Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate

Jaleh Malakooti, Ricardo Sandoval, Vanchad C. Memark, Pradeep K. Dudeja, and Krishnamurthy Ramaswamy

Department of Medicine, Section of Digestive and Liver Diseases, University of Illinois, and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois

Submitted 11 January 2005 ; accepted in final form 7 June 2005


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The apical membrane Na+/H+ exchanger isoforms NHE2 and NHE3 are involved in transepithelial Na+ absorption in the intestine. However, they exhibit differences in their pattern of tissue expression and regulation of their activity by various molecular signals. To study the mechanisms involved in the transcriptional regulation of these genes, we characterized cis-acting elements within the human NHE2 promoter that regulate NHE2 promoter expression in C2BBe1 cells. A small DNA region (–85/+249) was involved in the regulation of basal transcriptional activity of the NHE2 promoter as determined by transient transfection assays. RT-PCR analysis showed that NHE2 mRNA was upregulated in response to phorbol 12-myristate 13-acetate (PMA). Results from actinomycin D-treated cells indicated that the regulation of the NHE2 gene by PMA occurs in part at the transcriptional level. Furthermore, PMA treatment led to a 100% increase in promoter activity through elements located on the –415/+249 DNA fragment. A PMA-induced nuclear factor that bound to the NHE2 promoter was identified as the transcription factor Egr-1. We identified two PMA response elements in the –415/+1 promoter region that bind to Sp1 and Sp3 in untreated nuclear extracts and to Egr-1 in PMA-treated nuclear extracts. In cotransfection experiments, Egr-1 was able to transactivate the NHE2 promoter. Our data indicate that Egr-1 may play a key role in regulated expression of the human NHE2 gene.

Na+/H+ exchanger isoform 2; transcriptional regulation; PMA response element; Sp1; Sp3


NA+/H+ EXCHANGERS (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of an extracellular Na+ for an intracellular H+. These proteins are involved in a variety of physiological processes such as Na+, bicarbonate, and water absorption and regulation of intracellular pH and cell proliferation (10, 38, 52). Of the eight isoforms identified to date, three isoforms, NHE1, NHE2, and NHE3, have been identified and characterized in the human intestinal tract. NHE1 is localized to the basolateral membrane of polarized cells, whereas NHE2 and NHE3 are predominantly located on the brush border membrane (19).

Although extensive studies have been carried out investigating posttranslational regulation of NHE gene products and their interaction with cytoplasmic regulatory proteins (42, 53, 54), data on the transcriptional regulation of NHE isoforms are just emerging. Studies focused on the NHE1 isoform have demonstrated the regulation of the NHE1 gene at the transcriptional level by external acid, growth factors, serum, and retinoic acid (41, 55, 56). In addition, transcription factors COUP-TFI, COUP-TFII, AP-1, and AP-2 have been shown to be involved in transcriptional regulation of the NHE1 promoter (15, 16, 20). Among the other NHE isoforms, the promoter sequences for human NHE2 (hNHE2), rat NHE2 (29, 36), and NHE3 isoforms (9, 21, 31) have been cloned. Recently, the stimulatory effect of hypertonocity and transcription factor Sp1 on rat NHE2 promoter expression has also been described in mIMCD-3 cells (4, 5).

Previous studies have demonstrated a stimulatory effect of acute phorbol 12-myristate 13-acetate (PMA) treatment on Na+ uptake in different NHE2-transfected cell lines (21, 35, 47). These studies have demonstrated that the activity of the cloned NHE2 gene product is increased by PMA through a change in maximal velocity of the transporter, and the PMA stimulatory effect was shown to be via a specific PMA-sensitive subdomain in the COOH terminus of the NHE2 polypeptide (37). The PMA-sensitive subdomain maps to a similar location in the COOH-termini of both NHE2 and NHE3 proteins; however, PMA inhibits NHE3 but activates NHE2 transport activity (37). The question of whether PMA was also involved in the transcriptional activation of the NHE2 gene is addressed in the present study.

Previously, we (29, 30) reported the isolation and characterization of hNHE2 cDNA and its genomic organization and promoter region. These studies revealed that the NHE2 promoter lacks TATA and CCAAT boxes, is highly GC rich, and contains a number of Sp1 binding sites as well as a GC box and CACCC motifs (29). The existence of multiple Sp1 binding sites in close vicinity of transcription initiation sites in TATA-less promoters have been attributed to the involvement of Sp1 in recruiting TATA binding protein (40) and fixing the transcription start site at these promoters (8, 27).

In the present study, we examined the functional characteristics of the hNHE2 promoter under basal and PMA-induced conditions in the C2BBe1 cell line. We show that PMA treatment results in increased endogenous hNHE2 mRNA expression and leads to elevated NHE2 promoter-reporter gene activity in transiently transfected cells. Further studies demonstrated that the transcription factor Egr-1 is induced by PMA in C2BBe1 cells. Two PMA response elements (PRE) that may mediate the transcriptional activation of the hNHE2 promoter in response to PMA were identified. By gel mobility shift assays (GMSA) and transient transfections using wild-type and mutated PREs, we show not only Egr-1 interactions with PREs but also the effects of such binding on NHE2 promoter-reporter gene activity. Cotransfection experiments with an Egr-1 expression vector demonstrated that Egr-1 is capable of transactivating NHE2 expression, providing further evidence that this transcription factor is involved in PMA-inducible expression of the hNHE2 gene.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
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 GRANTS
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Reagents, molecular techniques, antibodies, and plasmids. All chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical (St. Louis, MO); restriction endonucleases and other modifying enzymes were from New England Biolabs (Beverly, MA), GIBCO-BRL (Gaithersburg, MD), or Promega (Madison, WI). The luciferase assay system was from Promega. DNA manipulations, including restriction enzyme digestion, ligation, plasmid isolation, and transformation, were carried out by standard methods (3). Anti-human Sp1, Sp2, Sp3, and Egr-1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Plasmid constructs. All constructs used for transient transfections and functional analyses were generated using pGL2-Basic (Promega), which contains a promoterless luciferase reporter gene. Plasmids pJM3.3-N2 (–3 kb/+318) and pJM-415/+249 have been described previously (29). To construct 5'-truncation plasmids with a common 3'-end, the following approaches were taken: p-861/+249 was made by digesting p-861/+318, a pCR II (Invitrogen) derivative, with the restriction enzymes SacI, which cuts in the polylinker of the vector, and AatII, which cuts the NHE2 promoter fragment internally. After gel purification, this DNA fragment was cloned into p-415/+249, which was cut with the same restriction enzymes. Two pCR II derivative constructs, p-296/+318 and p-85/+318, were generated by cloning PCR amplification products of the forward primers (5'-CGCTGGTTCCTTCACTGGATGGACG-3' and 5'-GTGTGCCGGCCAGGTCCCCTTGTCC-3', respectively) and the common reverse primer (5'-TTCCATGGGTGCCGCCGGTTGCCGCCAAGGGG-3') using pJM3.3-N2 as a template. To construct p-296/+249 and p-85/+249, DNA fragments containing sequences –296/+249 and –85/+249 were released from the respective clones by digestion with SacI, which cuts the polylinker of the vector, and SmaI at position +249. These DNA fragments were then cloned into pGL2-Basic, which was digested with HindIII, and the ends were filled in with Klenow and digested again with SacI to generate p-296/+249 and p-85/+249.

To mutate the distal PRE at the 5'-Egr-1 site (M1), at the 3'-Egr-1/Sp1 site (M2), or all these sites (M3), the forward primers (mutated nucleotides are underlined) 5'-AGGAGCCTAATGGGGCGGGGGAGGAGG-3', 5'-AGGAGCCTGCGGGGGCGAATGAGGAGG-3', and 5'-AGGAGCCTGCGGGGAAATGGGAGGAGG-3', respectively, in conjunction with a common reverse primer, 5'-CCGCGGGGGCGATGGCTGCCCTCC-3', were synthesized and used in PCR amplification reactions using p-415/+249 as a template. All forward primers contained an XhoI recognition site at their 5'-end. The PCR products were then digested with XhoI and the internal AatII, gel purified, and cloned back into p-415/+249 digested with the same restriction enzymes. p-345/+249, which carried wild-type DNA sequences, was constructed as above with a forward primer containing the wild-type nucleotide sequences starting at bp –345.

The Egr-1 expression vector (pAC-hEgr-1) containing human Egr-1 cDNA was provided by Dr. John Monroe, University of Pennsylvania.

Cell culture, transfections, and reporter assays. The C2BBe1 cell line, a subclone of Caco-2 human colonic epithelial cells, was cultured and maintained as described previously (29). Transient transfection assays were conducted using LipoFectamine 2000 Reagent (Invitrogen). Briefly, C2BBe1 cells were seeded at a density of 1.5 x 105 cells/well in 12-well culture dishes and transfected the following day. A total of 2 µg DNA was used for each transfection at a ratio of 4:1 for experimental construct versus pSV-{beta}gal. For PMA treatments, transfected cells were synchronized by 24-h incubation in media containing 0.5% FBS, fresh media supplemented with PMA (100 nM) was then added, and incubation continued for 16 h. Forty-eight hours posttransfection, cells were washed with PBS and lysed using a kit from Promega. Luciferase activity was assayed using a TD 20/20 luminometer (Promega) and normalized to {beta}-galactosidase activity. The relative luciferase activity was then calculated by dividing the normalized luciferase activity of the experimental constructs by the normalized luciferase activity obtained from promoterless pGL2-Basic. All transfection studies were performed in triplicate and repeated at least three times. For experiments involving treatment with the transcription inhibitor actinomycin D (5 µM) or the protein synthesis inhibitor cycloheximide (15 µg/ml), C2BBe1 cells were first incubated in medium containing low serum for 24 h, and subsequently actinomycin D or cycloheximide were added 1 h before the addition of PMA and then in combination with PMA for the duration of treatment.

RNA extraction and RT-PCR analysis. Total RNA was extracted by the RNAzol method (Tel-Test; Friendswood, TX) according to the manufacturer's directions. PCR was performed in a Perkin-Elmer/Cetus DNA Cycler with thermostable DNA polymerase rTth (Boehringer Mannheim; Indianapolis, IN) according to the manufacturer's directions. For RT-PCR experiments, 5 µg of total RNA were reverse transcribed using SuperScriptII reverse transcriptase (Invitrogen) according to the supplier's instructions. The cDNA synthesis was primed by an oligo-(dT) and performed at 42°C for 60 min. For PCR analysis, Egr-1- or NHE2-specific primers were utilized in separate amplification reactions, and, initially, cDNA was amplified for 17 cycles with the experimental primers; at this point, control GAPDH-specific primers were added to each reaction, and amplification continued for 16 additional cycles. A 1/10 volume of the PCR was resolved on 1.5% agarose gels. Gels were stained with ethidium bromide, and relative DNA amplification was determined by densitometric analysis using an Alpha Imager 1220 Documentation and Analysis System (Alpha Innotech). GAPDH was used as an internal control for dendistometric analysis.

Preparation of nuclear extracts and GMSA. Nuclear proteins were prepared essentially as previously described (3), and protein contents were determined using the Bio-Rad Protein Assay kit with BSA as the standard. All oligonucleotide probes for GMSA were synthesized by Life Technologies (Invitogen). Complementary oligonucleotides were made double stranded by heating to 95°C for 5 min and slow cooling to 25°C in Tris-EDTA buffer [10 mM Tris·HCl (pH7.5) and 1 mM EDTA]. Double-stranded oligonucleotides were end labeled with [{gamma}-32P]ATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (New England Biolabs). Protein-DNA binding reactions were performed in binding buffer [50 mM Tris·HCl (pH 7.5), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 1 µg/sample poly (dI.dC)-poly (dI.dC), and 4% glycerol] and 30,000 counts/min (cpm) of the probe. Reactions were initiated by the addition of nuclear proteins (5 µg) to [{gamma}-32P]ATP end-labeled double-stranded oligonucleotides probes. The reaction mixtures were incubated for 20 min at room temperature before electrophoresis on a native 5% polyacrylamide gel in 0.5x Tris borate-EDTA running buffer (45 mM Tris borate and 1 mM EDTA; pH 8.3). For competition assays, a 100-fold molar excess of nonradioactive oligonucleotides was added to the GMSA reaction mixture and incubated at room temperature for 10 min before the addition of the radioactive probe. Supershift assays were performed by the addition of 1 µl of the appropriate antibody (Santa Cruz Biotechnology) after the initial 20-min incubation with the labeled probe and then further incubation for 30 min at room temperature. Gels were dried and visualized by autoradiography.

SDS-PAGE and Western immunoblot analysis. Nuclear extracts were prepared from C2BBe1 cells at different time points after PMA (100 nM) treatment. A total of 10 µg nuclear extracts was fractionated on an 8% SDS-polyacrylamide gel and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore; Bedford, MA). The membranes were blocked in 5% nonfat dry milk in 10 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature, washed with Tris-buffered saline containing 0.1% Tween 20, and subsequently incubated with primary antibody for 1 h at room temperature. The antibody-antigen interactions were detected by an enhanced chemiluminescence system (ECL Plus, Amersham Pharmacia Biotech) in conjunction with a horseradish peroxidase-conjugated secondary antibody. As a control for the amount of protein loaded per lane, blots were stripped and rehybridized with anti-tubulin antibody (Santa Cruz Biotechnology).


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of PMA treatment on NHE2 mRNA expression. It has previously been demonstrated that PMA-treated cells show increased NHE activity that was attributed to the NHE2 isoform (22, 35, 47). To examine whether PMA affects NHE2 mRNA expression, we performed RT-PCR experiments. C2BBe1 cells were serum starved for 24 h and then treated with 100 nM PMA. The cells were harvested at the indicated time points after PMA treatment, and total cell RNA was obtained from untreated and PMA-treated cells and subjected to reverse transcription and subsequent PCR amplification using hNHE2-specific primers. As shown in Fig. 1A, NHE2 mRNA expression increased with time after PMA treatment, reaching a maximum level after 6 h, and returned to baseline by 24 h. To examine the mechanism by which PMA treatment leads to increased NHE2 mRNA expression, we pretreated C2BBe1 cells with the transcription inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide; the cells were then incubated in the presence or absence of PMA, and, subsequently, RT-PCR analysis was performed. As shown in Fig. 1B, lanes 4 and 7, the simultaneous presence of actinomycin D and PMA did not result in enhanced NHE2 mRNA levels, suggesting that the stability of NHE2 mRNA was not prolonged. Cycloheximide treatment blocked the PMA-induced increase in NHE2 mRNA as well (Fig. 1B, lane 11). Therefore, these observations indicate that the upregulation of hNHE2 mRNA expression by PMA is mediated at the transcriptional level and requires de novo protein synthesis.



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Fig. 1. Expression of Na+/H+ exchanger (NHE)2 mRNA in C2BBe1 cells in response to phorbol 12-myristate 13-acetate (PMA) treatment. RT-PCR was performed on 5 µg of total RNA isolated from PMA-treated and untreated C2BBe1 cells with an oligo-(dT) as primer and SuperScript II reverse transcriptase. One-tenth of the reverse transcription product was used as a template in each PCR with gene-specific primers to human NHE2 (forward primer 5'-ACTATTCGACCACTGGTGGAG-3' and reverse primer 5'-ACTTATCATCCCAGTCTCTGCC-3') and GAPDH (forward primer 5'-ATGGCACCGTCAAGGCTGAGG-3' and reverse primer 5'-GGCATGGACTGTGGTCATGAG-3') as an internal control. A: time course of PMA treatment. C2BBe1 cells were grown to confluence and then serum deprived for 24 h before the experiment. Cells were either treated with the vehicle (untreated) or 100 nM PMA (treated). The duration of the PMA treatments is shown on the top of the gel. B: effect of actinomycin D and cycloheximide on PMA-induced NHE2 mRNA expression. C2BBe1 cells were preincubated with 5 µM actinomycin D for 30 min (lanes 3,4, 6, and 7) and then incubated in the absence (lanes 3 and 6) or presence (lanes 4 and 7) of 100 nM PMA. Lanes 2 and 6 are cells treated with PMA only for 2 and 6 h, respectively. Lanes 1 and 8 represent the untreated cells. In lanes 10 and 11, C2BBe1 cells were preincubated with 15 µg/ml cycloheximide for 60 min and then treated with 100 nM PMA for 6 h in the presence of the inhibitor (lane 11). Lane 9 represents cells grown in presence of PMA for 6 h.

 
Identification of the PMA response region within the human NHE2 promoter. We next examined the effect of PMA treatment on the transcriptional regulation of the NHE2 promoter. C2BBe1 cells were transiently transfected with 5'-truncated NHE2 promoter-reporter constructs and cultivated in serum starvation media for at least 24 h. The cells were then treated with 100 nM PMA or vehicle for 16 h, harvested, and lysed. Luciferase activity was measured in the cell lysates of PMA-treated and untreated cells. PMA treatment resulted in a two- to threefold increase in luciferase activity of p-861/+249 compared with the control (Fig. 2A), indicating that a PRE(s) was located in this promoter fragment. p-415/+249 also responded to PMA with a two- to threefold increase in luciferase activity. The promoer-repoter constructs containing the sequences from bp –296 to +249 also displayed a minor increase (10–20%) in luciferase activity in response to PMA. These data indicated that the cis element(s) responsible for the PMA effect was located on the DNA region downstream from position –415.



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Fig. 2. A: effect of PMA treatment on transcriptional activity of the 5'-deletion constructs of NHE2 promoter in C2BBe1 cells. The 5'-truncated NHE2 promoter-reporter constructs were transiently transfected into C2BBe1 cells, and the cells were either not treated or treated with 100 nM PMA for 16 h before being lysed for luciferase assays. The bar graph represents the relative luciferase (Luc) activity of the deletion constructs in the presence (solid bars) or absence (shaded bars) of 100 nM PMA. The activity of the promoterless vector, pGL2-Basic, is assigned a value of 1.0, and the activity of the other promoter-reporter constructs are expressed as the relative fold increase. Values are means ± SE; n = 3. B: identification of the putative PMA response elements (PREs) in the human NHE2 promoter region. The putative distal (D-PRE) and proximal PRE (P-PRE) and their nucleotide sequences are shown. The overlapping Sp1 and Egr-1 binding sites are indicated in boxes. The indicated sequences were utilized as probes in DNA binding assays.

 
The nucleotide sequences of the promoter region downstream from bp –415 were scanned by MatInspector application (http://www.genomatix.de) to identify the potential transcription binding sites. Two overlapping Sp1/Egr-1 motifs were detected (Fig. 2B). Sequences similar to regions containing the overlapping Sp1/Egr-1 motifs have been shown to be the target of PMA response factors in different systems (11, 12, 23, 24, 46, 51). Consistent with our functional analysis (Fig. 2A), the potential distal PRE containing the overlapping Sp1/Egr-1 motif is located at bp –339 to –324. This element is composed of two Egr-1 binding sites that overlap by three nucleotides and an Sp1 consensus sequence that overlaps with both of the Egr-1 binding sites (Fig. 2B). The putative proximal PRE is situated at bp –32 to –19 and contains three potential overlapping Sp1 sites, of which the 3' element also overlaps with an Egr-1 binding site (Fig. 2B). The overlapping configuration of these elements suggests that the binding of each transcription factor can be mutually exclusive.

PMA treatment induces Egr-1 mRNA and protein expression in C2BBe1 cells. The sequences of both the distal and proximal PREs in the promoter region of the hNHE2 gene are highly homologous to motifs identified in the promoter region of other genes that are a target of PMA-induced transcription factor Egr-1. To explore the possibility that the NHE2 gene may also be regulated by Egr-1, we examined the kinetics of Egr-1 mRNA and protein expression in response to PMA treatment in C2BBe1 cells. As shown in Fig. 3A, the Egr-1 mRNA level was increased dramatically by 1 h post-PMA treatment, and the elevated level of mRNA expression persisted for up to 4 h and returned to basal levels by 16 h after PMA treatment. Egr-1 protein abundance in C2BBe1 nuclear extracts also increased in response to PMA treatment and was maximal by 2 h after exposure to PMA. The elevated levels of Egr-1 protein reduced to the basal level by 16 h after PMA treatment, as determined by Western blot analysis (Fig. 3B) and compared with tubulin protein expression.



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Fig. 3. Induction of Egr-1 mRNA and protein expression in response to PMA treatment in C2BBe1 cells. A: RT-PCR was performed on 5 µg of total RNA isolated from PMA-treated and untreated C2BBe1 cells with an oligo-(dT) as primer and SuperScript II reverse transcriptase. One-tenth of the reverse transcription product was used as a template in each PCR with gene-specific primers to Egr-1 and GAPDH as an internal control. The duration of the PMA treatments is indicated on the top of the gel. B: Western blot. Confluent C2BBe1 cells were maintained in serum-reduced media for 24 h and then treated with PMA (100 nM) for the indicated periods of time, and nuclear extracts were prepared. Ten micrograms of nuclear proteins were resolved on an 8% SDS-PAGE, electroblotted on to Immobilon-P membranes, and detected with anti-Egr-1 antibody. The same membrane was stripped and rehybridized with anti-tubulin as a control for the amount of protein loaded in each lane.

 
Transcription factors Sp1, Sp3, and Egr-1 interact with distal and proximal PREs of the hNHE2 promoter. The results of functional analysis of the 5'-deletion constructs (Fig. 2A) implicated the distal PRE as the major site involved in PMA responsiveness of the hNHE2 promoter. Therefore, we focused our further studies to characterize this motif in more detail. To identify the specific nuclear factors that may interact with the distal PRE, we used a double-stranded oligonucleotide probe corresponding to bp –345 to –319 and nuclear proteins from untreated and PMA-treated cells in GMSAs. This probe harbors the potential distal PRE. When incubated with the nuclear extracts from cells grown under basal growth conditions (DMEM + 10% FBS), four DNA-protein complexes (C1-C4) were formed with this probe (Fig. 4A, lane 1). The binding specificity of complexes was determined by competition assays where all complexes were removed when an unlabeled probe was used as a competitor and a nonspecific unlabeled oligonucleotide did not compete with any of the complexes (data not shown). Because the distal PRE contained a potential binding site for Sp1, we examined whether any of the detected DNA-protein complexes were related to Sp1. For this purpose, an excess of unlabeled oligonucleotide containing the Sp1 consensus binding sequence was used as a competitor (Fig. 4A, lane 2). This oligonucleotide eliminated all complexes except for C3 and a diffused smeary band migrating under C3, suggesting that Sp1 family members interact with this DNA region.



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Fig. 4. Constitutive and PMA-inducible DNA binding activity of C2BBe1 nuclear factors to the distal PRE. A: constitutive DNA binding activity of C2BBe1 nuclear factors to the distal PRE. A representative gel mobility shift assay (GMSA) is shown. These experiments were performed using an oligonucleotide corresponding the distal PRE as an end-labeled probe and nuclear proteins from C2BBe1 cells. The interacting proteins are shown on the left. A total of 5 µg of nuclear proteins was combined with 30,000 counts/min of probe per reaction and, after 20-min incubation at room temperature, were resolved on a 5% nondenaturing polyacrylamide gel and visualized by autoradiography. Competition experiments with 100-fold molar excess of Sp1 consensus sequence is shown in lane 2. Supershift assays were performed by the addition of 1 µl of anti-Sp1, anti-Sp3, and anti-Egr-1 antibodies to the nuclear extract-probe complexes and a 30-min further incubation at room temperature before gel electrophoresis (lanes 3–5, respectively). B: Supershift experiments were performed with nuclear extracts from PMA-treated cells. Nuclear proteins were coupled to end-labeled probe and incubated with anti- Sp1, anti-Sp2, anti-Sp3, and anti-Egr-1 antibodies (lanes 2–5, respectively). SS, supershifted bands. C: constitutive and PMA-inducible DNA binding activity of C2BBe1 nuclear factors to the distal PRE probe. Because of the high level of Egr-1 expression in PMA-treated cells, in theses experiments 5 µg of nuclear proteins from untreated cells (lanes 1–3) or 2.5 µg from PMA-treated cells (lanes 4 and 5) were coupled with the distal PRE probe. Egr-1 was detected by supershift assays in lanes 2, 3, and 5 in the presence of anti-Egr-1 antibody. In lane 2, anti-Egr-1 antibody was added to nuclear proteins 30 min before the addition of the end-labeled probe and further incubated for 20 min at room temperature. D: nuclear proteins were purified from untreated cells and incubated with the 32P-labeled distal PRE probe. Competition experiments were performed with the unlabeled distal PRE (lane 2), proximal PRE (lane 4), and Sp1 consensus oligonucleotide (lane 3; the sequence of the top strand is 5'-ATTCGATCGGGGCGGGGCGAGC-3'). The identities of the proteins in these complexes were determined by supershift experiments using Sp1, Sp3, and Egr-1 antibodies (lanes 5–7). NF-X, unknown transcription factor.

 
To identify the protein components of these complexes, supershift experiments were performed using anti-Sp1, anti-Sp3, and anti-Egr-1 antibodies (Fig. 4A, lanes 3–5, respectively). Incubation of nuclear extracts with Sp1 antibody completely blocked the formation of C1 and identified the protein present in C1 as Sp1; anti-Sp3 antibody removed C2 and the fastest migrating complex C4, therefore identifying the protein component of C2 and C4 as Sp3; anti-Sp2 failed to generate a supershift or to eliminate any of the complexes (data not shown); and a faint and diffused band that migrates under C3 (lane 2) was removed with anti-Egr-1 antibody, thus suggesting that this broad band may represent the Egr-1 transcription factor (also see Fig. 4C, lanes 2 and 3). The identity of the protein in C3 is not known at this time and is designated as NF-X. Therefore, we conclude that in constitutive basal conditions, Sp1, Sp3, NF-X, and low levels of Egr-1 are the proteins that interact with the distal PRE.

To assess whether PMA treatment leads to differential interactions of nuclear factors with the distal PRE, nuclear proteins from PMA-treated cells were used in GMSA. As shown in Fig. 4B, a single DNA-protein complex was detected. This PMA-induced DNA binding protein was identified as Egr-1 by supershift experiments with anti-Egr-1 antibody (Fig. 4, B and C, lanes 5). Supershift analysis using anti-Sp1, anti-Sp2, and anti-Sp3 did not affect DNA-protein complex formation (Fig. 4B, lanes 2–4). Therefore, it appears that binding of PMA-induced Egr-1 to this region interferes with binding of Sp family members with the distal PRE motif.

As a part of another study, we have previously shown that transcription factors Sp1 and Sp3 bind to the NHE2 promoter region from bp –37 to –14 at basal growth conditions (J. Malakooti and K. Ramaswamy, unpublished data). This region contains the proximal PRE. To confirm whether the same proteins interact with both the distal and proximal PREs, competition experiments were performed with end-labeled probe containing the distal PRE and unlabeled oligonucleotides harboring the distal PRE (Fig. 4D, lane 2) or proximal PRE (Fig. 4D, lane 4) and control Sp1 oligonucleotide containing the Sp1 consensus sequence (Fig. 4D, lane 3). These studies revealed that the same proteins bind to both the distal and proximal PREs as both distal and proximal PREs competed out all DNA-protein complexes. Antibody supershift studies confirmed that these proteins include Sp1 (lane 5), Sp3 (lane 6), and Egr-1 (lane 7). As in Fig. 4A, Egr-1 antibody removed the faint broad band migrating just under NF-X.

Expression of Sp1 and Sp3 was not affected by PMA treatment. To determine whether the diminished interactions of Sp1 and Sp3 with the distal PRE was the result of decreased Sp1 expression or its reduced DNA binding activity, we investigated the kinetics of Sp1 expression by Western blot analysis. Nuclear extracts were obtained from untreated C2BBe1 cells or cells treated with PMA for a period of 1–16 h. Ten micrograms of nuclear proteins were subjected to SDS-PAGE, and, after electroblot analysis, Sp1 and Sp3 expression was detected with anti-Sp1 and anti-Sp3 antibodies. As shown in Fig. 5A, the expression levels of both Sp1 and Sp3 remained unaltered throughout of the time course of the PMA treatment period employed in this experiment. However, during the same PMA treatment regimen, Egr-1 expression was induced to maximal level by 2 h post-PMA treatment (Fig. 3B). Furthermore, to evaluate whether the DNA binding activity of Sp1/Sp3 was compromised as a consequence of PMA treatment or the status of serum in growth media, the DNA binding activities of Sp1 and Sp3 in nuclear extracts prepared from PMA-treated and untreated cells or nuclear proteins from cells grown media supplemented with 0.5% and 10% serum (Fig. 5B) were determined. No significant change in the DNA binding affinity of either Sp1 or Sp3 was observed in these studies. Thus these data demonstrate that PMA treatment of C2BBe1 cells does not affect the expression of Sp1 and Sp3 proteins or their DNA binding affinity.



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Fig. 5. Expression of Sp1 and Sp3 proteins and their DNA binding affinity are not affected by PMA. A: immunoblot. Confluent C2BBe1 cells were maintained in serum-reduced media for 24 h and then treated with PMA (100 nM) for the indicated periods of time, and nuclear extracts were prepared. Ten micrograms of nuclear proteins were resolved on an 8% SDS-PAGE, electroblotted onto Immobilon-P membranes, and detected with anti-Sp1 and anti-Sp3 antibodies as indicated. B: GMSA. Nuclear extracts were prepared from untreated or PMA-treated cells (as indicated on the top of the gels). A total of 5 µg of nuclear proteins was combined with double-stranded end-labeled probes containing the Sp1 consensus DNA binding site or proximal PRE and, after a 20-min incubation at room temperature, were resolved on 5% nondenaturing polyacrylamide gels.

 
Egr-1 binding to the distal PRE displaces prebound Sp1 and Sp3 transcription factors. To explore the mechanism of interaction of PMA-induced Egr-1 with the distal PRE, we studied the DNA binding affinity of overexpressed Egr-1 in competition assays. In these experiments, either nuclear proteins from untreated cells were combined with different concentrations of PMA-treated nuclear proteins as a source of Egr-1 (Fig. 6, lanes 1–3) or PMA-treated nuclear proteins were challenged with unlabeled oligonucleotides carrying the Egr-1 consensus sequence (Fig. 6, lanes 4 and 5). The results of these studies showed that the increasing concentration of PMA-induced Egr-1 leads to a reduction in binding of Sp1/Sp3 to the probe. In contrast, inhibition of the Egr-1 interaction with the probe results in Sp1/Sp3 DNA-protein complex formation. Therefore, these studies indicate that Egr-1 and Sp1 family members compete for binding to the overlapping Egr-1/Sp1 binding sites on the distal PRE.



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Fig. 6. PMA-induced Egr-1 interferes with the binding of Sp1 and Sp3 to the distal PRE. Nuclear proteins from untreated cells (lanes 1–3) were mixed with 2.5 and 5 µg of nuclear proteins from PMA-treated cells (lanes 2 and 3, respectively) and then coupled with the 32P-labeled distal PRE. In another experiment, nuclear proteins (5 µg) from PMA-treated cells were incubated with the radiolabeled probe (lane 4) or with a competing oligonucleotide containing the Egr-1 consensus sequence to block Egr-1 DNA binding activity before an incubation with the 32P-labeled distal PRE (lane 5). DNA-protein complexes were separated by polyacrylamide gel electrophoresis and visualized by autoradiography.

 
Evaluation of the functional importance of the distal PRE by mutational analysis. The distal PRE is composed of two overlapping Egr-1 binding sites that also overlap with an Sp1 binding sequence (Fig. 7A). To determine more precisely whether nuclear factors interacting with the overlapping Egr-1 or Sp1 binding sites are necessary for NHE2 gene activation in response to PMA treatment, mutations in the distal PRE were generated. In these mutants, the 5'- or 3'-Egr-1 consensus sequences of the distal PRE were disrupted by nucleotide substitutions, as shown in Fig. 7A.



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Fig. 7. Gel mobility shift experiments with the mutated distal PRE. A: nucleotide sequences of oligonucleotides containing mutations in the distal PRE (M1–M3) are shown, and the base substitutions are underlined. B: GMSA was performed using nuclear extracts purified from untreated cells and after incubation with the following end-labeled probes: distal PRE (lanes 1–7), M1 (lane 8), M2 (lane 9), and M3 (lane 10). Unlabeled competitor oligonucleotides are indicated at the top and were used at 100-fold molar excess. C: GMSA was performed using nuclear proteins from PMA-treated cells and after an incubation with the following end-labeled probes: distal PRE (lanes 1 and 2), M3 (lanes 3 and 4), M1 (lanes 5 and 6), and M2 (lanes 7 and 8). Anti-Egr-1 antibody was used in the supershift assays (lanes 2, 4, 6, and 8). Positions of the known DNA-protein complexes are shown on the left.

 
First, the mutated double-stranded oligonucleotides were used as competitors in GMSA to test whether they can compete with the formation of any of the DNA-protein complexes that were formed with nuclear proteins from untreated C2BBe1 cells and the wild-type probe (Fig. 7B). Oligonucleotide M1, which contains a three-nucleotide substitution in the 5'-Egr-1 binding site, competed out all DNA-protein complexes except for NF-X (Fig. 7B, lane 7). This suggested that the M1 mutant, which has lost the 5'-Egr-1 binding site, could interact with all proteins that bind to the wild-type probe except for NF-X. To test directly the ability of the M1 oligonucleotide to bind to these proteins, GMSA was performed with nuclear extracts prepared from untreated C2BBe1 cells and end-labeled M1 probe (Fig. 7B, lane 8). The results of this experiment indicated that the M1 probe can interact with Sp1 and Sp3 but not NF-X, confirming the data obtained from competition assays. In contrast, when the M2 oligonucleotide, which contains a 3-bp substitution in overlapping 3'-Egr-1 and Sp1 binding sites, was used as unlabeled competitor (Fig. 7B, lane 6), it was unable to compete with Sp1, Sp3, and NF-X. This indicated that the base substitutions in the M2 oligonucleotide resulted in not only disruption of the 3'-Egr-1 binding site but also eliminated the binding site for Sp1-related proteins. This was confirmed by using the M2 oligonucleotide as a labeled probe in GMSA with nuclear extract from C2BBe1 cells (Fig. 7B, lane 9), where only one DNA-protein complex was formed with the M2 probe that migrated slightly faster than NF-X band. This band comigrates with Egr-1 binding activity. The use of the M3 oligonucleotide as cold competitor resulted in diminished interactions of Sp1 and Sp3 with the probe (Fig. 7B, lane 5), suggesting that these transcription factors were still able to bind to this probe to some extent. DNA nucleotide sequences at the 3'-end of M3 (GGGAGGAGG) showed high degrees of similarities to the Sp1 consensus binding sequence (Fig. 7B, lane 10) and may be the sites of DNA-protein interactions. When nuclear proteins from PMA-treated cells were coupled with the end-labeled wild-type, M1, M2, and M3 probes, a single DNA-protein complex was generated with wild-type, M1, and M2 probes (Fig. 7C, lanes 1, 5, and 7), which was supershifted with anti-Egr-1 antibody (Fig. 7C, lanes 2, 6, and 8). The M3 probe did not interact with any protein from PMA-treated nuclear extracts (Fig. 7C, lanes 3 and 4). This confirmed that the wild-type distal PRE as well as M1 and M2 mutant oligonucleotides could bind to PMA-induced Egr-1 protein independently. However, the intensity of the complex formed with these probes was consistently in order of wild-type sequence > M2 > M1 (Fig. 7C), suggesting that in the absence of Egr-1 binding to the 3'-Egr-1 cis element, this nuclear factor has a higher affinity for interaction with the 5'-Egr-1 motif.

The distal PRE is essential for basal and PMA-induced NHE2 promoter activity. To further define the relative contribution of the components of the distal PRE to PMA-induced hNHE2 promoter activity, we introduced the nucleotide substitutions of the M1, M2, and M3 mutants (Fig. 7A) into the wild-type NHE2 promoter construct, p-345/+249. Because the Sp1 binding site overlaps with both Egr-1 sites, we were unable to generate a mutation in the Sp1 site without disrupting the integrity of Egr-1 binding sites. Wild-type and mutated constructs were transiently transfected into C2BBe1 cells, and reporter gene activity was measured and analyzed. Under nonstimulated conditions, the p-345/+249 construct displayed promoter activity that was comparable to that of p-415/+249 (Fig. 2) and increased threefold after PMA treatment (Fig. 8A). The basal expression of the promoter construct that contains the M1 mutation was not significantly different from that of the wild-type construct, whereas in PMA-stimulated conditions it showed a slight decrease in the luciferase activity. Base substitutions in the 3'-Egr-1 binding site, which also disrupted the overlapping Sp1 binding site, reduced the basal luciferase activity about 10% compared with the wild-type untreated construct, whereas the PMA-induced activity showed a 40–50% decrease compared with the PMA-treated wild-type plasmid. In contrast, mutations that disrupted the overlapping Egr-1 binding sites (M3) and eliminated binding of Egr-1 while allowing reduced levels of Sp1 interaction (Fig. 7) resulted in a 40% decrease in basal luciferase activity and abolished PMA-induced promoter activity. However, the extent of the increase for PMA-induced reporter activity was comparable in mutant M1 and M2 (2.4 and 2.1, respectively), and the M3 mutant exhibited a 1.3-fold increase in response to PMA. Our results showing that M3 mutation did not completely suppress PMA-induced reporter gene activity support the results of Fig. 4: that the proximal PRE is also involved in the PMA response, albeit to a much lesser extent. These data suggest that PMA-induced Egr-1 protein binding to either 5' or 3' binding sites may compensate to some extent for the lack of interactions with the other response element. A drastic decrease in luciferase activity with the M3 mutation suggests that binding of Egr-1 to these sites is necessary for maximal activation of the hNHE2 promoter in basal and stimulated conditions.



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Fig. 8. A: effect of PMA on reporter genes driven by the wild-type NHE2 promoter and constructs containing mutations in the distal PRE. C2BBe1 cells were seeded in 12-well plates and transfected the next day with 1.6 µg NHE2 promoter constructs in conjunction with 0.4 µg pSV-{beta}gal. Transfected cells were placed in serum-reduced media at least for 24 h before PMA treatment (100nM, 16 h) and harvested 48 h posttransfection, and cell lysates were prepared according to the instructions of Promega and processed as indicated in METHODS. Data are presented as luciferase activities of untreated cells transfected with the wild-type NHE2 promoter construct as 100%. B: Egr-1 transactivates the NHE2 promoter. C2BBe1 cells were cotransfected with 0.8 µg of NHE2 promoter-reporter construct (p-415/+249) and an equal amount of phEgr-1 expression vector or an empty vector. A total of 2 µg DNA was use for each transfection. pSV-{beta}gal was used as an internal control for transfection efficiency in all experiments. All transfections were done in triplicate and repeated at least 3 times. Data from 1 representative experiment are shown.

 
Egr-1 overexpression induces NHE2 promoter-reporter gene expression. To establish the transactivational importance of the Egr-1 transcription factor for regulation of NHE2 gene expression, we cotransfected the p-415/+249 NHE2 promoter-reporter construct with a human Egr-1 expression vector or a control empty vector into C2BBe1 cells. Indeed, as shown in Fig. 8B, cotransfection of the Egr-1 expression vector led to a twofold increase in luciferase activity compared with mock cotransfected cells, which showed no increase in reporter activity. These studies demonstrated that the expression of Egr-1 alone is sufficient to stimulate NHE2 promoter activity in C2BBe1 cells. However, when Egr-1 cotransfected cells were treated with PMA, luciferase activity in response to simultaneous overexpression of Egr-1 and the addition of PMA did not show a significant synergistic effect. This may suggest that the overexpressed level of Egr-1 in transfected cells is sufficient to transactivate the NHE2 promoter but not to the level of stimulation by PMA.


    DISCUSSION
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 ABSTRACT
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To analyze the regulatory mechanisms involved in the human NHE2 gene expression, we examined the promoter region of the gene. We (29) have previously shown that the NHE2 promoter contains neither a TATA nor a CAAT box (29). As typical of all other TATA-less and CAAT-less genes, the promoter region of NHE2 is highly GC rich and, in addition to other transcription factor binding sites, contains multiple Sp1 binding sites and GC boxes. These motifs are targeted by Sp1 transcription factor family members as well as by other transcription factors that interact with GC-rich sequences such as Krüppel-like proteins, MAZ, ZBP-89, G10BP-1, Egr-1, and WT1 (7, 44, 45, 50). We have characterized the region spanning these sites for their interactions with nuclear factors and their potential role in regulated expression of the NHE2 promoter. By GMSA and supershift experiments, we identified Sp1 and Sp3 as trans-acting factors that bind to the sequences immediately upstream from the NHE2 transcription initiation site.

The Sp1 transcription factor has been characterized as a ubiquitously expressed factor that regulates basal transcription activity; however, recent findings indicate that Sp1 binding and transactivation can be modulated by a variety of stimuli including various growth factors and hormones (34, 39). Sp1 is also implicated in tissue-specific (26, 57), developmental (32), and hormonal regulation of gene expression (39). The Sp1 family of transcription factors displays similar domain structures comprised of a COOH-terminal domain that contains three zinc fingers that is required for DNA binding and an NH2-terminus transactivation domain. Because the DNA binding domain is highly conserved among different family members, they interact with the same consensus sequence, and, as such, they may compete for the same binding site. Both Sp1 and Sp3 have been reported to be able to act as activators or repressors of transcription depending on their relative cellular environment (48). In cotransfection studies using Drosophila SL2 cells, we have shown that overexpression of both Sp1 and Sp3 exhibits positive effects on basal transcriptional activity of the hNHE2 promoter and that Sp3 consistently induces NHE2 promoter activity to a greater extent than that of Sp1 (unpublished data). This is in contrast to the effects of Sp1 and Sp3 on the rat NHE2 promoter, where a positive regulatory role Sp1 and an inhibitory role for Sp3 have been reported (5). In this study, we have shown that the level of Sp1 and Sp3 protein expression under the conditions used is not affected by PMA treatment in C2BBe1 cells. In addition, as a control for changes in DNA binding affinity of Sp1 and Sp3 upon PMA treatment, we showed that Sp1 and Sp3 in nuclear extracts prepared from untreated or PMA-treated cells bind to a commercially available oligonucleotide that contains an Sp1 consensus sequence with the same intensity.

We examined transcriptional regulatory mechanisms associated with the PMA-induced stimulation of hNHE2 expression. Acute PMA treatment has been shown to increase NHE2 exchanger activity (22, 35, 47). Using RT-PCR experiments, we demonstrated that PMA treatment leads to increased endogenous NHE2 mRNA expression. Experiments with actinomycin D and cycloheximide treatments established that hNHE2 mRNA stability is not affected by PMA treatment and enhanced mRNA expression is controlled at the transcriptional level and requires de novo protein synthesis.

Through 5'-deletion analysis of the hNHE2 promoter in transiently transfected C2BBe1 cells, two PREs were identified. These elements, which are comprised of overlapping Sp1/Egr-1 binding sites, were also reported in the promoter region of some PMA responsive genes (1, 12, 25, 45, 51). We showed by DNA binding experiments that in nuclear proteins from untreated cells, a probe (bp –345 to –319) spanning the distal PRE could serve as a binding site for transcription factors Sp1, Sp3, and an unknown nuclear factor that we termed NF-X. The NF-X DNA binding factor does not appear to be Sp1 related because it was not removed by a competitor oligonucleotide that contained the Sp1 consensus sequence, nor did specific antibodies against Sp1, Sp2, and Sp3 affect it. In addition, the mutant oligonucleotide M1 was capable of binding to Sp1 and Sp3 but not to NF-X. In the presence of M1 competitor, the oligonucleotide containing base substitutions in the 5'-Egr-1 binding site, NF-X was the only clearly visible factor that bound to the wild-type probe, suggesting that disruption of the 5'-Egr-1 binding site had also abolished the binding site for NF-X. Therefore, these two proteins may share an overlapping binding site. Several other zinc finger proteins have been shown to have similar binding site specificity for the GC box (48). The identity of NF-X at this time is unclear.

We found that, similar to endothelial and some other cell types (2, 33), the Egr-1 transcription factor is also highly induced in response to PMA treatment in C2BBe1 cells. An increased level of Egr-1 expression was observed by RT-PCR and Western blot analysis. Egr-1 is induced by a number of stimuli including mitogenic stimuli and signals for proliferation, development, and differentiation (17). Our data indicate that, although the level of protein expression and DNA binding activity of Sp1 family members are not affected by PMA treatment (they bind to the proximal PRE in PMA-treated nuclear extracts and the Sp1 consensus sequence with high affinity), Sp1 and Sp3 interaction with the distal PRE is minimal in the presence of PMA-induced Egr-1. Ackerman and colleagues (1) first reported competition between Sp1 and Egr-1 for the adenosine deaminase (ADA) gene promoter, where overexpression of Egr-1 resulted in displacement of Sp1 and repression of ADA expression. In contrast, an induction of tissue factor gene expression by PMA in Hela cells or by shear stress in epithelial or endothelial cells is mediated through induction of Egr-1 and subsequent displacement of Sp1 from the overlapping Egr-1/Sp1 site (28, 43). Similarly, Khachigian et al. (24) demonstrated that upon cell injury, newly synthesized Egr-1 displaces the prebound Sp1 factor and occupies the overlapping Sp1/Egr-1 binding site of the promoter, leading to induction of PDGF-B gene expression. Our DNA-protein binding experiments with the wild-type distal PRE probe revealed that binding of PMA-induced Egr-1 to the overlapping Egr-1/Sp1 site excludes interactions of both Sp1 and Sp3 with this sequence element, whereas, in nuclear proteins from untreated cells, both Sp1 and Sp3 interact with the same probe. We demonstrated that this binding was competitive as it resulted in a gradual reduction of Sp1/Sp3 interaction with the probe when cell extracts from PMA-treated cells were added to nuclear proteins from untreated cells. Moreover, when binding of PMA-induced Egr-1 to the probe was prevented by competition with the unlabeled Egr-1 consensus sequence, Sp1/Sp3 bound to the probe (Fig. 6). Thus these data confirmed that PMA treatment does not interfere with the DNA binding affinity of Sp1/Sp3 and suggests that conditions that result in higher levels of Egr-1 expression, such as PMA treatment, may prevent simultaneous interaction of Egr-1, Sp1, and Sp3 with the NHE2 promoter at this region. This potentially could be not only due to an increase in the concentration but also an increase in DNA binding affinity of Egr-1 resulting in this transcription factor to be the predominant DNA binding protein that interacts with the overlapping Egr-1/Sp1 binding site at this location. Therefore, taking these results into consideration, a mechanism similar to PDGF-A and tissue factor appears to be responsible for hNHE2 expression in response to PMA.

Sp1 expression has also been reported to be upregulated in response to PMA (6, 13). For example, Tanaka et al. (49) reported that Sp1 mRNA and protein expression were upregulated by PMA in calf pulmonary artery endothelial cells, leading to an inducible expression of Mn-SOD; however, Sp1 expression increased gradually and was maximum after 6–12 h of PMA treatment. In the same study, Egr-1 was induced both at mRNA and protein levels at a much earlier time and had no effect on Mn-SOD promoter activity. We showed that at the time intervals tested, Sp1 expression or DNA binding activity did not differ significantly between untreated and PMA-treated C2BBe1 cells. However, we cannot rule out the possibility of early activation of Sp1 (within 0–30 min) and its indirect effect on PMA-induced NHE2 mRNA expression by affecting processes such as overexpression of Egr-1, as the Egr-1 promoter is regulated by Sp1.

Immediate-early genes such as c-fos, c-jun, and Egr-1 have been described as "third messengers" that act as connectors between signal transduction pathways and downstream target genes and, as such, propagate the response to various stimuli. Recent studies have also shown that infection of human epithelial cells with enteropathogenic Escherichia coli (EPEC), which is a major cause if infantile diarrhea, results in activation of extracellular signal-regulated kinases leading to the induction of a number of genes including the transcription factor Egr-1 (14). Although the changes in signaling pathways and their subsequent effects on the expression of the target genes may contribute to the onset of diarrhea, Egr-1 target genes in intestinal epithelial cells have not yet been identified, and the functional consequence of Egr-1 induction in response to EPEC infection are not clear. Because NHE2 and NHE3 genes are expressed in human intestinal epithelial cells, our finding that hNHE2 and hNHE3 (J. Malakooti and K. Ramaswamy, unpublished data) are direct targets of Egr-1 is of interest and may aid in the determination of the possible contribution of these isoforms to diarrhea associated with EPEC infection. We (18) have recently shown that acute EPEC infection in Caco-2 cells leads to an increase in NHE2 and a reduction in NHE3 transport activities. Further investigations need to be carried out to understand how these alterations can cause the changes in sodium and fluid transport in EPEC infection.

In summary, the data presented in this study show that the expression of the hNHE2 gene in response to PMA is regulated in part at the transcriptional level. In addition, our results define a potential mechanism for the PMA-induced upregulation of hNHE2 expression in the intestinal epithelial cell line C2BBe1 and identify cis elements in the NHE2 promoter region that may mediate the PMA response. We demonstrated that the Egr-1 transcription factor is induced by PMA in C2BBe1 cells and binds to PREs in the NHE2 promoter. In PMA-treated cells, NHE2 mRNA expression showed a delay in transcription activation, which may suggest a requirement for Egr-1 mRNA and protein synthesis and activation before NHE2 expression. Furthermore, cotransfection experiments with an Egr-1 expression vector led to transactivation of hNHE2 promoter-driven reporter gene activity in the C2BBe1 cell line. Taken together, our results suggest that transcription factor Egr-1 may be involved in regulated expression of the hNHE2 gene in C2BBe1 cells.


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 ABSTRACT
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33349 and DK-67990 and by the Department of Veterans Affairs.


    ACKNOWLEDGMENTS
 
Present address of V. C. Memark: Stritch School of Medicine, Loyola University-Chicago, Maywood, IL 60153.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Malakooti, Univ. of Illinois-Chicago, Dept. of Medicine, Section of Digestive and Liver Diseases, 840 S. Wood St., Chicago, IL 60612 (e-mail: malakoot{at}uic.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.


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