(Received for publication, March 7, 1994; and in revised form, October 3, 1994)
From the
We have isolated and characterized regions important for
expression of the mouse Na/H
exchanger gene. A 1.1-kilobase fragment upstream of the
5`-untranslated region contains specific DNA motifs characteristic of
promoter and enhancer elements including a TATA box, two CAAT boxes, an
SP-1 site, a cyclic AMP response element-binding site, and an AP-2-like
site. This 1.1-kilobase fragment directs transcription of a luciferase
reporter gene in mouse fibroblasts (NIH 3T3) and human Hep G2 cells.
Deletion or mutation of an AP-2-like site 100 base pairs from the start
site of transcription resulted in loss of most of the reporter plasmid
activity. In addition, cotransfection of an AP-2 expression plasmid and
the mouse promoter/luciferase plasmid increased the amount of
Na
/H
exchanger-directed transcription
in AP-2-deficient Hep G2 cells. Moreover, mobility shift analysis
indicated that a putative AP-2-binding site is capable of binding
purified AP-2 protein and a specific protein from nuclear extracts of
NIH 3T3 cells. The results show that the transcription factor AP-2 may
play an important role in regulation of transcription of the mouse
Na
/H
exchanger gene.
The Na/H
exchanger is a
mammalian plasma membrane protein that mediates the exchange of
intracellular protons for extracellular sodium. It is involved in pH
regulation(1) , control of cell volume(2) , and is
stimulated by growth factors(3) . Several different isoforms of
the protein exist which have been designated NHE-1 to NHE-4. The NHE-1
isoform is the widely distributed ``housekeeping'' isoform of
the family present in most, if not all, mammalian cells(4) .
Although the mechanism of regulation of protein levels is extremely
important, only a few studies have examined the human NHE-1
gene(5, 6) . Miller et al.(5) demonstrated that the 5`-flanking region of the human
NHE-1 gene contains a number of putative sites for DNA-binding
transcription factors, any of which could regulate the exchanger gene
transcription. Of these sites, only AP-1 has been implicated to have a
possible role in activating the NHE-1 gene(7) . Recently, a
more in-depth study of the human NHE-1 gene has analyzed the proximal
regulatory elements of the promoter(6) . To date, however,
there has been no direct identification of any specific transcription
factor responsible for regulating the NHE-1 promoter.
Expression of
the message and activity of the Na/H
exchanger has been shown to vary greatly depending on the state
of differentiation (8) and a variety of other
stimuli(9, 10, 11) . The regulation of the
NHE-1 gene, however, involves the link between DNA-binding protein
motifs located in promoter and enhancer sequences and the general
transcription machinery. Various cis elements of the gene may be able
to activate transcription through responses to certain extracellular
messages. One such element is the transcription factor AP-2.
Transcriptional activation by AP-2 involves the 52-kDa AP-2 protein
binding to a specific DNA motif found in the cis-regulatory region of
the gene(12) . AP-2 activity is regulated in a cell
type-specific manner (13) and is induced by phorbol esters,
retinoic acid, and cAMP(12, 13, 14) .
Additionally, mRNA levels of AP-2 have been shown to increase
dramatically upon differentiation indicating that the expression of
AP-2 is regulated during differentiation(14) .
In this study
we demonstrate, for the first time, a specific region within the
Na/H
exchanger gene that is involved
in regulating transcription. Moreover, we have shown that this DNA
motif interacts with AP-2 or an AP-2-like transcription factor. We
report the sequence of the upstream region of the mouse NHE-1 gene, the
identification and characterization of an AP-2 site, and the importance
of this region for NHE-1 gene transcription.
To examine if the
Na/H
exchanger gene could directly
bind purified AP-2 protein, we isolated and end labeled a DNA fragment
of the gene from bp -171 to +22 of the promoter. This
fragment contained the putative AP-2-binding site and the surrounding
nucleotides. Gel mobility shift assay was performed as described above.
Two positive clones were obtained by library screening with
NHE-1 probes. Clone 3-1 contained most of the coding region along
with the entire 3`-untranslated region, and clone 3-3 included a
section of the coding region, the 5`-untranslated region, and
approximately 10 kb upstream from the 5`-untranslated region. A 2.2-kb
fragment from clone 3-3 was sequenced in both directions using
exonuclease digestions. The deduced sequence of the first 40 amino
acids of the mouse 5`-coding region was compared with other
Na/H
exchangers (NHE 1-4) from
a variety of species. It was 90 to 83% identical to the other NHE-1
isoforms with the hamster showing the greatest identity. No significant
identity was seen with the other isoforms of the exchanger (NHE 2-NHE
4, not shown). The sequence of the mouse promoter/enhancer region is
shown in Fig. 1. It contains putative recognition sites for
several transcription factors. This includes a TATA box, two CAAT
boxes, an SP-1 site, a CREB site, and an AP-2 site (Fig. 1). The
sequence TATAAA in the mouse NHE-1 gene is identical to the well
characterized consensus sequence for the mammalian TATA box. Two CAAT
boxes are present at sites -407 to -401 and -568 to
-562 along with an SP-1 site at position -600 to
-592. Both CAAT and SP-1 are characteristic binding sites for
transcriptional regulatory proteins seen in many eukaryotic
promoters(19, 20) . The CREB site, at position
-789 to -784, is a putative cAMP response element that can
activate transcription upon cAMP or Ca
stimulation(21) . The site may be of significance since
it has been shown that cAMP can modulate exchanger activity
acutely(22) . Finally, a putative binding site for one of the
more well known transcription factors, AP-2, is located -111 to
-94 and was also a candidate for transcriptional regulation.
Figure 1: Nucleotide sequence of the promoter/enhancer region of the mouse NHE-1 isoform. Nucleotides for the coding strand are numbered beginning with the first start site of transcription, shown as +1. Start sites for transcription are denoted with arrows at positions +1 and +5. Horizontal lines indicate putative binding sites for DNA-binding proteins, SP-1, CAAT, CREB, TATA, and AP-2. The first base pairs of the plasmids pMP+AP2 and pMPAP2 are indicated by the + and -, respectively.
The start sites for transcription were demonstrated to occur at base pairs +1 and +5 (Fig. 1) and occur at nucleotides that are 26 and 30 bp from the TATA box. The two start sites and their distance from the TATA box are identical to that reported for the human promoter(5) . Our initial experiments tested whether a 1.1-kb sequence upstream from the start site was indeed a promoter, and if so its relative ability to direct transcription in mouse NIH 3T3 cells. When the level of luciferase activity from pXP-1MP was compared to pXP-1, a 73-fold increase in activity was seen (Fig. 2A). Transfection experiments also (Fig. 2B) showed that pXP-1MP induced 23.8-fold less luciferase activity than the Rous sarcoma virus RSV promoter located 5` to the luciferase gene (pRSVLUC). This indicates that the promoter for the mouse NHE-1 gene is relatively weak at directing transcription in comparison to pRSVLUC. These results could provide an explanation for the low levels of exchanger protein and mRNA present in mammalian tissues(4) . Another set of experiments examined the relative ability of an 8-kb fragment of the NHE-1 promoter/enhancer region to direct transcription. This fragment did not stimulate transcription greater than the 1.1-kb fragment (not shown).
Figure 2:
NHE-1 promoter activity in mouse NIH 3T3
fibroblasts. A, mouse fibroblasts were transiently transfected
with either a 1.1-kb fragment of the mouse
Na/H
exchanger (NHE-1)
promoter/enhancer region linked to a luciferase reporter gene (pXP-1MP) or with the reporter plasmid without the 1.1-kb
insert (pXP-1). B, fibroblasts were transfected with
either pXP-1MP as in A or with pRSVLUC. C, fragments
of the promoter were inserted upstream of luciferase gene in the vector
pXP-1. pXP-1MP contains from -1085 to +22 of the mouse
promoter/enhancer region. pMP+AP2 contains the region between
-125 and +22, and pMP-AP2 contains the region between
-92 and +22. The boxed sequence indicates the
region containing the putative AP-2 site that is deleted in pMP-AP2,
and the bold letters denote the putative AP-2 site. D, mouse fibroblasts were transiently transfected with either
pXP-1MP, pMP+AP2, or pMP-AP2. pSV-
-galactosidase was used as
an internal control. For all transfection experiments the results
reported were obtained from at least three independent experiments each
carried out in triplicate using at least two different DNA preparations
for each plasmid. pSV-
-galactosidase was used as an internal
control.
To examine which of
the identified consensus sequences are involved in regulation of NHE-1
transcription, we constructed several different plasmids containing
varying fragments of the promoter/enhancer region. We initially removed
all base pairs upstream of the AP-2 site from pXP-1MP. This construct
was named pMP+AP2. The second construct, pMP-AP2, was identical to
pMP+AP2 except for the further deletion of the AP-2 site. We then
examined the ability of these two plasmids to direct transcription of
the luciferase gene. They were transfected into NIH 3T3 cells along
with pSV--galactosidase which was used to normalize for any
differences in transfection efficiency. Panels C and D in Fig. 2are comparisons between the plasmid containing
the entire sequenced region (pXP-1MP), the plasmid containing the
AP-2-binding site (pMP+AP2) and the plasmid with the AP-2-binding
site removed (pMP-AP2). Deleting all the putative transcription factor
binding sites except AP-2 caused some decrease in NHE-1 transcription (Fig. 2D). Further removal of the AP-2 containing
region, however, decreased transcription 6-fold (Fig. 2D).
To confirm the importance of the 33-bp deleted region, we introduced several mutations into the AP-2 consensus sequence of the pMP+AP2 construct. The substitutions are shown in Fig. 3A. When the mutant construct no. 1 (pMP(MUT)AP2) is transfected into NIH 3T3 cells we see a 7.9-fold decrease in the level of luciferase activity (Fig. 3B). This decrease is comparable to the levels seen when the entire AP-2 region is deleted. To localize the nucleotides involved in this activity, two other mutations were generated (Fig. 3B). Mutation no. 2 involved a 5 base pair modification of the putative AP-2 site but had little effect on the activity of the promoter. When another two mutations were added to these existing five (mutation no. 3) a small decrease in luciferase activity was seen. The mutational analysis indicates that the region at approximately -106 to -95 may be important for regulating transcription in NIH 3T3 cells. To confirm the data from the mutational analysis, we performed DNase I footprinting experiments to determine if the specific protein from NIH 3T3 nuclear extracts could interact with the DNA sequence containing this AP-2 consensus site. A nuclear protein protected a region corresponding to -106 to -95 of the mouse NHE-1 promoter region (Fig. 4). This region lies within the AP-2 consensus site which is located in the 33-bp fragment of DNA.
Figure 3: Effect of mutant constructs on NHE-1 promoter activity in NIH 3T3 cells. A, comparison of the wild-type and three mutant constructs of the 33-bp sequence containing the AP-2 site. All the mutations are indicated by asterisks, and the numbers correspond to the position of the nucleotides relative to the first transcription initiation site. The numbers also represent the region of DNA which is protected by AP-2 protein from nuclear extracts of NIH 3T3 cells during DNase I footprint analysis. The level of luciferase activity in NIH 3T3 cells for the wild-type and the mutations are shown to the right and are expressed as percent of the pMP+AP2 construct. B, mouse fibroblasts were transiently transfected with either pMP+AP2 or pMP(MUT)AP2. Harvest and transfections were as described for Fig. 2.
Figure 4:
DNase I footprinting analysis of the mouse
NHE-1 promoter. DNase I footprint analysis was performed with the
-171 to +22 fragment of the NHE-1 promoter (lanes
1-5). The naked fragment of DNA was treated with DNase I
(0.1 units (lane 1) and 0.2 units (lanes 2 and 3)) for 10 s at room temperature. The fragment of DNA was also
treated with DNase I (1.0 units (lane 4) and 2.0 units (lane 5)) for 2 min at room temperature after incubation with
nuclear extracts from NIH 3T3 cells. Lane M represents the
[-
P]dATP end-labeled molecular weight
markers whose lengths are shown to the left. The protected region of
DNA is indicated to the right as an open box, and the numbers correspond to the position of the nucleotides in the
DNA sequence.
NIH 3T3 cells have one of the most abundant AP-2 levels of a number of murine cell lines and tissues(13, 23) . Hep G2 are human liver hepatoma cells which have been shown to be deficient in AP-2 mRNA and have little, if any, AP-2 binding and transcription activity(14) . To confirm that AP-2 protein or an AP-2-like protein regulates transcription of the mouse NHE-1 gene Hep G2 cells were transfected with pMP+AP2 and cotransfected with either a plasmid expressing AP-2 protein (pRSVAP2) or the identical plasmid that cannot express AP-2 protein (pRSV-AP2). The results (Fig. 5) show that cotransfection with an AP-2 expression plasmid causes a large 2-fold increase in the activity of the NHE-1 promoter. When the mutated construct pMP(MUT)AP2 is transfected into Hep G2 cells along with pRSVAP2 there is no increase in activity of the mouse NHE-1 promoter as compared to the wild-type containing plasmid (Fig. 5).
Figure 5: Effects of AP-2 on NHE-1 promoter activity in human Hep G2 cells. Human liver hepatoma cells (Hep G2) were transiently transfected with either pMP+AP2 or pMP(MUT)AP2 and cotransfected with either an AP-2 expression plasmid (pRSVAP2) or the same expression plasmid with the AP-2 gene deleted (pRSV-AP2) as indicated. Harvest and transfections were as described for Fig. 2.
To investigate the role of AP-2 protein as an enhancer of NHE-1 transcription in more detail, we examined the ability of the AP-2 motif to bind AP-2 protein. A short double-stranded oligonucleotide (MPAP2a, b) of 24 base pairs was synthesized that consisted of the sequence 5`-TTC CTT CCC TGG GCG ACA GGG GCC-3`. Gel retardation assays showed that this oligonucleotide bound to the purified AP-2 protein (Fig. 6A, lane 1). Competition experiments were done to show specific binding of AP-2, using unlabeled MPAP2a, b oligonucleotide as the competitor and an unlabeled SP-1 oligonucleotide as a non-competitor. Competitor concentrations of 6, 12, and 50 times blocked the protein-DNA interaction while 100 times excess of non-competitor had no effect (Fig. 6A, lanes 2-5, respectively). To further characterize the protein binding ability of the mouse AP-2 consensus site, nuclear extracts from NIH 3T3 cells were used. The results with purified AP-2 protein were similar to those seen in experiments with nuclear extracts. Nuclear extracts of NIH 3T3 cells bound to the AP-2 containing synthetic oligonucleotide (MPAP2a, b) and 12 and 25 times excess unlabeled competitor oligonucleotide decreased the binding (Fig. 6B, lanes 2 and 3), while 100 times excess of non-competitor did not (lane 4). Some binding of smaller size was apparent, but competition experiments revealed that the interaction was nonspecific (lanes 2 and 3). We also examined whether a 193-bp fragment cut directly from the promoter could bind purified AP-2 protein. The results are shown in Fig. 6C. An intense band was seen indicative of AP-2 binding to the labeled fragment of the gene. In the absence of the protein, the band was not apparent. The synthetic oligonucleotides containing the sequence for the mutated mouse putative AP-2 binding site (MUTAP2a, b) were used in another bandshift assay. The oligonucleotides MUTAP2a, b only bound small amounts of protein from nuclear extracts of NIH 3T3 cells (Fig. 6D, lane 2) in comparison to MPAP2a, b (Fig. 6D, lane 1). In addition, the unlabeled mutated oligonucleotides were unable to compete with MPAP2a, b confirming their reduced ability to bind to the protein. Finally, AP-2 was specifically removed from the nuclear extract by immunoprecipitation with anti-AP-2 antibody. This resulted in greatly reduced binding to MPAP2a, b by the nuclear extracts (Fig. 6E).
Figure 6:
DNA-mobility shift binding assay and
competition analysis of the mouse NHE-1 AP-2 site. The labeled 24-base
pair oligonucleotide MPAP2a, b (positions -117 to -94) was
incubated with purified human AP-2 protein or nuclear extracts for 10
min at room temperature. The binding mixtures were analyzed by
electrophoresis on 4% polyacrylamide gels as described under
``Experimental Procedures.'' A, lane 1,
purified AP-2 protein (1.4 µg) alone added to the binding
reactions; lanes 2-4, competitions with 6-, 12-, and
50-fold excess of unlabeled MPAP2a, b respectively; lane 5,
competition with 100-fold excess of a nonspecific sequence competitor,
SP-1. B, nuclear extracts from NIH 3T3 were used for mobility
shift binding assay with MPAP2a, b and 5 µg of NIH 3T3 cell nuclear
extract as described in A. Lane 1, nuclear extract
alone added to the binding reactions; lanes 2 and 3,
competitions with 12- and 25-fold unlabeled MPAP2a, b, respectively; lane 4 shows competition with 100-fold excess of a nonspecific
sequence competitor SP-1. C, a fragment of
Na/H
exchanger genomic DNA containing
the AP-2 consensus sequence was isolated and labeled with
[
-
P]ATP as described under
``Experimental Procedures.'' Purified AP-2 protein was
incubated with the fragment, and the results were analyzed as described
in A. D, the labeled 24-base pair oligonucleotide
MUTAP2a, b or MPAP2a, b (positions -117 to -94) were
incubated with 5 µg of NIH 3T3 nuclear extract and analyzed as
above. Lane 1 is the wild-type oligonucleotide MPAP2a, b,
while lane 2 is the mutant oligonucleotide. Lane 3 is
a competition assay containing labeled MPAP2a, b and 25-fold excess of
cold MUTAP2a, b. E, DNA mobility shift of MPAP2a, b with
untreated nuclear extracts (lane 1), without nuclear extracts (lane 2), or with AP-2 immunodepleted nuclear extracts (lane 3).
Previous studies have shown that the level of
Na/H
exchanger mRNA and transcription
itself can be increased due to a wide variety of treatments including
chronic acid loading (9, 10, 11) and
treatments causing cell differentiation(8) . To date, however,
there have been few studies on the NHE-1 promoter. One of these studies
identifies the regions of the human promoter/enhancer region which are
capable of binding nuclear factors (6) , while the second
indirectly examines the role of AP-1(7) . It was suggested that
AP-1 could play a role in activating the antiporter especially during
acidosis mediated increases in antiporter activity. Alternatively, it
was suggested that some other protein kinase C-dependent pathway could
mediate the effects of acidosis through AP-1 (7) . To date,
however, there has been no direct examination of the role of specific
transcription factors on the actual NHE-1 promoter.
Our analysis suggests that base pairs -106 to -95 play an important role in regulation of expression. Mutation of these base pairs reduced both in vivo transcription and binding of nuclear extracts to this region. DNA footprinting analysis also localized the binding of a protein to this region. It is likely that the transcription factor AP-2 or a closely related AP-2-like protein is involved in binding to this region. The evidence supporting this is that cotransfection of Hep G2 with an AP-2 expression plasmid increased NHE-1 transcription levels. In addition purified AP-2 protein bound to this sequence and removal of AP-2 protein from nuclear extracts by immunoprecipitation greatly reduced their ability to bind to the AP-2 consensus sequence. However, it is possible that an AP-2-like protein could be responsible for some of the observed effects. Future experiments will attempt to purify the protein binding to this region of the gene. It is of note that base pairs -95 to -105 are perfectly conserved in sequence and location in the human gene (5) while more upstream distal regions are not. This may indicate an important regulatory function. It should be noted that AP-2 is not the only transcription factor involved in regulation of NHE-1 expression, and other regions have also recently been suggested to be of importance(6) .
We examined the
mouse NHE-1 promoter because of the widespread availability of a number
of useful mouse cell lines. Our results show that we have isolated the
NHE-1 isoform since our probes for screening the genomic library
originated from the NHE-1 isoform and the clone hybridized under
relatively high stringency. We also show that the promoter for the
housekeeping isoform of the Na/H
exchanger family is activated by the transcription factor AP-2 or
an AP-2-like transcription factor. Although housekeeping genes such as
NHE-1 are normally not acutely regulated, there are examples of such
promoters that are activated by cis elements. One such example is the
second promoter of the acetyl-CoA carboxylase gene (24) whose
activation is mediated through an AP-2-like sequence(25) . This
example may be analogous to the NHE-1 promoter. Both AP-2 and NHE-1
transcription rate increase in some models of cellular differentiation.
For example, during retinoic acid induced differentiation of human
leukemic cells (HL-60), there is an 8.3-fold increase in NHE-1
transcription(8) . Additionally, retinoic acid-induced
differentiation of human NT2 teratocarcinoma cells shows increased AP-2
mRNA and protein expression levels(14) . The relationship
between increased AP-2 levels during differentiation induced by
retinoic acid and the increase in NHE-1 transcription seems to be an
important issue. Experiments are currently underway which may help to
explain this relationship.
The results from this paper have, for the
first time, directly examined the role of a specific transcription
factor in the regulation of the Na/H
exchanger gene. We have suggested that this transcription factor
is AP-2 or an AP-2-like protein, and we have located the region of the
gene where it is able to bind. While the extent to which this protein
regulates the Na
/H
exchanger in
specific cellular events is not yet known, the identification of a DNA
binding protein motif has provided enough information to warrant
further interest. AP-2 is involved in the regulation of a number of
cellular events including growth and differentiation(14) . The
involvement in these events is an interesting characteristic AP-2 has
in common with the Na
/H
antiporter(4, 8) . Future studies will focus on
these specific cellular events and how they work together to control
regulation of Na
/H
exchanger levels
and growth and differentiation of the cell.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L37525[GenBank].