Mutations in PKD1 (encoding for Polycystin-1, PC1) are found in 80-85% of patients with ADPKD. We tested the hypothesis that changes in actin dynamics result from PKD1 mutations through dysregulation of compartmentalised centrosomal RhoA signalling mediated by specific RhoGAP (ARHGAP) proteins resulting in the complex cellular cystic phenotype. Initial studies revealed that the actin cytoskeleton was highly disorganised in PKD1 patient-derived cells and was associated with an increase in total and centrosomal RhoA activation and ROCK signalling. Using cilia length as a phenotypic readout for centrosomal RhoA activity, we identified ARHGAP5, 29, 35 as essential regulators of ciliation in normal human renal tubular cells. Importantly, a specific decrease in centrosomal ARHGAP35 was observed in PKD1 null cells using a centrosome-targeted proximity ligation assay and by immunofluorescence labelling. Finally, we demonstrate that another ROCK inhibitor (hydroxyfasudil) reduced cyst expansion in both human PKD1 3D cyst assays and an inducible Pkd1 mouse model. In summary, we report a novel interaction between PC1 and ARHGAP35 in the regulation of centrosomal RhoA activation and ROCK signalling. Targeting the RhoA/ROCK pathway inhibited cyst formation in vitro and in vivo indicating its relevance to ADPKD pathogenesis and for developing new therapies to inhibit cyst initiation.
Andrew J. Streets, Philipp P. Prosseda, Albert C.M. Ong