title: Nephronophthisis-associated CEP164 regulates cell cycle progression, apoptosis and epithelial-to-mesenchymal transition.
abstract: We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Gisela G
authors.initials: GG
authors.lastname: Slaats
authors.affiliation: Division of Geriatrics & Palliative Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Amiya K
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Lucas L
authors.initials: LL
authors.lastname: Falke
authors.affiliation: Nephrology Division, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Charlestown, Massachusetts, United States of America.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Le Corre
authors.affiliation: Department of Medical Oncology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Indra A
authors.initials: IA
authors.lastname: Shaltiel
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Glenn
authors.initials: G
authors.lastname: van de Hoek
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Klasson
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Marijn F
authors.initials: MF
authors.lastname: Stokman
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Ive
authors.initials: I
authors.lastname: Logister
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Marianne C
authors.initials: MC
authors.lastname: Verhaar
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Roel
authors.initials: R
authors.lastname: Goldschmeding
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Tri Q
authors.initials: TQ
authors.lastname: Nguyen
authors.affiliation: Nephrology Division, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Charlestown, Massachusetts, United States of America.
authors.forename: Iain A
authors.initials: IA
authors.lastname: Drummond
authors.affiliation: Division of Nephrology, Boston Children's Hospital, Massachusetts, United States of America; Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America.
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Hildebrandt
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Rachel H
authors.initials: RH
authors.lastname: Giles
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D002923:Cilia / Q000235:genetics* / Q000473:pathology; D004249:DNA Damage / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition; D005355:Fibrosis / Q000235:genetics* / Q000473:pathology; D055785:Gene Knockdown Techniques; D006801:Humans; D052177:Kidney Diseases, Cystic / Q000235:genetics* / Q000473:pathology; D008868:Microtubule Proteins / Q000096:biosynthesis / Q000235:genetics*; D034741:RNA, Small Interfering; D015398:Signal Transduction; D015027:Zebrafish
chemical_list: C524694:CEP164 protein, human; D008868:Microtubule Proteins; D034741:RNA, Small Interfering