Abstract 3479: RFX1 Transcription Factor Differentially Interacts with Adducin Gly460Trp Variants to Regulate Alpha 1 Na,K-ATPase Gene (ATP1A1) Expression
The Gly460Trp mutation in alpha-adducin (ADD1), a heterodymeric cytoskeletal protein, is associated to arterial hypertension and overall increased tubular Na reabsorpion likely mediated by an increased number of basolateral Na-K pumps in kidney tubular cells. Previous studies showed a specific interaction between ADD1 and the regulatory factor X box (RFX1), a ubiquitously expressed nuclear factor that works as a multi- protein complex regulating the expression of a number of cellular genes. We examined whether the Na-K pump (ATP1A1) gene expression is mediated by RFX1 protein and differentially modulated by ADD1 Gly460Trp variants. Two RFX1 binding sites in the human ATP1A1 proximal promoter (−1022+251) were identified using the zPicture tool (http://zpicture.dcode.org) and confirmed, in vitro and in vivo, by gel shift and chromatin immunoprecipitation assays, respectively. ADD1 hypertensive variant (460Trp) enhanced both the coimmunoprecipitation and the complex formation in gel shift assay of RFX1, compared to the normotensive one (460Gly). Cotransfection studies in different renal epithelial cell lines indicated that RFX1 downregulated ATP1A1 promoter activity from 20% to 50%, as measured by luciferase assay. HK2 (human kidney) cell line was stably transfected with ADD1– 460Gly and ADD1– 460Trp variants, three clones for each variant were selected and ADD1 overexpression levels quantified in Western blot (Mock:1, ADD1–460Gly:2.9 ± 0.27, ADD1– 460Trp:2.6 ± 0.53). The ATP1A1 promoter activity increased two-fold (p<0.01) in ADD1 hypertensive clones (Mock: 151, ADD1– 460Gly:143 ± 21.2, ADD1– 460Trp:291 ± 16.7) and was partially attenuated, but in statistically significant fashion (−26%; p<0.05) after cotransfection of RFX1 (Mock 150.4, ADD1– 460Gly 130.7 ± 23.2, ADD1– 460Trp 215 ± 22.5). RFX1 is a repressor of ATP1A1 gene transcription through functional binding elements identified in the promoter. ADD1 hypertensive variant enhanced the activity of ATP1A1 promoter, such effect was partially antagonized by RFX1. Further studies, including the knockdown approaches, are necessary to elucidate the mechanism by which ADD1 and RFX1 cooperate in regulating ATP1A1 expression.