Format

Send to

Choose Destination
PLoS Genet. 2014 Oct 23;10(10):e1004594. doi: 10.1371/journal.pgen.1004594. eCollection 2014 Oct.

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

Author information

1
Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
2
Division of Geriatrics & Palliative Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
3
Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
4
Nephrology Division, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Charlestown, Massachusetts, United States of America.
5
Department of Medical Oncology, University Medical Center Utrecht, Utrecht, The Netherlands.
6
Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
7
Division of Nephrology, Boston Children's Hospital, Massachusetts, United States of America; Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America.

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.

PMID:
25340510
PMCID:
PMC4207587
DOI:
10.1371/journal.pgen.1004594
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center