Human amiloride-sensitive epithelial Na+ channel gamma subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation.

The mRNA for the epithelial Na(+) channel gamma subunit (gammaENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na(+) deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5' flanking elements of the human gammaENaC gene. The proximal 5' flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human gammaENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human gammaENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody 'supershift' assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5' flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA ('H-DNA') structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines.

Glucocorticoid induction of epithelial sodium channel expression in lung and renal epithelia occurs via trans-activation of a hormone response element in the 5'-flanking region of the human epithelial sodium channel alpha subunit gene.

In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na+ transport is associated with increased expression of the epithelial Na+ channel alpha subunit (alphaENaC). In H441 lung cells, 100 nM dexamethasone increases amiloride-sensitive short-circuit current (3.3 microA/cm2 to 7.5 microA/cm2), correlating with a 5-fold increase in alphaENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of alphaENaC, the human alphaENaC 5'-flanking region was cloned and tested in H441 cells. By deletion analysis, a approximately 150-base pair region 5' to the upstream promoter was identified that, when stimulated with 100 nM dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na+ transport, malphaENaC expression and luciferase expression from alphaENaC genomic fragments were also increased by 100 nM dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated alphaENaC gene transcription. We conclude that glucocorticoids stimulate alphaENaC expression in kidney and lung via activation of a hormone response element in the 5'-flanking region of halphaENaC and this response, in part, is the likely basis for the up-regulation of Na+ transport in these sites.

The structure of the rat amiloride-sensitive epithelial sodium channel gamma subunit gene and functional analysis of its promoter.

Prolonged dietary Na+ depletion and chronic administration of adrenal steroids increase steady-state mRNA levels of the gamma subunit of the epithelial sodium channel (gammaENaC) in rat colon. This increase correlates with a marked increase in transepithelial Na+ transport and is thought to occur via transcriptional regulation. To begin to evaluate these mechanisms in detail, we determined the organization of the rat gammaENaC gene. A rat genomic library was screened and overlapping lambda clones that together span the gene (approximately 36 kb) and contain at least 1 kb of 5' flanking genomic DNA were isolated. As in the human gene, the rat gammaENaC gene contains 13 exons and a CpG island at the 5' end of the gene. A single transcription start site was identified in rat kidney by nuclease protection assay defining a 5' untranslated region of 126 nt. The translation initiation codon was identified within the second exon and the entire 3' UTR (approximately 1 kb) was within the last exon. 800 bp of 5' flanking sequence, as well as the 3.4 kb first intron, were sequenced and analyzed for transcriptional regulatory motifs. Analogous to the human gammaENaC gene [Thomas, C.P., Doggett, N.A., Fisher, R., Stokes J.B., 1996. Genomic organization and the 5' flanking region of the gamma subunit of the human amiloride-sensitive epithelial sodium channel. J. Biol. Chem. 271, 26 062--26 066], two GC boxes were seen at -30 and -61 to the transcription start site. In addition, putative AP-1, AP-2, CRE, Sp1 and GATA-1 and GRE motifs were identified elsewhere in the 5' flanking region or the first intron. Two mammalian-wide interspersed repeats and a rodent-specific B1 repeat were also identified within the first intron. Fragments containing the putative GRE motifs coupled to luciferase did not confer a glucocorticoid-stimulated response in two cell lines that contained a functional glucocorticoid receptor. However, a 76 nt rat gammaENaC 5' flanking fragment (-76 to +68) directed expression of luciferase in the epithelial cell lines H441 and FRTL5, suggesting that this minimal region that contained both GC boxes was sufficient for promoter activity.