title: AF17 facilitates Dot1a nuclear export and upregulates ENaC-mediated Na+ transport in renal collecting duct cells.
abstract: Our previous work in 293T cells and AF17(-/-) mice suggests that AF17 upregulates expression and activity of the epithelial Na(+) channel (ENaC), possibly by relieving Dot1a-AF9-mediated repression. However, whether and how AF17 directly regulates Dot1a cellular distribution and ENaC function in renal collecting duct cells remain unaddressed. Here, we report our findings in mouse cortical collecting duct M-1 cells that overexpression of AF17 led to preferential distribution of Dot1a in the cytoplasm. This effect could be blocked by nuclear export inhibitor leptomycin B. siRNA-mediated depletion of AF17 caused nuclear accumulation of Dot1a. AF17 overexpression elicited multiple effects that are reminiscent of aldosterone action. These effects include 1) increased mRNA and protein expression of the three ENaC subunits (α, β and γ) and serum- and glucocorticoid inducible kinase 1, as revealed by real-time RT-qPCR and immunoblotting analyses; 2) impaired Dot1a-AF9 interaction and H3 K79 methylation at the αENaC promoter without affecting AF9 binding to the promoter, as evidenced by chromatin immunoprecipitation; and 3) elevated ENaC-mediated Na(+) transport, as analyzed by measurement of benzamil-sensitive intracellular [Na(+)] and equivalent short circuit current using single-cell fluorescence imaging and an epithelial Volt-ohmmeter, respectively. Knockdown of AF17 elicited opposite effects. However, combination of AF17 overexpression or depletion with aldosterone treatment did not cause an additive effect on mRNA expression of the ENaC subunits. Taken together, we conclude that AF17 promotes Dot1a nuclear export and upregulates basal, but not aldosterone-stimulated ENaC expression, leading to an increase in ENaC-mediated Na(+) transport in renal collecting duct cells.
authors.affiliation: Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Wu
authors.forename: Lihe
authors.initials: L
authors.lastname: Chen
authors.forename: Qiaoling
authors.initials: Q
authors.lastname: Zhou
authors.forename: Wenzheng
authors.initials: W
authors.lastname: Zhang
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001692:Biological Transport; D004268:DNA-Binding Proteins / Q000502:physiology*; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000502:physiology*; D021122:Protein Subunits; D012333:RNA, Messenger / Q000032:analysis; D012964:Sodium / Q000378:metabolism*; D014018:Tissue Distribution; D015854:Up-Regulation
chemical_list: D004268:DNA-Binding Proteins; D053503:Epithelial Sodium Channels; C486273:Mllt6 protein, mouse; D009363:Neoplasm Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D012964:Sodium; C481527:Dot1l protein, mouse; D008780:Methyltransferases; D011495:Histone-Lysine N-Methyltransferase
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