Promoter analysis of the gene encoding the beta -subunit of the rat amiloride-sensitive epithelial sodium channel

Harry Robert Bremner1, Tanya Freywald1, Hugh M. O'Brodovich1,2,3, and Gail Otulakowski1,2

1 Canadian Institutes of Health Research Group in Lung Development, Lung Biology Programme, Research Institute, Hospital for Sick Children, and Departments of 2 Paediatrics and 3 Physiology, University of Toronto, Toronto, Ontario M5G 1X8, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The amiloride-sensitive epithelial Na+ channel (ENaC), found in the apical membrane of Na+-absorptive epithelia, is made up of three differentially regulated subunits: alpha , beta , and gamma . We undertook a study of the 5'-end of the gene encoding the beta -ENaC subunit in the rat. 5'-Rapid amplification of cDNA ends and RNase protection assays indicated multiple transcription start sites over a 50-bp region. Sequencing 1.3 kb of the 5'-flanking DNA revealed putative binding sites for PEA3, Sp1, activator protein (AP)-1 and Oct-1 but neither a TATA box nor consensus sites for steroid hormone receptor binding. Transient transfections of reporter constructs driven by beta -ENaC 5'-flanking DNA in the representative epithelial cell lines Madin-Darby canine kidney, MLE-15, and Caco-2 revealed a negative element present between positions -424 and -311 that affected basal transcription rates. Gel shift assays showed protein-DNA binding activity of an AP-1 consensus site in this region; however, mutation of the AP-1 site did not abrogate the repressive activity of the region in transient transfections. Deletion of two clusters of Sp1 consensus binding sites between -1 and -51 bp and between -169 and -211 bp indicated that the proximal cluster was essential to basal promoter activity in transfected cell lines. In a comparison of these data with those in published studies on alpha - and gamma -ENaC promoters, the beta - and gamma -subunit promoters appear to be more similar to each other than to the alpha -promoter.

ion transport; Sp1; transcription; gene expression


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ABSTRACT
INTRODUCTION
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THE EPITHELIAL SODIUM CHANNEL (ENaC) is an amiloride-sensitive Na+-permeant ion channel that is found in the apical membrane of most absorptive epithelia (reviewed in Ref. 13). The ENaC was cloned from the distal colon of salt-deprived rats and was found to contain three homologous subunits: alpha , beta , and gamma  (6, 7). The channel is expressed in the distal nephron of the kidney, lung alveoli and airways, distal colon, skin, salivary and sweat gland ducts, and taste buds (13). In all of these organs and tissues, the channel has a significant role in salt and fluid homeostasis. All three ENaC subunits are required for maximal channel activity in Xenopus oocyte expression systems (7).

In the kidney, the ENaC contributes to electrolyte balance and is thus involved in blood pressure control. Loss-of-function mutations cause the genetic hypotensive syndrome pseudohypoaldosteronism type I (9, 14), whereas gain-of-function mutations result in an inherited form of hypertension known as Liddle's syndrome (5, 15, 34). Genetic knockouts of the beta - or gamma -subunit of the mouse ENaC resulted in severe defects in renal Na+ and K+ transport, leading to death from hyperkalemia within 2 days after birth (4, 24). In the lung, active Na+ transport across the epithelium helps both in the reabsorption of fetal lung fluid at birth and in keeping the adult alveolar spaces free of fluid (23). Pharmacological inhibition of Na+ channels slowed the clearance of fetal lung fluid after birth (25), whereas knockout of the alpha -subunit resulted in mice that were unable to clear their fetal lung liquid and died from respiratory distress shortly after birth (16). Pseudohypoaldosteronism type I is not associated with respiratory distress at birth, but patients have minimal or absent Na+ absorption from airway surfaces and a volume of airway surface liquid more than twice the normal value (17). In the distal colon, the ENaC is expressed in surface epithelial cells (but not in crypts) and has not been detected in any other part of the intestine (11, 30). Under basal conditions, only alpha -subunit mRNA is expressed at a detectable level in the colon, whereas the beta - and gamma -subunits are induced with exposure to mineralocorticoid (20, 30).

ENaC mRNA concentrations are subunit and tissue specific during fetal development and adult life. Promoter studies of the ENaC genes have so far been reported for the alpha - and gamma -subunits of both rats and humans (2, 10, 27, 32, 37, 38). The three alpha -subunit studies describing the rat and human genes (10, 27, 32) reported the absence of a TATA box and the presence of GC boxes near the transcription start site. Studies of both the human and rat gamma -subunits (2, 37) also reported the absence of a TATA box in their promoters and the presence of GC boxes (Sp1 consensus sites) near their transcription start sites. In one study (2), deletion of the two GC boxes of the human gamma -ENaC severely compromised promoter activity. ENaC subunits display tissue-specific responses to glucocorticoid stimulation; glucocorticoid stimulation of adrenalectomized animals produces an increase in mRNA levels of only the alpha -subunit in the kidney and lung, whereas in the colon, only the beta - and gamma -subunit mRNA levels are increased (1, 8, 12, 22, 30, 35); transient transfections with alpha - and gamma -ENaC promoters have identified an active glucocorticoid response element (GRE) among the upstream promoter elements in the alpha -ENaC promoter (10) but not in the gamma -ENaC promoter (37).

The genomic organization of the alpha - and gamma -ENaC genes from both rats and humans have been reported along with initial characterizations of their promoters (2, 10, 21, 27, 32, 37, 38). The organization of the gene encoding the human beta -ENaC has been reported (31) and is highly homologous to the other two subunits. We present here the first report describing the sequence and transcriptional activity of the beta -ENaC promoter, using a clone isolated from a Sprague-Dawley rat genomic DNA library.


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Cell culture. Madin-Darby canine kidney (MDCK) epithelial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Human colon adenocarcinoma cells (Caco-2) were maintained in alpha -MEM without ribonucleosides and deoxyribonucleosides and with 20% FBS. Mouse distal lung epithelial cells (MLE-15) were maintained in HITES medium with 2% FBS. HITES medium consists of RPMI 1640 medium supplemented with 5 µg/ml of insulin, 10 µg/ml of transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM beta -estradiol, 10 mM HEPES, and 2 mM L-glutamine. MLE-15 cells were a gift from Dr. J. Whitsett (Children's Hospital Medical Center, Cincinnati, OH); MDCK and Caco-2 cell lines were obtained from American Type Culture Collection (Manassas, VA). All lines were maintained in the presence of 100 U/ml of penicillin G and 100 µg/ml of streptomycin sulfate.

Rapid amplification of cDNA ends. Rat total RNA was isolated from whole kidneys with TRIzol reagent (Life Technologies, Burlington, ON). RNA ligation-mediated (RLM) rapid amplification of cDNA ends (RACE) was used to amplify cDNAs from the rat beta -rENaC (beta -rENaC) mRNA (First Choice RLM-RACE kit, Ambion, Austin, TX). The manufacturer's protocol was followed with the gene-specific primer beta sp4 (5'-GGCTGGACTTCGGAGCCAGAATCT-3') and the kit's outer adapter primer. The amplified cDNAs were cloned into pGEM-T easy for sequence analysis of multiple subclones.

Library screening. A commercial rat genomic DNA library from kidney cells of a 16-mo-old male Sprague-Dawley rat in a Lambda FIX II vector (Stratagene, La Jolla, CA) was screened for genomic sequences encoding exon I and the 5'-flanking region of beta -rENaC. A probe specific to the 5'-end of the beta -rENaC gene was generated by PCR with a beta -rENaC RACE product as a template. lambda -Recombinants (1.0 × 106) were screened as previously described (27).

RNase protection assay. Due to the small size of exon I, the DNA template for the RNA probe was constructed by joining genomic and cDNA sequences (see Fig. 2A). The genomic fragment was amplified with primers beta LEI [5'-CTGCTCCAATGTGCAGTGATGGCAGCTAA-3'; nucleotides (nt) +91 to +63], and beta RPAG5' (5'-CCCAAGCTTAGTCCCCTGTCGTTGCTTT-3'; nt -239 to -221), and a lambda -clone containing exon I of beta -rENaC as a template. The cDNA fragment was amplified with primers beta 5'EI (5'-ACCACCTTAGCTGCCATC-3'; nt +57 to +74) and beta RPAcDNA3' (5'-CCCCTCGAGATGCGTTTGGGGCCGTGTGT-3'; nt +233 to +214), with a 5'-RACE product as a template. (Underlined sequences represent nonhomologous regions, including restriction enzyme recognition sites for subcloning.) The full-length RNase protection assay (RPA) template was generated by PCR from the overlapping cDNA and genomic PCR products. The 490-bp product was digested with MspI, blunted, and digested with XhoI; the resulting 279-bp fragment was cloned into pBluescript II KS(-); and the sequence was verified. This DNA template was transcribed in vitro following standard protocols (3) with 8,000 µCi/mmol (10 µCi/µl) of [alpha -32P]CTP (ICN) and T3 RNA polymerase (Ambion). The resulting transcript is 309 bp in length, of which 266 bp are homologous to beta -rENaC. The probe was gel purified before RPA with the RPA III kit (Ambion) as per the manufacturer's instructions. Rat total RNA was isolated from primary cultures of fetal distal lung epithelial (FDLE) cells, whole lungs, and whole kidneys with TRIzol reagent (Life Technologies). Protected fragments were analyzed with 8% PAGE followed by autoradiography with X-OMAT film (Kodak, Rochester, NY).

Sequencing. All manual sequencing was carried out with the T7Sequencing kit (Amersham Pharmacia Biotech) and deoxyadenosine 5'-[alpha -35S]thiotriphosphate following the manufacturer's protocol. Automated sequencing was carried out with the ThermoSequenase fluorescent-labeled primer cycle-sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech) and the M13Rev and M13Fwd(-38) primers (LI-COR, Lincoln, NE) following the manufacturer's protocol. Reactions were run on the LI-COR DNA sequencer model 4000L, and data were collected with the software LI-COR BaseImagIR data collection version 4.

beta -rENaC promoter sequence analysis. To locate consensus transcription factor binding sites, beta -rENaC 5'-flanking DNA was analyzed with FINDPATTERNS (Wisconsin sequence analysis package) against the TFSITES database and the MatInspector version 2.2 search engine (28) (http://transfac. gbf.de/cgi-bin/matSearch/matsearch2.pl).

Reporter constructs. Cloned genomic DNA fragments containing portions of the putative beta -rENaC promoter were inserted upstream of the secreted alkaline phosphatase (SEAP) gene in the promoterless expression vector pSEAP2-Basic (Clontech, Palo Alto, CA). A combination of naturally occurring restriction sites and fragments generated by PCR were used to assemble reporter constructs (see Fig. 4A).

The construct pTKSEAP2 was built by extracting the thymidine kinase (TK) promoter from the pTKLUC plasmid (a gift from S. Hollenberg, Oregon Health Sciences University, Portland, OR, and V. Giguere, McGill University Health Centre, Montreal, PQ, Canada) and inserting it into the pSEAP2-Basic vector multiple cloning site. beta negTK and beta negalpha 548 were constructed by inserting the 114-bp Eco47III-SphI fragment from the beta -rENaC 5'-flanking DNA into TKSEAP2 or alpha 548SEAP2 (from Ref. 27), respectively (see Fig. 5A). Orientation of these constructs was confirmed by sequencing. A further variation of the beta 424SEAP2 construct was created in which the consensus activator protein (AP)-1 site (TGACACA) at -335 to -341 bp was mutated to CTGCACT by sequential PCR steps and confirmed by sequencing. This construct was called beta -424(mAP-1) (see Fig. 6B). Deletion constructs of the Sp1 site clusters (see Fig. 7A) were generated by PCR, cloned into pGEM-T easy for sequence verification, and subcloned to the reporter vector pGL3-Basic (Promega).

Transient transfection. MDCK, MLE-15, and Caco-2 cells were seeded on Costar six-well plates (Corning, Corning, NY) and transfected with LipofectAMINE (Life Technologies). SEAP2 reporter constructs were cotransfected with a Rous sarcoma virus (RSV) promoter-driven beta -galactosidase (beta gal) expression vector (RSVbeta gal; an internal control for transfection efficiency). Transfection, harvesting, and SEAP and beta gal assays were carried out as previously described (27). For assays with the pGL3 reporters, the pGL3 construct was cotransfected with the internal control plasmid pRL-TK (Promega). Firefly and Renilla luciferase activities were measured with the dual-luciferase reporter assay kit (Promega).

DNA mobility shift assay. A variety of double-strand DNA fragments derived from the -424- to -311-bp region of the beta -rENaC promoter was used to attempt to identify the nuclear proteins binding to this region. These included a 34-bp oligonucleotide (nt -343 to -310; designated GSAL3) and an 18-bp double-strand DNA fragment (nt -347 to -330; designated negbeta ) with a putative AP-1 consensus sequence. These probes were created by annealing complementary in vitro synthesized oligonucleotides. Standard protocols for mobility shift DNA-binding (gel shift) assays were followed, with nuclear extracts prepared from MDCK, MLE-15, and Caco2 cells (3). Binding reaction mixtures contained 10 mM Tris · HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 2 µg of poly(dI-dC), 20 µg of BSA, 0.05% Nonidet P-40, 8 µg of nuclear protein extract, and 20,000 counts/min of 32P end-labeled probe. Three antibodies were used in the gel shift assays: 1) c-Jun/AP-1 (D) × TransCruz, 2) p-c-Jun (KM-1) × TransCruz, and 3) Oct-1 (C-21) × TransCruz (Santa Cruz Biotechnology, Santa Cruz, CA). Binding reactions were incubated for 30 min at room temperature followed by electrophoresis for 4 h at 80 V on 5% acrylamide nondenaturing gels in 1.0× Tris-borate-EDTA. Dried gels were exposed to X-OMAT film overnight.

Statistical analysis. Reporter gene activities in transiently transfected cells are presented as mean ± SE from n = 3 or 6 wells as indicated in Figs. 4-6, and the significance between the means of different groups was calculated with an analysis of variance (Instat software by GraphPad, San Diego, CA). A probability (P) of <0.05 was considered significant. All transfections were performed at least twice with triplicate wells.


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Transcription start site analysis. To define the promoter of beta -rENaC, the 5'-end of the mRNA needed to be precisely identified. This was done with RLM-RACE, a modification of the 5'-RACE procedure designed to amplify cDNA only from full-length, capped mRNA (Fig. 1). When RLM-RACE was performed on total RNA from rat kidneys, a single product of ~500 bp was detected. This product was dependent on cleavage of the pyrophosphate linkage between the cap structure and the mRNA (Fig. 1A, compare lanes 1 and 2), demonstrating its specificity to the 5'-end of the decapped mRNA. After subcloning of the amplification mixture, 30 isolates were analyzed and sequenced. Twenty-two subclones contained sequences homologous to beta -rENaC, all of which were contiguous. beta -rENaC sequences extended to 56 nt upstream of the published cDNA sequence (GenBank accession no. X77932). Multiple end points were detected, suggesting that multiple transcription start sites are used within a 50-bp region (Fig. 1B). The most commonly found end point was an A residue at position +42. The sequence surrounding this position closely matches the consensus initiator sequence commonly found in TATA-less promoters (19).


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Fig. 1.   Determination of the transcription start site of rat epithelial Na+ channel beta -subunit (beta -rENaC) with RNA ligation-mediated (RLM) rapid amplification of cDNA ends (RACE). A: agarose gel showing beta -rENaC cDNA amplified from kidney RNA by RLM-RACE. Lane 1, amplification product after a single round of PCR; lane 2, no amplification is seen in the absence of cap cleavage by tobacco acid pyrophosphatase; lane 3, minus template negative control. A 100-bp DNA ladder (Life Technologies) was used as a molecular mass marker. B: sequence obtained from RLM-RACE products. , 5'-Termini of 22 independent subclones. Vertical line between nucleotides (nt) 56 and 57, 5' boundary of the published beta -rENaC cDNA sequence (GenBank accession no. X77932). Box, initiating methionine codon of the beta -rENaC open reading frame. Vertical arrows, major transcription start sites determined by RNase protection assay (RPA; +1, +21 and +42 bp in Fig. 2B). Consensus initiator sequence YYANWYY is commonly found in TATA-less promoters (19).

A single lambda -clone that contained beta -rENaC exon I was isolated from the Sprague-Dawley rat genomic library. Sequence analysis showed that exon I was only 103 bp in length. (Additional clones confirmed that exon II contained a sequence beginning at position 104; data not shown.)

To confirm the multiple start sites suggested by RLM-RACE, RPA was carried out on rat total RNA from three sources: FDLE cells, adult lungs, and adult kidneys. Due to the small size of exon I, which would be prone to inefficient precipitation in the assay procedure, a probe was created that fused beta -rENaC cDNA (exon I and partial exon II, including the translation start codon) to the 5'-flanking genomic DNA (Fig. 2A). In agreement with the RLM-RACE data, multiple protected fragments were detected by RPA (Fig. 2B), the most prominent of which (193 and 214 nt in length) are of the size predicted for transcripts starting at nt +21 and +42, the most commonly detected initiation positions in the RLM-RACE clones. The longest protected species, at 233 nt, is fainter but consistent with the few longer clones found by RLM-RACE. We have designated the T residue initiating the longest RLM-RACE clone as position +1 but note that positions +21 and +42 are more heavily favored. The pattern of protected fragments was similar among the three RNA sources tested.


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Fig. 2.   RPA confirmation of multiple start sites of the beta -rENaC gene. A: template for riboprobe synthesis fused beta -rENaC gene 5'-flanking sequence (line) to cDNA (exons 1 and 2), eliminating intron 1. Solid boxes, vector sequences. Nos. on top, bp. B: protected products were run on an 8% polyacrylamide sequencing gel. Lanes 1-3, protected species from fetal distal lung epithelial (FDLE) cell, adult lung, and adult kidney total RNAs, respectively; lanes 4 and 5 contained yeast RNA controls in the presence and absence, respectively, of RNase treatment. M, RNA markers (Ambion Century Plus marker set); ACGT, DNA sequencing ladder used for single-base pair determination of size.

Promoter sequence and cis-element consensus sequences. Isolation of the lambda -clone beta 27.1.1 and its subsequent subcloning and Southern blotting with the exon I probe revealed a 2-kb XbaI fragment that contained the first exon. Sequencing of this fragment showed that it contained 1.2 kb of the 5'-flanking sequence of the beta -rENaC gene. No TATA box was found, which is consistent with the observed multiple start sites and consensus initiator sequence. Although mRNA induction studies in the colon (12, 35) showed that beta -rENaC mRNA levels were increased by glucocorticosteroids, no consensus GRE was found. Many other cis-element consensus sites were observed as indicated in Fig. 3. The sequence contained consensus AP-1 sites, PEA3 elements, Oct-1 elements, a nuclear factor-kappa B element, and numerous Sp1 sites.


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Fig. 3.   Sequence of beta -rENaC 5'-flanking region. Boxes, transcription factor consensus binding sites, with names above the sequence. NF-kappa B, nuclear factor-kappa B; AP-1, activator protein-1. Arrow, transcription start site (+1). This sequence has been submitted to GenBank with accession no. AF380338.

Characterization of beta -rENaC promoter activity. The longest beta -rENaC promoter constructs, which terminate 5' at -1,221 bp, and the shortest, which terminate 5' at -311 bp (Fig. 4A), were tested for transcriptional activity in transfected cells. beta -1221a and beta -311a both terminate 3' at the BstEII site at +22 bp, whereas beta -1221 and beta -311 extend in the 3' direction to +100 bp, covering nearly all of exon I. It was observed that beta -1221 produced approximately threefold more activity than beta -1221a and that beta -311 produced three- to fivefold more activity than beta -311a (Fig. 4B). This phenomenon was observed consistently in each of the three epithelial cell lines, MDCK, MLE-15, and Caco-2. The negative control, the empty pSEAP2-Basic vector, showed minimal activity. From this point on, all constructs were created with the 3' terminus at +100 bp.


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Fig. 4.   Transcriptional activity of beta -rENaC promoter fragments. A: beta -rENaC promoter-secreted alkaline phosphatase (SEAP) reporter constructs. Thick line, genomic DNA with restriction enzyme sites used to generate promoter constructs indicated above and below. Arrow, transcription start site (+1). Thin lines, position and extent of genomic DNA included in SEAP reporter constructs (names at left, indicating distances upstream from transcription start site). B: transcriptional activity of beta -rENaC promoter constructs containing varying amounts of exon I (n = 3 experiments). SEAP activity, normalized to the beta -galactosidase (beta gal) internal control, was plotted from transient transfections in each of 3 epithelial cell lines, Madin-Darby canine kidney (MDCK; a), MLE-15 (b), and Caco-2 (c). The constructs terminated 5' at -1,221 and -311 bp and 3' at either +22 (beta -1221a and beta -311a) or +100 (beta -1221 and beta -311) bp. The empty pSEAP2-Basic vector (pSEAP2) was also transfected as a negative control. * Significant difference between a construct terminating at +100 bp and its 3'-truncated version, P < 0.05. C: activity of beta -rENaC promoter constructs varying at 5'-end (n = 6 experiments). SEAP activity, normalized to activity of cotransfected Rous sarcoma virus (RSV) beta gal, was plotted from transient transfections in MDCK (a), MLE-15 (b), and Caco-2 (c). The 6 constructs, beta -311, beta -424, beta -542, beta -611, beta -934, and beta -1221 were transfected into each cell line alongside the promoterless pSEAP2-Basic vector as a negative control. * Significant difference between beta -311 and beta -424, P < 0.05.

Among constructs extending up to 1,221 bp upstream from the transcription start site, the beta -311 promoter fragment consistently showed the highest activity in each cell line (Fig. 4C). Reporter gene activity observed in construct beta -424 showed a significant decrease of 25-40% when the promoter sequence was extended to include an additional 114 bp upstream (P < 0.05). Longer promoter fragments exhibited a further gradual decline in transcriptional activity that did not reach significance. The pattern suggests the presence of a negative regulatory element between -424 and -311 bp in the beta -rENaC promoter.

Effect of beta -rENaC (-424- to -311-bp) element on heterologous promoters. The 114-bp fragment derived from the beta -rENaC -424- to -311-bp flanking DNA containing the putative negative element was tested in two heterologous promoters (Fig. 5A) in MLE-15 cells. This fragment was inserted in front of an alpha -rENaC promoter fragment, alpha 548, and in front of a TK promoter. The beta -rENaC (-424- to -311-bp) element suppressed activity of the alpha -rENaC promoter by ~25% (Fig. 5C). In contrast, the TK promoter activity was enhanced by the beta -rENaC (-424- to -311-bp) element (Fig. 5D).


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Fig. 5.   Activity of the beta -rENaC (-424- to -311-bp) region in heterologous promoters. A: beta -rENaC (-424- to -311-bp) element was inserted immediately upstream of an alpha -rENaC promoter fragment (alpha 548) or a thymidine kinase (TK) promoter. SEAP activity, normalized to activity of cotransfected RSVbeta gal, was plotted from transient transfections in MLE-15 cells. B: extending the beta -rENaC promoter from -311 to -424 bp reduces SEAP reporter activity. C: insertion of this 114-bp beta -rENaC element (-311 to -424 bp) in front of the alpha -rENaC promoter construct (alpha 548) similarly resulted in a decrease in reporter activity (beta negalpha 548). D: insertion of the 114-bp beta -rENaC element in front of the TK promoter resulted in an increase in reporter activity (beta negTK; n = 6 experiments). All promoters were inserted into the promoterless pSEAP2-Basic vector. * Significant difference between constructs with and without the beta -rENaC (-424- to -311-bp) element, P < 0.05.

Role of AP-1 consensus element within the beta -rENaC (-424- to -311-bp) element. As indicated in Fig. 3, the -424- to -311-bp region contained putative consensus AP-1 and Oct-1 sites, both of which have previously been reported to be able to function as repressors or activators of transcription depending on context (18, 33, 36). Using mobility shift experiments, we were able to demonstrate that the AP-1 element in the 114-bp beta -rENaC sequence was capable of specifically binding Jun proteins in vitro (Fig. 6A, lanes 1-13). Using antibodies directed against Oct-1, we have not been able to demonstrate Oct-1 binding to the putative Oct-1 consensus sequence within the beta -rENaC (-424- to -311-bp) element; the major DNA-protein complexes formed with a probe (GSAL3) encompassing both consensus sites appear to involve Jun but not Oct-1 proteins (Fig. 6A, lanes 14-21). However, mutation of the AP-1 consensus element within the context of the beta -424 promoter did not restore transcriptional activity in SEAP reporter constructs (Fig. 6B). Effectiveness of the mutation within the AP-1 consensus element was assessed with further gel shifts; the mutated sequence failed to compete with the wild-type sequence for binding to nuclear protein (Fig. 6C).


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Fig. 6.   A: DNA mobility shift analysis of nuclear protein interactions with DNA fragments derived from the beta -rENaC (-424- to -311-bp) element. Nuclear extracts from 3 cell lines, MDCK (D), MLE-15 (L), and Caco-2 (C), were incubated with 2 different 32P-labeled DNA probes. negbeta , an 18-bp fragment corresponding to nt -347 to -330 of the beta -rENaC promoter, encompassed the putative AP-1 site. GSAL3, a 34-bp oligonucleotide corresponding to nt -343 to -310, encompassed both the putative AP-1 and Oct-1 sites. Arrow indicates shifted protein-DNA complexes. Lanes 1, 14, and 18, negative controls in the absence of nuclear extract. With negbeta as probe, DNA-protein interactions with all 3 nuclear extracts (lanes 2-4) were specifically competed out with a 200× excess of unlabeled negbeta (lanes 5-7). Antibodies directed specifically against c-Jun supershifted approximately half of the DNA-protein complexes (lanes 8-10), whereas antibodies directed against the DNA binding site of all Jun species blocked the protein binding almost completely (lanes 11-13). With GSAL3 as probe, DNA-protein interactions (lane 15) were specifically competed out by a 200× excess of GSAL3 (lane 16) but were not reduced or shifted by antibodies directed against Oct-1 (lane 17). The majority of binding seen with the GSAL3 probe appeared to still be AP-1 related; the effect of antibodies against Jun on GSAL3-protein complexes (lanes 20 and 21) was similar to that seen with negbeta as a probe (lanes 8-13). Similar results were obtained with extracts from MDCK or Caco-2 cells on GSAL3 probe (data not shown). -, Absence; +, presence. B: effect of mutation of AP-1 consensus site on beta -rENaC promoter activity in transfected MLE-15 cells (n = 3 experiments). The consensus AP-1 binding sequence was mutated as described in MATERIALS AND METHODS. SEAP activity, normalized to activity of cotransfected RSVbeta gal, was plotted from transient transfections in MLE-15 cells. * Both the wild type (beta -424) and mutated [beta -424(mAP-1)] constructs showed significantly less activity than beta -311, P < 0.02. C: mutated version of the AP-1 consensus site fails to compete with wild-type sequence for binding to nuclear proteins. Nuclear extracts from MDCK, MLE-15, and Caco-2 cells were incubated with 32P-labeled negbeta . DNA-protein complexes (lanes 1-3) were specifically competed out with a 200× excess of unlabeled negbeta but were not affected by a 200× excess of unlabeled mouse AP-1 (mAP-1), an 18-bp oligonucleotide containing the same mutation as the beta -424(mAP-1)SEAP construct.

Effect of deletion of Sp1 consensus elements. The activity of TATA-less promoters is frequently dependent on Sp1 sites in the proximal promoter region. The beta -rENaC 5'-flanking sequence displays two clusters of Sp1 sites, at -211 to -170 and -51 to -1 bp. These sequences were deleted, singly and in combination, in the context of the beta -311 promoter and inserted into the pGL3-Basic luciferase reporter (Fig. 7A). Transient transfections of these constructs in all three epithelial cell lines showed that the proximal Sp1 cluster was essential for promoter activity, whereas deletion of the distal cluster had no effect (Fig. 7B).


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Fig. 7.   Effect of deletion of Sp1 consensus sites on beta -rENaC promoter transcriptional activity. A: beta -rENaC promoter fragments linked to firefly luciferase reporter. Top line, beta -rENaC genomic DNA. Arrow, transcription start site (+1). Nos. on bottom, bp. Open boxes, clusters of consensus Sp1 binding sites. Thin lines, genomic sequences inserted into pGL3 reporter; dotted lines, deleted sequences. B: firefly luciferase activity, normalized to Renilla luciferase internal control, was plotted from transient transfections in each of MDCK (a), MLE-15 (b), and Caco-2 (c) cells.


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ABSTRACT
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When RLM-RACE was used to amplify the 5'-end of beta -rENaC cDNAs, only one major product was detected by gel electrophoresis. However, sequencing of independent subclones revealed multiple start sites over a 50-bp region, with a favored position within a consensus initiator sequence at +42 bp. All RACE-derived sequences were contiguous with each other, the reported cDNA sequence, and our 5'-flanking genomic sequence (Fig. 3), with no indication of alternative splicing or additional upstream exons. To confirm the functional existence of multiple start sites, we performed RPA using RNA from rat tissues and cultured rat FDLE cells. The longest protected fragment in RPA agreed with the most upstream transcription start site predicted by RLM-RACE. Two other clusters of protected fragments were also noted, again indicating multiple start sites with favored positions at +21 and +42 bp. Although RPA would suggest that the +21-bp position is favored over the +42 bp most commonly found in RLM-RACE, the sizes of these fragments overall agree well between the two methods.

Our studies indicate that the beta -rENaC promoter fits the criteria of a TATA-less promoter. In the 5'-flanking DNA that we obtained, we did not detect a TATA box. A GC-rich area upstream from +1 bp contained multiple Sp1 sites characteristic of a TATA-less promoter (19). Such genes typically initiate transcription at multiple sites over a region of 20-200 bp. We detected multiple start sites over a 50-bp region (Figs. 1 and 2). Our results in Fig. 4B, in which promoter constructs ending at +22 bp exhibited significantly less activity than those extending to +100 bp, suggest that these multiple start sites make an important contribution to the overall promoter activity.

The promoters of each of the homologous alpha -, beta -, and gamma -ENaC genes share a certain degree of similarity. They all lack a TATA box but contain multiple consensus PEA3 and Sp1 elements (2, 10, 27, 38). For example, although the three subunits are all TATA-less, the gamma -subunit gene resembles the beta -rENaC gene in that it also contains a GC-rich start site containing multiple Sp1 consensus sites (2, 38). The importance of the Sp1 sites at the transcription start site of the human gamma -ENaC were investigated by Auerbach et al. (2). Consistent with our studies of beta -ENaC (Fig. 7), transcriptional activity from a minimal human gamma -ENaC promoter was dependent on these sites. In alpha -ENaC promoters, the Sp1 consensus elements are found considerably further upstream; their functionality has not yet been confirmed (27, 32). It is also of interest to note that Sp1/Sp3 sites have been demonstrated to be important for both basal and hyperoxia-induced activity in the promoter of the beta 1-subunit of Na-K-ATPase (40, 41). Transepithelial ion transport requires both apical Na channels (ENaC) and basolateral Na pumps (Na-K-ATPase), and thus some mechanism to coordinately regulate expression of both these types of ion transporters may exist. Like Na-K-ATPase, ENaC mRNA expression has also been shown to be regulated by oxygen concentration (29), although we have not explored the role of the beta -rENaC Sp1 consensus sites in this phenomenon.

Second, we detected a negative regulatory element between positions -311 and -424 bp in the beta -rENaC promoter. Similarly, Auerbach et al. (2) have shown a negative regulatory element between positions -1248 and -1525 of the human gamma -ENaC gene. Analysis of the beta - and gamma -ENaC "negative" sequences indicates that they share a number of consensus transcription factor binding sites, including AP-1 and Oct-1 sites. Inserting the beta -rENaC -424- to -311-bp element into heterologous promoters suggested that it was context specific; it was successful in suppressing an alpha -rENaC minimal promoter, but it enhanced the activity of a TK promoter. This could be consistent with Oct-1 and/or AP-1 playing a role because both have been reported to be able to function as repressors or enhancers depending on context (18, 33, 36). One factor that may have contributed to this could be the fact that alpha -rENaC, like beta -rENaC, has a TATA-less promoter, whereas the TK promoter possesses a TATA box. We have noted a significant decrease in transcriptional activity mediated by an upstream region of the rat alpha -ENaC promoter (26, 27). However, the magnitude of the effect is much less than that observed here for the beta -rENaC negative element. No such negative element has been reported in studies of the human alpha -ENaC promoter (10, 27, 32).

A final point of comparison lies in the steroid inducibility of the ENaC subunits. Previous studies have shown the alpha -ENaC promoters of both human and rat to contain functionally active GREs fitting a classic consensus sequence (10, 27, 32), whereas neither human nor rat gamma -ENaC promoters contain a consensus GRE or respond to dexamethasone in such experiments (37, 38). No consensus GREs were found in the 1,221 bp of the 5'-flanking DNA of the beta -rENaC gene we examined, although one may exist further upstream.

Overall, the data described here for the beta -rENaC promoter (Sp1 sites surrounding the transcription start, presence of an upstream negative element, and absence of classic consensus GREs) suggest that the beta - and gamma -subunits may be coordinately regulated. In contrast, although the alpha -subunit promoter is also a TATA-less promoter, its Sp1 sites are located further upstream, it does not appear to contain a strong negative element upstream, and it does contain classic, consensus GREs. We suggest that the beta - and gamma -subunit genes are more similar to each other than they are to the alpha -subunit gene and may share a common mechanism of steroid induction in the colon, distinct from that operational on alpha -ENaC in kidneys and lungs. In this light, it is of interest to note the observation that the beta - and gamma -ENaC genes are tightly linked in both humans and rodents; Voilley et al. (39) found that the human beta - and gamma -subunit genes were found within the same 400-kb genomic fragment. This tight linkage indicates that these genes are physically close enough to theoretically physically share regulatory DNA elements. A more extensive characterization of this region, to finely map regulatory elements relative to each of the subunit genes, will be necessary to prove such a hypothesis. The work in our present report, representing the first description of the beta -rENaC promoter region, forms a starting point for such studies.


    ACKNOWLEDGEMENTS

For advice and technical assistance, we thank Brent Steer, Bijan Rafii, Christopher Fladd, Yanxia Wen, and Vicky Hannam. We also acknowledge Dr. B. Rossier for rat epithelial sodium channel beta -subunit cDNA, Dr. J. Whitsett for the MLE-15 cell line, and Dr. V. Giguere for the Rous sarcoma virus beta -galactosidase construct and the thymidine kinase promoter.


    FOOTNOTES

This work was supported by the Canadian Institutes of Health Research Group in Lung Development (Project 8).

Address for reprint requests and other correspondence: G. Otulakowski, Lung Biology Research, Hospital for Sick Children Research Institute, 555 University Ave., Toronto, Ontario M5G 1X8, Canada (E-mail: gotulak{at}sickkids.on.ca).

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 8 March 2001; accepted in final form 15 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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