation, and DNA sequence of the cognate responsive element. There
are 4 half-site sequences segregated into two clusters (each containing
two half-sites) within the 1.4-kb 5`-flanking region of Nhe3.
The distal cluster is located between nucleotides -1273 and
-1230, and the more proximal cluster is found between nucleotides
-479 and -454. Each cluster contains a half-site that
exhibits either a 5/6 match (positions -1273 and -459) or
6/6 match (positions -1235 and -479) to the conserved
hexanucleotide core element. However, homology to the entire 15-bp
palindromic sequence is low. Although GREs can function as independent
elements, it is now becoming apparent that the maximal activity of many
GREs is greatly influenced by the presence of additional cis-elements/trans-acting factors. For example, both
NF-I (78) and the ubiquitous transcription factor OCT-1/OTF-1 (53) participate in the glucocorticoid regulation of the mouse
mammary tumor virus promoter. In addition, the AP-1 complex has been
found to influence the ability of the glucocorticoid receptor to
stimulate or inhibit gene transcription(79) . In many respects,
these factors can be viewed as components of a larger composite hormone
responsive unit that creates the potential for greater flexibility in
the hormonal regulation of gene transcription. Interestingly, potential
NF-I, OCT-1/OTF-1, and AP-1 binding sites are also located in the
general vicinity of the GRE clusters in the rat Nhe3 gene,
although their functional importance, if any, remains to be determined.
As mentioned previously, glucocorticoids elevate
Na/H
exchanger NHE3 activity and/or
mRNA abundance in ileum(29) , renal proximal
tubules(27, 28) , and OK cells (56) . The
studies described in this report extend these observations by
demonstrating that the transcriptional activity of the 5`-flanking
region of the rat Nhe3 gene fused to the luciferase reporter
gene is activated by glucocorticoids in transiently transfected OK and
LLC-PK
cells. This suggests that the observed in vivo induction of native NHE3 mRNA by glucocorticoids is most probably
mediated primarily at the transcriptional level. More detailed studies
will be required to precisely delineate the specific cis-acting elements involved.
In summary, we have isolated and characterized the 5`-flanking promoter region and exon-intron organization of the rat Nhe3 gene. In addition, we have demonstrated that this gene can be transcriptionally activated by glucocorticoids. This information provides the framework for further investigations on the mechanisms involved in the chronic regulation of this gene by glucocorticoids as well as other stimuli and its possible involvement in pathophysiological conditions such as systemic acidosis(69) , hypertension(80, 81) , and congenital secretory diarrhea(82) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49386[GenBank].