Nephronophthisis-associated CEP164 regulates cell cycle progression, apoptosis and epithelial-to-mesenchymal transition

• Published in: PLoS genetics Vol. 10; no. 10; p. e1004594
• Main Authors: Slaats, Gisela G, Ghosh, Amiya K, Falke, Lucas L, Le Corre, Stéphanie, Shaltiel, Indra A, van de Hoek, Glenn, Klasson, Timothy D, Stokman, Marijn F, Logister, Ive, Verhaar, Marianne C, Goldschmeding, Roel, Nguyen, Tri Q, Drummond, Iain A, Hildebrandt, Friedhelm, Giles, Rachel H
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.10.2014; Public Library of Science (PLoS)
• Subjects: Animals; Apoptosis; Apoptosis - genetics; Biology and Life Sciences; Cell cycle; Cell Cycle - genetics; Cilia - genetics; Cilia - pathology; DNA damage; DNA Damage - genetics; Epithelial-Mesenchymal Transition; Fibrosis - genetics; Fibrosis - pathology; Gene Knockdown Techniques; Genetic aspects; Genetic regulation; Genetic research; Health aspects; Humans; Kidney diseases; Kidney Diseases, Cystic - genetics; Kidney Diseases, Cystic - pathology; Kidney tubules; Medicine and Health Sciences; Microtubule Proteins - biosynthesis; Microtubule Proteins - genetics; Mutation; Physiological aspects; RNA, Small Interfering; Signal Transduction; Studies; Zebrafish
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1004594

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.