Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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The rat Na+/H+ exchanger
isoform-2 (NHE-2) gene promoter lacks a TATA box and is very
GC rich. A minimal promoter extending from bp 36 to +116 directs
high-level expression of NHE-2 in mouse inner medullary
collecting duct (mIMCD-3) cells. Four Sp1 consensus elements were found
in this region. The introduction of mutations within these Sp1
consensus elements and DNA footprinting revealed that only two of them
were utilized and are critical for basal transcriptional activation in
mIMCD-3 cells. The use of Sp1, Sp3, and Sp4 antisera in electrophoretic
mobility shift assays demonstrated that Sp1, Sp3, and Sp4 bound to this
minimal promoter. We further analyzed the transcriptional regulation of NHE-2 by members of the Sp1 multigene family. In
Drosophila SL2 cells, which lack endogenous Sp1, the minimal
promoter cannot drive transcription. Introduction of Sp1 activated
transcription over 100-fold, suggesting that Sp1 is critical for
transcriptional regulation. However, neither Sp3 nor Sp4 was able to
activate transcription in these cells. Furthermore, in mIMCD-3 cells,
Sp1-mediated transcriptional activation was repressed by expression of
Sp3 and Sp4. These data suggest that Sp1 is critical for the basal promoter function of rat NHE-2 and that Sp3 and Sp4 may
repress transcriptional activation by competing with Sp1 for binding to core cis-elements.
Sp3; Sp4; Drosophila SL2 cells; sodium-hydrogen exchanger; mIMCD-3 cells
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INTRODUCTION |
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SODIUM-HYDROGEN EXCHANGERS are integral transmembrane proteins found in all mammalian cells, and they play roles in intracellular pH and cell volume regulation and in vectorial ion transport (20). Regulation of these cellular processes is vital for maintaining optimal cell function and viability. NHE-2 is the second cloned Na+/H+ exchanger; it is expressed in the stomach, kidney, uterus, intestine, adrenal gland, and, to a much lesser extent, trachea and skeletal muscle (4, 32). In the kidney, NHE-2 mRNA is expressed primarily in the inner medulla. In the stomach, NHE-2 is expressed in all three of the major gastric epithelial cell types, including mucous, zymogenic, and parietal cells (25).
Although the physiological function of NHE-2 is not well understood, NHE-2 likely plays important roles in the kidney and gastrointestinal tract. A recent study using NHE-2-null mice demonstrated that NHE-2 is involved in acid secretion by affecting viability of gastric parietal cells (25). In addition, NHE-2 was found to be involved in volume regulation of renal inner medullary cells (26). The regulation of NHE-2 by hormones, growth factors, and chronic extracellular stimuli has been studied. By using stably transfected fibroblasts expressing NHE-2, Levine et al. (17) and Tse et al. (30) showed that fibroblast growth factor and serum stimulate NHE-2 activity. It has also been shown that hyperosmolarity increases NHE-2 mRNA expression (1, 26), likely through a transcriptional mechanism (1). Despite these previous studies, the molecular mechanism of regulation of NHE-2 by different stimuli has not been precisely defined.
Sp1 is a ubiquitous transcription factor mostly associated with TATA-less, GC-rich promoters, and it is mainly involved in basal promoter activity by interacting with other trans-activation factors, which together may stabilize components of the transcriptional machinery (23). Sp1 consists of three contiguous zinc-finger domains that bind to the consensus sequence KRGGMGKRRY, which is referred to as a GC box (3, 15). Additional transcription factors (Sp2, Sp3, and Sp4), with structural and transcriptional properties similar to those of Sp1, have been cloned, and together they form an Sp1 multigene family (9, 11, 14, 27). Functional studies have shown that Sp1 and Sp4 generally act as transcriptional activators (5, 8, 10), while Sp3 acts as both a repressor and an activator (7, 10, 18, 31). The Sp1 multigene family members are important regulators of the cell cycle, differentiation, and development (22, 27, 31, 33).
We recently cloned the rat NHE-2 gene promoter
(19) and characterized hyperosmolarity regulation of the
NHE-2 gene in mouse inner medullary collecting duct
(mIMCD-3) cells. These experiments resulted in the identification of
two osmotic response elements in the rat NHE-2 proximal
promoter (1). In this promoter, we found that the typical
TATA and CAAT boxes were absent; however, a GC-rich region was
identified within 300 bp of the transcriptional initiation site. Four
Sp1 sites were found within 40 bp of the transcriptional initiation
site on the complementary strand. Such Sp1 sites may be especially
important in the NHE-2 promoter, because it has been
reported that the recognition of initiator elements by the
transcription factor TFIID is facilitated by Sp1 binding in TATA- or
CAAT-less promoters (13).
In the present study, we identify the minimal functional promoter of the rat NHE-2 gene, and we examine the potential roles of the members of the Sp1 multigene family in basal promoter activity. Functional analysis of promoter activity in mammalian and Drosophila cells demonstrates that Sp1, Sp3, and Sp4 have different effects on transcription of the NHE-2 gene, with Sp1 playing an activating role and Sp3 and Sp4 playing inhibitory roles. These findings will likely have significant future implications in understanding the molecular mechanism of NHE-2 gene regulation by many physiological factors.
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METHODS |
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Plasmid constructs.
Firefly luciferase expression vectors pGL3/promoter (with SV40 promoter
upstream of luciferase cDNA) and pGL3/basic (promoterless) were
purchased from Promega (Madison, WI). pGal/basic -galactosidase expression vector was purchased from Clontech (Palo Alto, CA). Full-length human Sp1 cDNA (pBS-Sp1-fl) was a generous gift from Dr.
Robert Tjian (12). Mammalian expression vectors pCMVSp3 and pCMVSp4 and Drosophila expression vectors pPacSp3 and
pPacSp4 were kindly provided by Dr. Guntram Suske (21, 28,
29). Mammalian expression vector pCMVSp1 and
Drosophila expression vector pPacSp1 were made by digesting
pBS-Sp1-fl with SacI and EcoRI, blunting both
ends by mung bean nuclease, and ligating into pPac vector or pCMV
vector. The pPac vector was prepared by digesting pPacSp3 with
BamHI, and the pCMV vector was made by digesting pCMVSp3
with NotI. Both vectors were then blunted by mung bean nuclease.
Transient expression assays in mammalian cells.
mIMCD-3 cells at passages 8-18 were seeded in
six-well plates and maintained in Ham's F-12-high-glucose DMEM (Irvine
Scientific) supplemented with 10% FCS. The same batch of FCS was used
in all experiments. When cells were 70% confluent, they were
cotransfected with 1 µg of luciferase reporter vector DNA and 0.1 µg of pRL-CMV vector (encoding renilla luciferase; used as an
internal standard) in six-well plates by a liposome-mediated method
(1). Dual luciferase activity was measured 48 h after
transfection. For Sp1 overexpression studies, 1 µg of pGal/basic or
pGal/36 bp construct was cotransfected with or without 1 µg of
pCMVSp1, pCMVSp3, or pCMVSp4.
-Galactosidase activity was
measured 48 h after transfection by a standard method. Each
experiment was repeated a minimum of three times with different
populations of cells on different days, and two wells were averaged
from each experiment to obtain n = 1.
Transient expression assays in Schneider Drosophila SL2 cells.
Drosophila SL2 cells (American Type Culture Collection,
Rockville, MD) were maintained at room temperature in Schneider cell culture medium (Life Technologies) supplemented with 10% FCS. The
cells were transfected by a liposome-mediated method when they reached
60% confluency. To investigate the role of Sp1, Sp3, and Sp4 on the
minimal promoter, 1 µg of pGal/basic or pGal/36 bp was
cotransfected with 2 µg of pPacSp1. Appropriate amounts of pPacSp3 or
pPacSp4 were additionally cotransfected to study the interaction
between Sp1 and Sp3 or Sp4. DNA was allowed to remain on the cells for
48 h, and the cells were then harvested and cell pellets were
resuspended in 100 µl of 1× passive lysis buffer. Equal amounts of
cellular protein were used for each
-galactosidase assay.
Nuclear extracts and electrophoretic mobility shift assay.
Nuclear extracts from mIMCD-3 cells were prepared as described
previously (1). Purified recombinant Sp1 protein was
purchased from Promega. Supershifting antibodies to Sp1 and Sp4 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Sp1 antibody
is against an internal domain of Sp1 and is non-cross-reactive with Sp2, Sp3, or Sp4. Sp4 antibody is against the COOH terminus of Sp4 and
is non-cross-reactive with Sp1, Sp2, or Sp3. Antibody against human Sp3
was purchased from Geneka (Montreal, PQ, Canada). This antibody
recognizes the DNA binding domain of Sp3 and results in a blocking
effect when used for gel shift assays according to the manufacturer's
manual. It may cross-react with Sp1 and Sp4, because the DNA binding
domains of these three members are highly conserved.
Two synthetic double-stranded oligonucleotides were designed that
contained the distal Sp1 sequence of the minimal NHE-2
promoter (41/
18 bp, 5'-CCGCGCCCGCCCCGCCCCCGTCCC-3') or the mutated Sp1 sequence (
41/
18 bp,
5'-CCGCGCCCGAACCGAACCCGTCCC-3', where bold
letters indicate mutated bases). These DNA fragments were labeled with
[
-32P]ATP. Four micrograms of mIMCD-3 nuclear extract
or 1 footprint unit of Sp1 was incubated with 0.1 pmol of labeled probe
in electrophoretic mobility shift assay (EMSA) binding buffer
containing 20 mM HEPES (pH 7.6), 1 mM EDTA, 10 mM
(NH4)2SO4, 1 mM dithiothreitol,
0.2% (wt/vol) Tween 20, 30 mM KCl, 5 µg/ml
poly-L-lysine, and 50 µg/ml poly(dI-C). After incubation
at room temperature for 15 min, the mixture was electrophoresed on a
6% polyacrylamide gel in 0.25× Tris-boric acid-EDTA buffer. The gel
was dried and exposed to X-ray film. For the competition experiments, a
100-fold molar excess of unlabeled probe was added to the reaction
mixture before the addition of labeled probe. For supershifting or
blocking experiments, 0.2 µg of antibodies against Sp1, Sp3, or Sp4
was incubated with nuclear extract for 20 min at room temperature
before addition of probe.
DNase I footprinting assays.
A core footprinting system (Promega) was used for DNase I footprinting.
Probe (109 to +116 bp) was prepared by
SalI/NcoI digestion of construct pGL3/
289
(
289 to +116 bp), and the digested probe was purified and labeled
with [
-32P]ATP by T4 polynucleotide
kinase. The labeled probe was further digested by BamHI and
precipitated by n-butanol to remove the labeled 3' end. The
labeled probe (2.5 × 104 cpm) was incubated with 1 footprint unit of purified Sp1 or 4 µg of mIMCD-3 nuclear proteins at
room temperature for 10 min in 50 µl of binding buffer [50 µg/ml
BSA, 10 µg/ml poly(dI-C), and 0.03% Nonidet P-40]. The probe was
then digested with 0.15 U of DNase I at room temperature for 1 min. The
samples were analyzed on a sequencing gel. Maxam-Gilbert sequencing
(24) was carried out and run with DNase I-digested probe
as a ladder.
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RESULTS |
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Identification of minimal promoter and basal cis-elements of the
rat NHE-2 gene.
Our previous study has narrowed down the functional promoter of rat
NHE-2 to 289 bp upstream of the transcriptional initiation site (19). In the present study, we made further
deletion constructs to determine the minimal promoter region. These
constructs were introduced into mIMCD-3 cells that endogenously express
NHE-2 (1). The construct containing 36 bp of
NHE-2 promoter sequence plus 116 bp of 5'-noncoding region
(pGL3/
36 bp) showed promoter activity similar to that of other longer
constructs (pGL3/
289 bp, pGL3/
110 bp, and pGL3/
65 bp; Fig.
1). However, a further deletion construct
that removed all sequences upstream of the transcriptional initiation
site (pGL3/+2 bp) was inactive, suggesting that 36 bp of upstream
sequences in the minimal promoter appear to be critical for basal
transcription of the rat NHE-2 gene.
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Sp1 binding to the NHE-2 minimal promoter.
The functional studies shown in Figs. 1 and 2 demonstrated that two
distal overlapping Sp1 sites of the minimal promoter seem to play a
major role in promoter function. To confirm the precise location of
protein binding, we characterized the minimal promoter by DNase I
footprinting (Fig. 3). A DNA fragment
containing the 109/+116 bp region was 5'-end-labeled, incubated with
various amounts of recombinant Sp1 or mIMCD-3 cell nuclear extracts,
and then digested with 0.15 U of DNase I. As shown in Fig.
3A, only the region corresponding to the distal (
36/
16
bp) consensus Sp1 sites was protected from DNase I digestion when
labeled probe was incubated with purified Sp1. The same region and an
additional 11 bp of 5'-flanking sequence and 3 bp of 3'-flanking
sequence (
47/
13 bp) were protected from DNase I digestion when
labeled probe was incubated with mIMCD-3 cell nuclear extract (Fig.
3B). The restricted binding of purified Sp1 to only the
distal consensus Sp1 sites is consistent with the functional studies
that showed that only these sites are involved in basal transcriptional
regulation of the rat NHE-2 gene.
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Proteins interacting with the NHE-2 minimal promoter in mIMCD-3
cell nuclear extracts.
EMSAs were used to identify and characterize potential protein binding
activity associated with the 37/
25 bp region. An oligonucleotide
encompassing the distal consensus Sp1 binding sequence of the minimal
promoter was used as a probe in the mobility shift assays. As shown in
Fig. 4, nuclear extracts from the mIMCD-3 cells and purified Sp1 protein led to the same band- shift pattern. The
presence of a 100-fold molar excess of unlabeled probe abolished the
interaction between the probe and nuclear proteins. Additionally, a
probe containing a double mutation of the distal consensus Sp1 binding
sites also did not generate a band shift.
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Regulation of the NHE-2 minimal promoter in mIMCD-3 cells by Sp
transcription factors.
Because members of the Sp transcription factor family share the same
binding sequence and given the results of the mobility supershift
experiments, we hypothesized that they may play important roles in
transcriptional regulation of the NHE-2 gene by interacting with the core Sp cis-elements. So we tested the effect of
overexpression of individual Sp factors on the minimal NHE-2
promoter in mIMCD-3 cells. In the initial study, we found that
overexpression of Sp1 increased luciferase activity of pGL3/basic
(Promega)-transfected cells but did not change -galactosidase
activity of pGal/basic-transfected cells (data not shown). Thus we
subcloned the minimal promoter (
36/+116 bp) into pGal/basic vector
and tested the functional relevance of Sp1, Sp2, Sp3, and Sp4 by
cotransfecting this minimal promoter construct (pGal/
36 bp) and Sp
expression vectors into mIMCD-3 cells. As shown in Fig.
6, cotransfection of 1 µg of Sp1 did
not significantly change
-galactosidase activity. Increasing the
levels of Sp2 also did not change promoter activity. However, 1 and 5 µg of Sp3 expression vector repressed promoter activity by 50 and
60%, respectively. Interestingly, Sp4, generally known as a
transcriptional activator, repressed promoter function similar to Sp3.
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Regulation of NHE-2 minimal promoter in Drosophila SL2 cells by Sp
transcription factors.
Sp1 and related factors are expressed in virtually all mammalian cells,
and this ubiquitous expression could affect the interpretation of
experimental results. Drosophila SL2 cells are an
established in vitro model that can be used to study gene regulation by
Sp transcription factors, since they lack endogenous Sp1 activity (5). We cotransfected the minimal promoter construct
pGal/36 bp along with Drosophila expression vectors
pPacSp1, pPacSp2, pPacSp3, and pPacSp4 into Drosophila SL2
cells. As shown in Fig. 7, pGal/
36 bp
alone was not able to drive transcription in these SL2 cells,
indicating that Sp1 is essential for the formation of the
transcriptional initiation complex. Addition of the Sp1 expression
vector drastically increased transcription in a dose-dependent manner,
resulting in an ~70-fold activation at 0.1 µg and 110-fold activation at 5 µg of pPacSp1. Sp2, Sp3, and Sp4 expression did not
significantly stimulate transcription of the minimal promoter.
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DISCUSSION |
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In this study, we analyzed the basal transcription of the rat
NHE-2 gene in the renal inner medullary collecting duct cell line mIMCD-3, which endogenously expresses NHE-2. Deletion
analysis of various lengths of the promoter indicated that nucleotides 36/+116 represent the minimal promoter. Results from site-directed mutagenesis studies demonstrated that two Sp1 elements in the minimal
promoter are essential for basal transcription. Additional evidence to
support this finding consisted of detection of DNA-protein complexes by
DNase I footprinting and the specific binding of purified Sp1 and
nuclear proteins to these two cis-elements. Sp1 is a strong
transcriptional activator of this promoter, acting through these sites.
Sp3 and Sp4 do not significantly activate this promoter but, rather,
compete with Sp1 for binding sites, leading to a reduction in
transcription. The repressive effect of Sp4 is of great interest,
because Sp4 generally functions as a transcriptional activator
(8, 10).
The most notable feature in the minimal promoter region is the presence of four clustered consensus Sp1 elements (Fig. 2). Therefore, these elements appeared to be suitable candidates for controlling basal promoter activity. Interestingly, when these elements were sequentially mutated, promoter suppression only occurred when the two distal elements were mutated. In these distal elements, each individual mutation caused 80% reduction of promoter activity, and the double mutation completely abolished promoter function, thus suggesting that they play a critical role in transcriptional regulation of the rat NHE-2 gene. Moreover, DNase I footprinting with purified Sp1 protein revealed regions of extended protection, which only covered the two distal Sp1 elements. DNase I footprinting with mIMCD-3 cell nuclear extracts revealed additional protected regions, including 11 bp of 5'-flanking region and 3 bp of 3'-flanking region of the distal Sp1 sites. However, on the basis of functional studies (Fig. 2) and the results of DNase I footprinting with purified Sp1, these additional protected regions may not be due to direct binding of Sp1 but, rather, spatial occupation of DNA by other transcriptional factors that interact with the Sp1-DNA complex.
One question was raised because the two distal Sp1 elements overlap each other: do these two Sp1 elements bind the same molecule of Sp1, or do they bind to two Sp1 molecules? It is believed that Sp1 is only able to bind simultaneously to adjacent Sp1 sites if the central portions of the elements are >10 nucleotides apart (6). Thus it is possible that only one Sp1 molecule binds to the NHE-2 minimal promoter.
Gel mobility shift and supershift assays provided in vitro evidence that Sp1, Sp3, and Sp4 are likely involved in basal regulation of the NHE-2 gene. By using purified Sp1 and mIMCD-3 cell nuclear extracts, we confirmed the DNase I footprinting observation that Sp1 specifically binds the distal Sp1 sites. Using antiserum to Sp1 and Sp4 in EMSAs caused a supershifted band, which indicated that Sp1 and Sp4 are present in mIMCD-3 cells and can bind to the distal Sp1 sites. Additionally, the Sp3 antiserum led to complete blocking of the shifted band, and this finding is likely due to the blocking of Sp3 protein binding and cross-reaction with Sp1 and Sp4 proteins (the DNA binding domains are highly conserved in Sp1, Sp3, and Sp4) (8, 28).
To determine whether Sp1 family members had any effect on NHE-2 promoter activity in a more physiologically relevant setting, we overexpressed Sp proteins with the minimal NHE-2 promoter in a mammalian system. In mIMCD-3 cells, which contain endogenous Sp1, overexpression of Sp1 did not significantly change the promoter activity (Fig. 6), probably because endogenous Sp1 masks the effect of exogenous Sp1. Sp2 did not affect transcription. This may be due to the DNA-binding specificity of Sp2 being different from that of other family members (14). As has been shown previously for other genes (10), Sp3 inhibited transcriptional activation by Sp1. Furthermore, in contrast to earlier reports that indicated that Sp4 is only an activating transcription factor (8, 9), Sp4 inhibited transcriptional activation by Sp1. More recently, however, one study indicated that Sp4 could also function as a negative regulator (16). The emerging picture, then, is that Sp4 is a bifunctional protein. The molecular mechanism underlying the dual-functional character of Sp4 remains to be elucidated.
To exclude the possible interference of endogenous Sp1, we overexpressed Sp family members with the minimal NHE-2 promoter in Drosophila SL2 cells, which lack endogenous Sp1 (5). The minimal promoter construct was not able to drive transcription in these cells. However, cotransfection of Sp1 strongly increased transcriptional activity. This demonstrates that Sp1 is a critical factor in transcriptional initiation of this promoter. We then tested whether Sp2, Sp3, and Sp4 could activate transcription in the absence of Sp1. None of them can drive transcription in Drosophila SL2 cells, suggesting that they lack the ability to form a functional transcriptional initiation complex. We also investigated the interaction of Sp1 with other family members. Similar to the results from mIMCD-3 cells, Sp2 did not interfere with Sp1-mediated transcription, while Sp3 and Sp4 significantly repressed transcriptional activation by Sp1. Because Sp3 and Sp4 cannot themselves drive transcription, Sp3 and Sp4 may compete with Sp1 for the same binding site and then abort the formation of the transcriptional initiation complex.
In summary, we have demonstrated that the binding of Sp1 to the minimal NHE-2 gene promoter is critical for controlling the basal transcriptional activity in mIMCD-3 cells. Sp3 and Sp4 can repress Sp1-mediated transcriptional activation. The interaction between Sp transcription factors may play an important role in NHE-2 gene regulation.
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ACKNOWLEDGEMENTS |
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We thank Carlos Enamorado and Adam K. Ghishan for help in making deletion constructs.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-DK-41274-10 and by the W. M. Keck Foundation.
Address for reprint requests and other correspondence: F. K. Ghishan, Dept. of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 September 2000; accepted in final form 22 November 2000.
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