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Am J Physiol Gastrointest Liver Physiol 293: G146-G153, 2007. First published March 22, 2007; doi:10.1152/ajpgi.00443.2006
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MUCOSAL BIOLOGY

Sp1 and Sp3 mediate NHE2 gene transcription in the intestinal epithelial cells

Ping Hua, Hua Xu, Jennifer K. Uno, Maciej A. Lipko, Jiali Dong, Pawel R. Kiela, and Fayez K. Ghishan

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

Submitted 26 September 2006 ; accepted in final form 7 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our previous studies have identified a minimal Sp1-driven promoter region (nt –36/+116) directing NHE2 expression in mouse renal epithelial cells. However, this minimal promoter region was not sufficient to support active transcription of NHE2 gene in the intestinal epithelial cells, suggesting the need for additional upstream regulatory elements. In the present study, we used nontransformed rat intestinal epithelial (RIE) cells as a model to identify the minimal promoter region and transcription factors necessary for the basal transcription of rat NHE2 gene in the intestinal epithelial cells. We identified a region within the rat NHE2 gene promoter located within nt –67/–43 upstream of transcription initiation site as indispensable for the promoter function in intestinal epithelial cells. Mutations at nt –56/–51 not only abolished the DNA-protein interaction in this region, but also completely abolished NHE2 gene promoter activity in RIE cells. Supershift assays revealed that Sp1 and Sp3 interact with this promoter region, but, contrary to the minimal promoter indispensable for renal expression of NHE2, both transcription factors expressed individually in Drosophila SL2 cells activated rat NHE2 gene promoter. Moreover, Sp1 was a weaker transactivator and when coexpressed in SL2 cells it reduced Sp3-mediated NHE2 basal promoter activity. Furthermore, DNase I footprinting confirmed that nt –58/–51 is protected by nuclear protein from RIE cells. We conclude that the mechanism of basal control of rat NHE2 gene promoter activity is different in the renal and intestinal epithelium, with Sp3 being the major transcriptional activator of NHE2 gene transcription in the intestinal epithelial cells.

Sp1; Sp3; NHE2 gene; intestine; epithelium

SODIUM-HYDROGEN EXCHANGERS (NHEs) are integral transmembrane proteins that exchange extracellular Na+ for intracellular H+ and are present in all mammalian cells. They have multiple cellular functions including intracellular pH homeostasis, cell volume regulation, and electroneutral NaCl absorption in epithelia (50). Among the nine Na+/H+ exchanger isoforms identified so far, NHE2, together with NHE3 and NHE8, is present on the apical membrane of epithelial cells of the small and large intestine. They all contribute to electroneutral sodium absorption (10, 46, 51). NHE2 is regulated by several factors including growth factors/protein kinase (34, 47), interferon-{gamma} (35), and other chronic extracellular stimuli (31, 33). NHE2 homozygous knockout mice exhibited marked alterations in gastric mucosal histology and function. NHE2–/– mice demonstrate a loss of net acid secretion coinciding with a reduced number of parietal and chief cells (36). The reduction of the number of zymogenic cells may be secondary to decreased viability of parietal cells, which although they develop normally undergo premature necrosis (36). This process is accompanied by progressive inflammation in the form of diffuse corporal gastritis that ranges from transmural neutrophilic infiltration to a profound atrophy consistent with chronic achlorhydria and is dependent on age and stage of inflammation (7). Although there is no apparent absorptive defect in adult NHE2–/– mice indicating that NHE2 may not play an important role in Na+ absorption (20, 36), our earlier observations suggested that in suckling rats NHE2 activity contributes as much as 41% of small intestinal Na+/H+ exchange (12). Recent studies show that NHE2 is the predominant NHE isoform found in murine colonic crypts. Moreover, lack of NHE2 expression is compensated by an alternative non-NHE3- or NHE1-dependent pathway (2, 22), thus raising a possibility that the role of NHE2 in the intestinal tract may have been underestimated.

Both rat and human Slc9a2 gene promoters have been cloned in an effort to understand the molecular mechanisms directing NHE2 gene transcription (30, 32). We have previously identified novel cis elements involved in transcriptional activation of NHE2 gene in response to osmotic stress in mouse inner medullary collecting duct cells (mIMCD3) (3) and we described the Sp-dependent mechanisms controlling basal activity of rat NHE2 gene promoter in renal epithelial cells (4).

Sp1 is one of the most well-characterized transcriptional activators and was originally identified to bind to and activate GC-rich promoter elements that are necessary for the regulation and expression of a variety of genes (18, 21). Sp1 is a founder member of the Sp transcription factor family, which also includes Sp2, Sp3, Sp4 (39), and Kruppel-like factors (KLFs) (9, 15, 41, 42). The Sp transcription factor family plays important roles in cell cycle regulation, hormonal activation, and development patterning (1, 14, 16, 44). Structurally, Sp transcription factors contain a conserved DNA binding domain composed of three zinc fingers close to the COOH terminus. The NH2-terminal regions of these proteins contain specific activation and repression domains that are capable of interacting with a variety of corepressors and/or coactivators, thus eliciting distinct transcriptional regulation (39). Thus far, Sp1 and Sp4 have been shown to be stimulatory (13, 23), whereas Sp3 acts as a bifunctional transcription factor that exerts additive or synergistic effects on gene activation or represses transcription driven by Sp1 or other transcription factors (5, 29). Consistent with these described roles of Sp1 and Sp3, the minimal promoter of the rat NHE2 gene identified in renal epithelial cells (nt –36 to +2) was activated by Sp1 and repressed by Sp3 (4). Interestingly, this promoter fragment was not sufficient to activate transcription in rat intestinal epithelial (RIE) cells (47) or Caco-2 cells (unpublished observations). This suggested a differential mechanism of transcriptional regulation of NHE2 gene expression in renal and intestinal epithelia, perhaps involving other upstream cis elements and/or other transcription factors. Therefore, the present study was conducted to identify the transcriptional mechanism of basal NHE2 gene expression in the intestinal epithelial cells and to determine the involvement of the Sp nuclear proteins. We found that the GC-rich region of the NHE2 promoter between nt –69 and nt –43 is an important determinant of the NHE2 promoter activity in the nontransformed RIE. Contrary to renal epithelial cells, we found that both Sp1 and Sp3 are important activatory factors interacting with the identified promoter region and regulating basal activity of NHE2 gene promoter. Moreover, Sp1 is a weaker transactivator than Sp3 and it can downregulate Sp3-mediated activity of NHE2 promoter, presumably by directly competing for the binding site. The described mechanism of transcriptional control of NHE2 gene expression underscores the differences in regulation of this transporter in the renal and intestinal epithelia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. RIE cells were a gift from Dr. Raymond Dubois (Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN). This cell line, originally described by Blay and Brown (6), represent nontransformed cells derived from rat intestinal epithelium; they have normal rat diploid number of chromosomes and provide a very good model of rat small intestinal epithelial cells. Endogenous expression of NHE1 and NHE2 has been described in RIE cells (47). They were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured at 37°C in a 95% air-5% CO2 atmosphere and passaged every 48–72 h. Media and other reagents used for cell culture were purchased from Irvine Scientific (Irvine, CA). Cells used for all experiments were from passages 6 to 13. Drosophila SL2 cells were purchased from American Type Culture Collection (Rockville, MD) and were maintained at room temperature in Schneider cell culture medium (Invitrogen, Carlsbad, CA) supplemented with 10% FCS and penicillin-streptomycin as above.

Plasmid constructs. Firefly luciferase reporter vector (pGL3/basic) and promoterless plasmid with Renilla luciferase (pRL-null) were purchased from Promega (Madison, WI). beta-Galactosidase reporter vector (pbetaGal/basic) was purchased from Clontech (Palo Alto, CA). pAc5.1A vector was purchased from Invitrogen. Drosophila expression vectors for Sp transcription factors (pPacSp1 and pPacUSp3) were generous gifts from Drs. Tjian and Suske, respectively.

A series of progressively shortened rat NHE2 promoter constructs was made by PCR and restriction enzyme digestion with GC-RICH PCR System (Roche Diagnostics, Basel, Switzerland) or Advantage-GC2 polymerase Mix kit (Clontech). Briefly, pGL3/–85, pGL3/–69, pGL3/–43, and pGL3/–36 were PCR amplified from the promoter construct pGL3/–110, by using primers with overhanging restriction enzyme sites for MluI and NcoI, and ligated into MluI/NcoI-digested pGL3/basic. Mutations in the rat NHE2 gene promoter were introduced by PCR-based site-directed mutagenesis with primers containing mutated base pairs. All promoter constructs ended at nt +116 relative to the transcription initiation site and were confirmed by automated DNA sequencing.

Transient transfection and reporter gene analysis. Cells were seeded in 24-well plates, and transfection was performed with Effectene (Qiagen, Valencia, CA) according to the manufacturer's protocol. For RIE cells, 0.2 µg of NHE2 gene promoter construct was cotransfected along with 20 ng of pRL-null plasmid (used as an internal control to normalize for transfection efficiency). Eighteen hours after transfection, the medium was removed and replaced with standard medium with 10% FBS. At 48 h after transfection, cells were harvested for reporter gene assay by use of the Dual Luciferase Assay kit (Promega). Luciferase activities were measured with a tube luminometer (Femtomaster FB 12, Berthold Detection System, Pforzheim, Germany) and expressed as normalized luciferase activity relative to the promoterless pGL3-basic vector.

Drosophila SL2 cells were seeded at 0.5 x 106 cells/well in 24-well plates and 24 h later were cotransfected with 0.2 µg of promoter constructs (pbetaGal/basic or pbetaGal/–69) and various amounts of pPacSp1 and pPacUSp3 (5, 10, 25, 50, 100, 150, and 200 ng). Cells were harvested 24 h after transfection. Cell were lysed in 100 µl of lysis solution and assayed for beta-galactosidase activity with Galacto-Star beta-galactosidase reporter gene assay system for mammalian cells (Applied Biosystems, Foster City, CA). beta-Galactosidase activity was normalized to protein concentration as determined with BCA protein assay kit (Pierce, Rockford, IL).

Nuclear extracts preparation and gel mobility shift assay. Nuclear extracts were prepared from RIE cells by a previously described method (48). Synthetic double-stranded oligonucleotides were designed to span the promoter region from nt –67 to nt –43. Oligonucleotides were labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase and purified on a microspin G-25 column (GE Healthcare, Piscataway, NJ), and 5 µg of nuclear proteins extracted from RIE cells were incubated for 20 min at 25°C with 32P-labeled oligonucleotide probes. Binding reactions were carried out in a total volume of 20 µl containing 10 mM HEPES (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 50 µg/ml poly d(I-C), and 1 ng of the labeled probe. After incubation, 2 µl of 0.1% bromophenol blue was added and the DNA-protein complexes were separated in nondenaturing 6% polyacrylamide gels in 0.5 x TBE buffer. Gels were subsequently dried and exposed to X-ray film. For competition experiments, 100-fold molar excess of unlabeled oligos was added to the reaction mixture before addition of the radiolabeled probe. For supershift assays, 4 µg of Sp1 or Sp3 antibody or an equivalent amount of nonspecific rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was incubated with the binding reactions at room temperature for 20 min before the addition of the labeled probe.

DNase I footprinting assay. DNase I footprinting was performed by a previously described method (49). Briefly, the DNA protein interaction reaction was performed at room temperature for 20 min by mixing 25 µg nuclear proteins with 100 ng pGL3/–69 plasmid DNA in gel mobility shift assay (GMSA) binding buffer. DNase I digestion was then performed according to the protocol from the Core Footprinting System (Promega). Primer extension was then used to amplify the DNA footprinting products. DNA sequencing was performed by using fmol DNA cycle sequencing system (Promega). pGL3/–69 plasmid DNA was used as the template for DNA sequencing. RV3 primer was used for both DNA footprinting and sequencing reactions. The RV3 primer is located at the 5' end of the subcloning site in the pGL3-Basic vector.

Statistical analysis. ANOVA and Fisher protected least significant differences post hoc test (StatView 5.0.1; SAS Institute, Cary, NC) were used to statistically compare the experimental data. P values of <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of the minimal promoter region of the rat NHE2 gene in the intestinal epithelial cells. Our previous studies identified a minimal Sp1-driven promoter fragment (–36/+116 nt) directing high expression of NHE2 in mouse renal epithelial cells mIMCD-3 (4). However, this minimal promoter region was not sufficient to drive active transcription of the reporter gene in RIE cells or in human colonic Caco-2 cells. To locate the minimal promoter region necessary for the basal activation of NHE2 gene expression in RIE cells, five promoter constructs containing various lengths of the rat NHE2 promoter (pGL3/–110, pGL3/–85, pGL3/–69, pGL3/–43, and pGL3/–36) were transiently transfected into RIE cells for reporter gene assay. As shown in Fig. 1, pGL3/–43 and pGL3/–36 were unable to drive NHE2 promoter activity. However, construct extending to –69 nt demonstrated significant promoter activity with no further increase in longer promoter constructs extending 5' to –85 or –110 nt. These results suggest that the GC-rich promoter region between nucleotides –69 and –43 is critical for the activation of NHE2 gene expression in intestinal epithelial cells.


Figure 1
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  		Fig. 1. Identification of the minimal promoter region of rat NHE2 gene in rat intestinal epithelial (RIE) cells. Firefly luciferase reporter constructs containing various lengths of the rat NHE2 promoter were cotransfected into RIE cells along with a promoterless pRL-null plasmid. Firefly and Renilla luciferase activities were measured 48 h after transfection. Firefly luciferase activity was normalized to the activity of Renilla luciferase and expressed as normalized luciferase activity relative to the promoterless pGL3-basic vector. The results are means ± SE from at least 3 independent experiments. *P < 0.001 for constructs pGL3/–110, –85, –69 vs. constructs pGL3/–36 and PGL3/–43.

 
Identification of the DNA-protein interactions within the –69/–43 nt of rat NHE2 gene promoter. To identify the cis element(s) involved in basal NHE2 gene transcription in intestinal epithelial cells, nuclear protein was extracted from RIE cells and GMSA were performed to detect protein-DNA interactions. Figure 2 depicts a prediction analysis of transcription factor binding sites identified with TRANSFAC 7.0 public database of eukaryotic transcription factors and a competition GMSA assay with progressive scanning mutations within this region. Compared with unlabeled wild-type probe (wt), mutated probes M2–M5 lost their ability to compete for binding to nuclear protein, suggesting a central role of the region located between –60 and –52 nt of the NHE2 gene promoter.


Figure 2
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  		Fig. 2. Prediction analysis and competition gel mobility shift assay (GMSA) for DNA-protein interactions within the –67/–43 nt promoter region. 32P-labeled probes covering the –67/–43 nt NHE2 promoter region were incubated with 5 µg of nuclear extract from RIE cells in the presence or absence of 100-fold molar excess of unlabeled wild-type (wt) probe or scanning mutant oligos (M1–M6). Bold characters indicate the mutated regions. KLF, Kruppel-like factors; NE, nuclear extracts from RIE cells; Comp, competitor.

 
In subsequent experiments, we targeted the four putative core binding sites for Sp transcription factors, arbitrarily named SpA through SpD. Figure 3A depicts a competition assay in which binding to the wild-type –67/–43 nt probe was effectively competed by an excess of unlabeled probe (wt), a consensus Sp binding site (Sp), and probes with sites A and D mutated. Mutation of the centrally positioned sites B and C partially eliminated the competition, whereas mutation in probe SpCf, which could be considered as targeting both closely spaced sites SpB and SpC, or flanking sites of site SpC, completely eliminated the ability of the probe to compete for binding (Fig. 3A). Consistent with the central role of site SpC in binding nuclear protein, GMSA assay with radiolabeled wild-type or mutant probes demonstrated a complete loss of binding for probe with mutated site SpC (Fig. 3B). Partial loss of binding capacity was observed for probes with mutated sites B and D, which, because of close spatial arrangement, may represent the effect of mutated flanking sites of the site SpC. When mutation Sp3f was introduced into the –69/+116 NHE2 reporter gene construct, it resulted in a complete loss of the promoter activity (Fig. 3C). Collectively, the data obtained from gel shift analysis strongly indicates that the core element SpC and its flanking sites (nt –56/–51) are critical for the activity of NHE2 promoter in the intestinal epithelial cells.


Figure 3
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  		Fig. 3. Mutational analysis of 4 putative Sp core binding sites within the –67/–43 nt promoter region. A: 32P-labeled probe covering –67/–43 nt NHE2 promoter region were incubated with 5 µg of nuclear extract from RIE cells in the presence or absence of 100-fold molar excess of unlabeled wt probe, Sp binding consensus sequence (Sp), oligos mutated to eliminate core Sp sites SpA, SpB, SpC, and SpD, or with mutated SpC flanking sites (SpCf). Bold characters indicate the mutated regions. B: GMSA binding reactions were performed with 32P-labeled probes: wt, and probes with mutated putative core binding sites SpA, B, C, and D. C: NHE2 gene promoter construct pGL3/–69 and a construct mutated as in SpCf were cotransfected with pRL-null plasmid, and promoter reporter gene assay was performed 48 h after transfection. Firefly luciferase activity was normalized to the activity of Renilla luciferase and expressed as normalized luciferase activity relative to the promoterless pGL3-basic vector. Results are means ± SE from at least 3 independent experiments. *Significant difference (P < 0.01) between these 2 constructs.

 
Identification of nuclear proteins involved in NHE2 promoter activity in RIE cells. Since the core element identified by GMSA analysis and functional studies bears typical features of a GC element, or a putative binding site for Sp family of transcription factors, control IgG, anti-Sp1 and or anti-Sp3 antibodies were included in the GMSA binding reactions. As shown in Fig. 4, the addition of anti-Sp1 antibody resulted in supershifting of the upper protein-DNA complex, consistent with Sp1-DNA binding. Addition of anti-Sp3 antibody produced a similar supershifted complex, and increased the intensity of the upper complex consistent with supershifting of the complexes with the lower molecular weight isoforms of Sp3 (Fig. 4). These experiments provide evidence, that both Sp1 and Sp3 transcription factors bind to –67/–43 nt region of the NHE2 gene promoter in intestinal epithelial cells.


Figure 4
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  		Fig. 4. Interactions between NHE2 gene promoter and Sp1/Sp3 transcription factors. 32P-labeled probes covering the minimal promoter region nt –67/–43 were incubated with 5 µg of nuclear extract from RIE cells in the presence of 4 µg of control immunoglobulins (IgG), anti-Sp1 ({alpha}Sp1), or anti-Sp3 ({alpha}Sp3) antibody (Ab). SS, supershifted DNA-protein complexes.

 
Functional analysis of the involvement of Sp1 and Sp3 in the basal NHE2 promoter activity. To evaluate the role of Sp1 and Sp3 in the regulation of NHE2 gene promoter activity, we cotransfected rat NHE2 gene minimal promoter construct pGal/–69 with Drosophila expression vectors pPacSp1 and/or pPacUSp3 into Sp-deficient Drosophila SL2 cells. As shown in Fig. 5, pGal/–69 alone was unable to drive reporter gene (beta-galactosidase) expression. Whereas previously obtained data with the shorter minimal promoter (–36/+116 nt) identified in renal epithelial cells (4) showed that only Sp1 activated reporter gene expression in similarly designed experiments, the minimal 5'-flanking sequence indispensable for NHE2 promoter activity on intestinal cells (–69/+116 nt) was strongly and dose dependently activated by both Sp1 (Fig. 5A) and Sp3 (Fig. 5B). Since Sp3 has been demonstrated to act as an inhibitor of Sp1-driven transcription of the short (–36/+116 nt) renal NHE2 gene promoter (4), we studied the functional interaction between Sp1 and Sp3 in regulating the activity of –69/+116 nt of NHE2 gene promoter. We cotransfected varying amounts of Sp1 and Sp3 expression plasmids with pbetaGal/–69 into Drosophila SL2 cells, and the obtained results were in striking contrast with those obtained with the minimal renal NHE2 promoter. In the presence of fixed amount of Sp3 expression plasmid (10 ng), Sp3-mediated NHE2 basal promoter activity was dose dependently inhibited with increasing amounts of cotransfected Sp1 (Fig. 6). Conversely, in the presence of a fixed amount of Sp1 expression plasmid (10 ng), the cotransfection with Sp3 further and dose dependently increased NHE2 promoter activity with all doses (Fig. 7).


Figure 5
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  		Fig. 5. Transactivation of basal intestinal NHE2 promoter by Sp1 and Sp3 in Drosophila SL2 cells. The NHE2 gene promoter construct pbetaGal/–69 was cotransfected with increasing amounts of Sp1 (A) or Sp3 (B) Drosophila expression plasmid into SL2 cells. beta-Galactosidase activity was assayed 24 h after transfection and was normalized by the protein concentration. Data are presented as means ± SE from at least 3 independent experiments. Statistical difference at P < 0.05.

 

Figure 6
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  		Fig. 6. Effect of Sp1 on Sp3-mediated activity of basal intestinal NHE2 promoter in Drosophila SL2 cells. NHE2 gene promoter construct pbetaGal/–69 was cotransfected with 10 ng of pPacUSp3 and with increasing amounts of pPacSp1. beta-Galactosidase activity was assayed 24 h after transfection and was normalized by the protein concentration. Data are presented as means ± SE from at least 3 independent experiments. Different symbols next to bars indicate statistical difference at P < 0.05.

 

Figure 7
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  		Fig. 7. Effect of Sp3 on Sp1-mediated activity of basal intestinal NHE2 promoter in Drosophila SL2 cells. NHE2 gene promoter construct pbetaGal/–69 was cotransfected with 10 ng of pPacSp1 and with increasing amounts of pPacUSp3. beta-Galactosidase activity was after transfection and was normalized by the protein concentration. Data are presented as means ± SE from at least 3 independent experiments. Different symbols next to bars indicate statistical difference at P < 0.05.

 
Confirmation of the protein-DNA interaction by DNase I footprinting. To further confirm the DNA-protein interaction region on the basal promoter of the rat NHE2 gene in the intestinal epithelial cells, DNase I footprinting assay was performed. As shown in Fig. 8, a region located at the –69/–43 nt of rat NHE2 promoter was protected by nuclear proteins isolated from RIE cells. This region was identified as the GCCCCGCC region (–58/–51 bp), which correlated with the region identified earlier by GMSA.


Figure 8
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  		Fig. 8. DNase I footprinting analysis of DNA-protein interactions within the proximal region of the rat NHE2 gene. A DNA-protein interaction reaction was performed at room temperature for 20 min in the presence of 20 µg of nuclear protein and 100 ng of pGL3/–69 plasmid DNA. DNase I digestion was performed after the binding reaction. Primer extension was performed to amplify DNase I footprinting products. A DNA sequencing reaction was performed to generate a sequencing ladder from pGL3/–69 plasmid DNA. The primers used for both DNase I footprinting and the DNA sequencing reaction are RV3 primer, which is located upstream of the subcloning site on pGL3-Basic vector. Protected regions are shown in sidelined sequences. G and C, sequencing reaction products.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our previous work has shown that the minimal promoter region required for activating rat NHE2 gene expression in mouse renal epithelial cells (mIMCD3) was located within 36 bp upstream of the transcription initiation site and that Sp1 was the main activator of this promoter, with Sp3 acting to inhibit Sp1-driven reporter gene expression (4). This promoter region, however, could not drive reporter gene expression in RIE (47) or Caco-2 cells (unpublished observations). In the present study, we further analyzed transcriptional regulation of the NHE2 gene and identified a minimal promoter region (–69/+116 nt) that activates NHE2 gene expression in intestinal epithelial cells.

To further focus on the region located upstream of the renal minimal promoter, GMSA identified DNA-protein interactions between nt –67 and nt –43 in this promoter region. Prediction analysis of the –67/–43 nt promoter region revealed the presence of four very closely spaced putative Sp binding sites (Fig. 2). A combination of GMSA, mutational, and functional analyses indicated that within this section the core element SpC and its flanking sites (nt –56/–51) are critical for the activity of NHE2 promoter in the intestinal epithelial cells. This putative Sp binding site is located on the complementary strand and has core and matrix similarity scores of 1.0 and 0.895, respectively. Mutations disrupting this site, particularly M3–M5 (Fig. 2), SpCm and SpCf (Fig. 3, A and B) reduced binding of nuclear protein to the wild-type –67/–43 nt probe, and mutation SpCf (CCCGCC->TTCGAA, –56/–51 nt) completely abrogated the activity of the minimal NHE2 promoter (–69/+116 nt) necessary for basal reporter gene expression in intestinal epithelial cells (Fig. 3C). We have further demonstrated that –67/–43 nt promoter region binds both Sp1 and Sp3 transcription factors (Fig. 4) and that this binding translates into dose-dependent activation of NHE2 promoter activity as demonstrated in transiently transfected SL-2 cells (Fig. 5). Further evidences provided by DNase I footprinting indicated that a GCCCCGCC region (–58/–51 bp) at the rat NHE2 promoter was protected by nuclear proteins isolated from RIE cells (Fig. 8) but was not protected by nuclear proteins isolated from kidney cells (4).

The obtained data established, therefore, that the more proximal Sp binding site identified as indispensable for NHE2 promoter activity in renal epithelium (nt –36/–25) is not utilized in intestinal epithelial cells. Moreover, in contrast to minimal renal NHE2 promoter, which was driven by Sp1 and inhibited by coexpressed Sp3, the 5'-flanking region of the NHE2 gene necessary for intestinal expression (–69/+116 nt) was stimulated by both Sp1 and Sp3 and seemingly to a greater extent by the latter transcription factor (Fig. 5). The weaker transactivation by Sp1 was particularly apparent when Sp3 was coexpressed with increasing amounts of Sp1 in SL-2 cells, which translated into a dose-dependent inhibition of Sp3-driven NHE2 promoter activity, presumably by a direct competition for their cognate cis element(s) (Fig. 6). It is important to note that the –69/+116 nt construct transfected into SL-2 cells contained Sp sites relevant for basal activity of NHE2 promoter in both renal and intestinal cells, and the observed reporter gene expression may reflect a combined effect of Sp transcription factors at both cis elements. However, contrary to the inhibitory effects of Sp3 on minimal renal NHE2 promoter in SL-2 cells, we observed positive additive effect of Sp3 in Sp1-driven expression of the reporter gene (Fig. 7), which suggests that in the context of a longer promoter construct required for intestinal epithelial gene expression the upstream Sp site (–58/–51 bp) plays a dominant role. Therefore, these observations allow us to conclude that NHE2 basal transcriptional activity in renal and intestinal epithelial cells is regulated by differential utilization of Sp binding sites and by different effects of Sp1 and Sp3.

GC/GT boxes are common regulatory elements found in promoters and are often involved in the activation and/or repression of gene expression and are associated with enhancers of housekeeping genes as well as tissue-specific genes (25, 37). A number of transcription factors have been identified to interact with these elements to either activate or repress gene expression (27, 41, 42). Sp1 family members and their structurally related relatives, KLF, are well documented to bind to GC-rich regions with their highly conserved COOH-terminal zinc finger regions (28, 41, 42). Sp3, one of the Sp-family members, recognizes GC-box virtually identical to the sequence of Sp1 binding site due to the structural similarity. Sp3 has been shown to act as an inhibitory transcription factor in Sp1-mediated transcription activation (4, 26, 40). Both Sp1 and Sp3 are ubiquitously expressed and their participation in tissue specific or lineage-specific gene expression is seemingly paradoxical. A number of phenomena could be attributed to this mechanism, including expression level and especially the ratio of Sp1 and Sp3 proteins, their posttranslational modifications, modification of their binding sites (e.g., methylation of CpG rich regions), and cooperative interactions with other transcription factors. High Sp1-to-Sp3 ratio was proposed to participate in endothelium-specific gene expression (24), and changes in this ratio in favor of Sp1 was implicated in induction of target genes in hypoxic myocytes (17). Posttranslation modifications of Sp1 and Sp3 proteins, such as phosphorylation (11), acetylation (8, 45), and sumoylation (19, 38, 43) may also participate in Sp-mediated tissue-specific gene expression. The effects of these modifications are not, however, easily classified and may result in stimulation or inhibition of their transactivation potential, dependent on the site of modification, gene of interest, and cell lineage. Which of these phenomena plays a role in the tissue-specific regulation of the basal NHE2 gene expression, particularly in the renal and intestinal epithelium, remains to be determined. On the basis of the data presented here and in an earlier report (4), and as proposed in a cartoon model depicted in Fig. 9, in renal epithelial cells an Sp binding site located at nt –36/–25 is utilized by Sp1 to drive basal levels of NHE2 expression and Sp3 acts as a transcriptional inhibitor by competing with Sp1 to inhibit the formation or recruitment of the transcriptional initiation complex. The more upstream element located at nt 56/–51 is neither required nor additive in renal epithelial cells (4). This element and not the proximal Sp site, however, is indispensable for NHE2 promoter activity in intestinal epithelial cells and is utilized by both Sp1 and Sp3 to promote gene transcription. Sp1, as a weaker transactivator, inhibits Sp3-driven transcription, presumably by direct competition for their common binding site.


Figure 9
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  		Fig. 9. Model utilization of GC boxes and interactions between Sp1 and Sp3 transcription factors within the proximal NHE2 gene promoter in transcription in renal (A) and intestinal (B) epithelial cells. Crossed boxed depict sites not utilized in respective epithelial cells. Different-sized arrows above the transcription initiation site symbolize transcription rate as influenced by the interacting Sp1 and Sp3 proteins at their cognate cis elements.

 
In summary, this and our previous studies (4) demonstrate differential utilization of GC boxes and regulatory potential of Sp1 and Sp3 transcription factors in the proximal rat NHE2 gene promoter in the renal and intestinal epithelial cells. These findings not only add to our understanding of basal and possibly regulated expression of NHE2 gene in the kidney and gastrointestinal tract but also highlight fundamental differences in transcriptional mechanisms governing tissue-specific regulation of gene expression on the basis of a limited number of coexpressed transcription factors in various epithelia.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This investigation was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5R01-DK41274.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. K. Ghishan, Dept. of Pediatrics, Steele Memorial Children's Research 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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