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      Transcription Factor Elf3 Modulates Vasopressin-Induced Aquaporin-2 Gene Expression in Kidney Collecting Duct Cells

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          Abstract

          Aquaporin-2 (AQP2) is a molecular water channel protein responsible for water reabsorption by the kidney collecting ducts. Many water balance disorders are associated with defects in AQP2 gene expression regulated by the peptide hormone vasopressin. Here, we studied roles of Elf3 (E26 transformation-specific (Ets)-related transcription factor 3) in AQP2 gene expression in the collecting duct cells (mpkCCD). Vasopressin increased AQP2 mRNA and protein levels without affecting AQP2 mRNA degradation, indicative of transcriptional regulation. Elf3 knockdown and overexpression, respectively, reduced and increased AQP2 gene expression under basal and vasopressin-stimulated conditions. However, the vasopressin-to-basal ratios of AQP2 gene expression levels remained constant, indicating that Elf3 does not directly mediate vasopressin response but modulates the level of AQP2 gene expression inducible by vasopressin. The Elf3-modulated AQP2 gene expression was associated with AQP2 promoter activity, in line with Elf3’s ability to bind an Ets element in the AQP2 promoter. Mutation in the Ets element reduced both basal and vasopressin-stimulated AQP2 promoter activity, again without affecting vasopressin-to-basal ratios of the AQP2 promoter activity. Lithium chloride reduced both Elf3 and AQP2 mRNA in the mpkCCD cells as well as in mouse kidney inner medulla. We conclude that Elf3 modulates AQP2 promoter activity thereby gauging vasopressin-inducible AQP2 gene expression levels. Our data provide a potential explanation to lithium-induced nephrogenic diabetes insipidus where lithium reduces Elf3 and hence AQP2 abundance.

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          Most cited references43

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          Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo

          Members of the large ETS family of transcription factors (TFs) have highly similar DNA-binding domains (DBDs)—yet they have diverse functions and activities in physiology and oncogenesis. Some differences in DNA-binding preferences within this family have been described, but they have not been analysed systematically, and their contributions to targeting remain largely uncharacterized. We report here the DNA-binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high-throughput microwell-based TF DNA-binding specificity assay, and protein-binding microarrays (PBMs). Both approaches reveal that the ETS-binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino-acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for a member of each class. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo.
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            Molecular physiology of water balance.

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              Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel.

              To regulate mammalian water homeostasis, arginine-vasopressin (AVP) induces phosphorylation and thereby redistribution of renal aquaporin-2 (AQP2) water channels from vesicles to the apical membrane. Vice versa, AVP (or forskolin) removal and hormones activating PKC cause AQP2 internalization, but the mechanism is unknown. Here, we show that a fraction of AQP2 is modified with two to three ubiquitin moieties in vitro and in vivo. Mutagenesis revealed that AQP2 is ubiquitinated with one K63-linked chain at K270 only. In Madin-Darby canine kidney cells, AQP2 ubiquitination occurs preferentially when present in the apical membrane, is transiently increased with forskolin removal or PKC activation, and precedes its internalization. Internalization kinetics assays with wild type (wt) and ubiquitination-deficient (K270R) AQP2 revealed that ubiquitination enhances AQP2 endocytosis. Electron microscopy showed that a translational fusion of AQP2 with ubiquitin (AQP2-Ub) localized particularly to internal vesicles of multivesicular bodies (MVBs), whereas AQP2-K270R largely localized to the apical membrane, early endosomes, and the limiting membrane of MVBs. Consistent with this distribution pattern, lysosomal degradation was extensive for AQP2-Ub, low for AQP2-K270R, and intermediate for wt-AQP2. Our data show that short-chain ubiquitination is involved in the regulated endocytosis, MVB sorting, and degradation of AQP2 and may be the mechanism used by AVP removal and PKC-activating hormones to reduce renal water reabsorption. Moreover, because several other channels are also (short-chain) ubiquitinated, our data suggest that ubiquitination may be a general mediator for the regulated endocytosis and degradation of channels in higher eukaryotes.
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                Author and article information

                Contributors
                Journal
                Front Physiol
                Front Physiol
                Front. Physiol.
                Frontiers in Physiology
                Frontiers Media S.A.
                1664-042X
                18 October 2019
                2019
                : 10
                : 1308
                Affiliations
                [1] 1Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University , Taipei, Taiwan
                [2] 2Department of Parasitology, College of Medicine, National Taiwan University , Taipei, Taiwan
                [3] 3Graduate Institute of Physiology, College of Medicine, National Taiwan University , Taipei, Taiwan
                Author notes

                Edited by: Bellamkonda K. Kishore, University of Utah Health Care, United States

                Reviewed by: Grazia Tamma, University of Bari Aldo Moro, Italy; Nirupama Ramkumar, The University of Utah, United States

                *Correspondence: Ming-Jiun Yu, mjyu@ 123456ntu.edu.tw

                This article was submitted to Renal and Epithelial Physiology, a section of the journal Frontiers in Physiology

                Article
                10.3389/fphys.2019.01308
                6813252
                31681015
                6e9d0691-8ea7-402f-b0d1-59f1220114d0
                Copyright © 2019 Lin, Ma, Kuo, Su, Wang, Chan, Su, Weng, Yang, Lin and Yu.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 30 April 2019
                : 30 September 2019
                Page count
                Figures: 7, Tables: 2, Equations: 0, References: 49, Pages: 13, Words: 0
                Funding
                Funded by: Ministry of Science and Technology 10.13039/100007225
                Award ID: 101-2320-B-002-038-MY3
                Award ID: 104-2320-B-002-064-MY3
                Categories
                Physiology
                Original Research

                Anatomy & Physiology
                elf3,ets,vasopressin,aquaporin-2,transcription,collecting duct
                Anatomy & Physiology
                elf3, ets, vasopressin, aquaporin-2, transcription, collecting duct

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