Transcriptional regulation of rat Na+/H+ exchanger isoform-2 (NHE-2) gene by Sp1 transcription factor

Liqun Bai, James F. Collins, Hua Xu, and Fayez K. Ghishan

Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

A series of progressively shorter NHE-2 promoter constructs in the pGL3/basic luciferase reporter vector were made by restriction enzyme digestion or PCR. Briefly, pGL3/-289 bp construct was made by subcloning an MluI-NcoI fragment of the NHE-2 promoter into the pGL3/basic vector (19). For other deletion constructs, pGL3/-110 bp, pGL3/-65 bp, pGL3/-36 bp, and pGL3/+2 bp, different lengths of NHE-2 promoter were PCR amplified, utilizing primers with overhanging restriction enzyme sites for KpnI and XhoI, and then ligated into KpnI/XhoI cut pGL3/basic. The 3' end of all deletion constructs was at +116 bp. The single and double mutations of Sp1 consensus sites were introduced by PCR-based site-directed mutagenesis (2). Briefly, the 36-bp promoter region was PCR amplified by primers containing mutated base pairs. The wild-type promoter was then replaced by PCR fragments containing different mutations using restriction digestion.

Because luciferase activity of pGL3/basic-transfected cells was increased by overexpression of Sp1 and beta -galactosidase activity of pGal/basic was not changed by overexpression of Sp1 (data not shown), we generated the pGal/-36 bp construct by moving the minimal promoter region (-36 to + 116 bp) into pGal/basic vector using KpnI and BglII. All constructs were confirmed by sequencing.

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. beta -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 beta -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 [gamma -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 [gamma -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Identification of the minimal rat Na+/H+ exchanger isoform-2 (NHE-2) promoter by deletion analysis. Deletion constructs were cotransfected with pRL-CMV vector, and dual luciferase activity of these constructs was measured in mouse inner medullary collecting duct (mIMCD-3) cells 48 h after transfection. Relative luciferase activity was calculated as follows: luciferase activity divide  renilla luciferase activity (internal control for transfection efficiency). Values are means ± SD of duplicate data from 3 experiments. *P < 0.001 vs. all other deletion constructs.

A search for transcription factor binding sites within this 36-bp region yielded four potential consensus binding sites for Sp1, with two of them overlapping (Fig. 2). To determine the role of these Sp1 consensus sequences in transcriptional activation of NHE-2, single and double mutations of these Sp1 sites were generated by site-directed mutagenesis (Fig. 2). Alteration of the distal (-35/-30 and -31/-25 bp) consensus Sp1 sites (mutants 1 and 2, respectively) reduced the promoter activity to <20% of that of wild-type promoter (pGL3/-36 bp). When both distal Sp1 binding sites were simultaneously mutated (mutant 5), the promoter activity was reduced to 8% of wild-type promoter. This value was essentially identical to that of the promoterless construct, pGL3/basic. However, alteration of the proximal (-18/-13 and -14/-9 bp) Sp1 binding sites by single (mutants 3 and 4) or double (mutant 6) mutation did not change promoter activity, indicating that the proximal Sp1 sites are not involved in basal transcriptional activity.


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Fig. 2.   Effect of Sp1 mutants on minimal promoter activity. The wild-type (WT) minimal promoter region identified in Fig. 1 is depicted with 4 putative Sp1 binding sites (2 of them overlapping each other). Six single or double mutants are indicated in bold letters. Values are means ± SD of duplicate data from 3 experiments. *P < 0.001 vs. wild-type minimal promoter construct.

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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Fig. 3.   DNase I footprinting analysis of protein interactions within the proximal region of the rat NHE-2 promoter. The probe of the proximal region of the NHE-2 promoter (containing the minimal promoter region) was labeled with [gamma -32P]ATP by T4 polynucleotide kinase. The purified probe was incubated with 1 or 4 footprint units (fpu) of recombinant Sp1 (A) or 1 or 4 µg of mIMCD-3 nuclear protein (B) and then treated with DNase I. A control reaction (control) was performed without nuclear protein or Sp1. G & A, Maxam and Gilbert sequencing reactions of the DNA fragment. *Protected regions (shown in bold sequences at top).

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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Fig. 4.   Electrophoretic mobility shift assay with purified mIMCD-3 nuclear extract and recombinant Sp1. 32P-labeled double-stranded oligonucleotides, covering the distal Sp1 sequences protected from DNase I in footprinting assays, were incubated with or without 4 µg of mIMCD-3 cell nuclear extract (IMCD) or 1 fpu of recombinant Sp1 (Sp1). The DNA-protein binding was performed with wild-type oligonucleotide (WT, -41/-18 bp, 5'-CCGCGCCCGCCCCGCCCCCGTCCC-3') or mutant oligonucleotide (Mut, -41/-18 bp, 5'-CCGCGCCCGAACCGAACCCGTCCC-3'; bold letters indicate mutated bases). Competition between labeled and unlabeled specific oligonucleotides at 100 molar excess is shown. Magnification, ×100.

The Sp1 consensus sequence is known to bind Sp1 and other related proteins, particularly Sp3 and Sp4. To confirm the specific binding of Sp proteins to the minimal promoter of the rat NHE-2 gene, we tested whether Sp1, Sp3, and Sp4 can interact with cis-elements in the minimal promoter. As shown in Fig. 5, binding of mIMCD-3 cell nuclear proteins to the probe caused a band shift, and the band was supershifted by Sp1 and Sp4 antibodies (antibodies raised against the transactivation domains, which still allow DNA binding). Additionally, the Sp3 blocking antibody was used to test the specific binding of Sp3 to the minimal promoter. The shifted band was completely blocked by Sp3 antibody. The Sp3 antibody also interacts with Sp1 and Sp4, since the DNA binding domains of these proteins are highly homologous. Furthermore, nonspecific rabbit IgG, used as a control, did not alter DNA-protein interactions.


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Fig. 5.   Interaction of the minimal promoter with Sp1 and related proteins. Gel mobility supershift and blocking-shift experiments were performed using the same wild-type oligonucleotide as in Fig. 4. End-labeled probe (0.1 pmol) was incubated with 4 µg of mIMCD-3 cell nuclear extract in the presence of 0.2 µg of supershifting antibody (Ab) for Sp1 and Sp4 or 2 µg of blocking Ab for Sp3. Rabbit IgG was used as a negative control.

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 beta -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 beta -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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Fig. 6.   Effects of Sp1, Sp2, Sp3, and Sp4 on transcription in mIMCD-3 cells. One microgram of pGal/-36 bp was cotransfected with or without 1 or 5 µg of various mammalian expression vectors encoding Sp transcription factors, all driven by the cytomegalovirus (CMV) promoter (pCMVSp1, pCMVSp2, pCMVSp3, and pCMVSp4). pGal/basic contained neither a promoter nor an enhancer and was used as a negative control. Ten micrograms of total nuclear protein were used for each beta -galactosidase assay. Values are means ± SD of duplicate data from 3 experiments. *P < 0.01 vs. pGal/-36 bp only.

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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Fig. 7.   Effects of Sp1, Sp2, Sp3, and Sp4 on transcription in Drosophila SL2 cells. One microgram of pGal/-36 bp was cotransfected with or without 0.1-5 µg of various Drosophila expression vectors encoding Sp transcription factors, all driven by the actin 5C promoter (pPacSp1, pPacSp2, pPacSp3, and pPacSp4). Ten micrograms of total nuclear protein were used for each beta -galactosidase assay. Values are means ± SD of duplicate data from 3 experiments.

To determine whether transcriptional activation by Sp1 could be affected by other Sp factors, we cotransfected the pGal/-36 bp construct and 1 µg of pPacSp1 with 1-5 µg of pPacSp2, pPacSp3, or pPacSp4 into Drosophila SL2 cells (Fig. 8). Sp1-mediated transcriptional activation was not changed by Sp2, while it was significantly repressed by Sp3 and Sp4. One microgram of Sp3 or Sp4 expression vectors led to a 40 and 35% decrease of transcriptional activity, respectively. Five micrograms of Sp3 or Sp4 reduced the activity to 50% of control values.


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Fig. 8.   Repression of Sp1-induced transcriptional activation by Sp3 and Sp4 in Drosophila cells. One microgram of pGal/-36 bp was cotransfected with 1 µg of pPacSp1 and with varying amounts of pPacSp2, pPacSp3 or pPacSp4 (1-5 µg). Ten micrograms of total nuclear protein were used for each beta -galactosidase assay. pGal/-36 bp plus pPacSp1 was used as control. Values are means ± SD of duplicate data from 3 experiments. *P < 0.01 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We thank Carlos Enamorado and Adam K. Ghishan for help in making deletion constructs.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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