Nephronophthisis-Associated <i>CEP164</i> Regulates Cell Cycle Progression, Apoptosis and Epithelial-to-Mesenchymal Transition

<div><p>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 <i>CEP164</i> in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of <i>CEP164</i> 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 <i>Cep164</i> siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type <i>CEP164</i>, but not disease-associated mutants. siRNA of <i>CEP164</i> 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 <i>CEP164</i> levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of <i>Cep164</i> or overexpression of dominant negative mutant allele <i>CEP164 Q525X</i> induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with <i>cep164</i> morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response <i>in vivo</i>. This study reveals a novel role for <i>CEP164</i> 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.</p>