Nephronophthisis-Associated CEP 164 Regulates Cell Cycle Progression , Apoptosis and Epithelial-to-Mesenchymal Transition

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, ubiquitinationbased 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 wildtype 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. Citation: Slaats GG, Ghosh AK, Falke LL, Le Corre S, Shaltiel IA, et al. (2014) Nephronophthisis-Associated CEP164 Regulates Cell Cycle Progression, Apoptosis and Epithelial-to-Mesenchymal Transition. PLoS Genet 10(10): e1004594. doi:10.1371/journal.pgen. 1004594 Editor: Susan K. Dutcher, Washington University School of Medicine, United States of America Received February 16, 2014; Accepted July 8, 2014; Published October 23, 2014 Copyright: 2014 Slaats et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from the European Community’s Seventh Framework Programme FP7/2009 under grant agreement no: 241955, SYSCILIA, the Dutch Kidney Foundation Consortium CP11.18 ‘‘KOUNCIL’’ and 13A3D103 (to RHG) and by grants from the National Institutes of Health (to FH) (DK068306 and DK088767). FH is an Investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist, and a Warren E. Grupe Professor. RG, TQN, and LLF are supported by the Netherlands Institute for Regenerative Medicine (NIRM, Grant No. FES0908). MCV is supported by the Netherlands Organisation for Scientific Research (NWO/ZonMw) Vidi-grant 016.096. 359. IAD and SLC were supported by NIH grant DK070263. GGS further acknowledges support from Foundation ‘De Drie Lichten’ in the Netherlands, the Dutch Kidney Foundation, and the EMBO fellowship program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: r.giles@umcutrecht.nl . These authors contributed equally to this work.