Abstract
The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5'-flanking sequence (5'FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5'FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5'FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5'FS are used as promoter, efficient transcription only occurs with 44 bp of 5'FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5'FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.
Highlights
NHE3 is an Na+/H+ exchanger member of the SLC9A family of transporters and is the predominant route for H+ secretion in the apical membrane of proximal tubules, where it plays a key role in fluid reabsorption and pH homeostasis [1]
OKP, a well-established cell culture, is a good proximal tubule model for studying NHE3 because, in addition to having a relatively stable phenotype, NHE3 is virtually the only Na+/H+ antiporter present in these cells, and many of the signaling molecules relevant for the regulation of its expression and activity are expressed in OKP cells
We have shown in the present study that endogenous opossum transcription factors bind with high or moderate affinity to DNA elements of the rat NHE3 promoter, suggesting a high level of identity between the DNA binding domains of opossum transcription factors and those of the corresponding transcription factors of the rat
Summary
NHE3 is an Na+/H+ exchanger member of the SLC9A family of transporters and is the predominant route for H+ secretion in the apical membrane of proximal tubules, where it plays a key role in fluid reabsorption and pH homeostasis [1]. In parallel to acute regulatory mechanisms of NHE3 activity, the expression of this protein is adjusted to different chronic physiological or pathological conditions. Comparing the 1.6 kb of the 5’-flanking sequence (5’FS) of rat and human NHE3 promoter, it was possible to detect 39% overall identity. 79%, was detected in the first 100 bp, where specificity protein 1/3 (Sp1/3), early growth response factor 1 (EGR-1), activator protein-2 (AP2), and TATA-like sequences appear at the same positions in both promoters [17]. Hormone receptor-responsive elements (e.g., triiodothyronine and glucocorticoid receptor binding sites) were identified in less restricted positions and orientation in the NHE3 gene promoters of these two species. Human and rat NHE3 gene promoters were analyzed in detail in the intestinal cell line Caco-2.
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