title: WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment.
abstract: Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 microg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44109-1998, USA.
authors.forename: Maribel
authors.initials: M
authors.lastname: Rico
authors.forename: Amitava
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Martha
authors.initials: M
authors.lastname: Konieczkowski
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Bruggeman
authors.forename: R Tyler
authors.initials: RT
authors.lastname: Miller
authors.forename: Shenaz
authors.initials: S
authors.lastname: Khan
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Schelling
authors.forename: John R
authors.initials: JR
authors.lastname: Sedor
mesh_terms: D000418:Albumins / Q000378:metabolism; D000918:Antibody Specificity; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D056970:Co-Repressor Proteins; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004058:Diffusion; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000096:biosynthesis; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D021381:Protein Transport; D011692:Puromycin Aminonucleoside / Q000494:pharmacology*; D056524:Retinoblastoma-Binding Protein 7; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014162:Transfection; D062826:Zonula Occludens-1 Protein
chemical_list: D000418:Albumins; D002352:Carrier Proteins; D056970:Co-Repressor Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C490746:RBBP7 protein, human; D056524:Retinoblastoma-Binding Protein 7; C503967:SYNPO protein, human; C503968:Synpo protein, mouse; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C484859:WTIP protein, human; D062826:Zonula Occludens-1 Protein; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine; D011692:Puromycin Aminonucleoside