Polycystin-1 Regulates Cilia-Dependent Hippo Signaling in ADPKD

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a characterized by the development of multiple fluid-filled cysts and massive enlargement of the kidneys, unltimatly leading to end-stage renal disease. Mutations in the PKD1 or PKD2 genes encoding Polycystin-1 (PC1) and Polycystin-2 (PC2) proteins is the primary cause of ADPKD. Limited treatment options are available for ADPKD, underscoring the need to identify new molecular targets and/or pathways for therapeutics development. PC1 and PC2 act in the primary cilia and we and others have shown a crosstalk of PKD1 with other G-protein coupled receptors (GPCRs). Studies implicate Hippo signaling in cysts formation, yet the molecular mechanisms linking PC1 and Hippo signaling, specifically in the primary cilia, is not understood. Methods: To further understand the interaction between cilia, PC1 and Hippo signaling we generated IFT88 and Pkd1 mutants by gene editing in mouse 3T3 fibroblast and inner medullary collecting duct (IMCD3) cells. Cells were growth arrested by serum starvation and hippo signaling was induced by the activation of the Lpar1 receptor by fetal bovine serum (FBS) or lysophosphatidic acid (LPA). Pathway activation was assayed by qRT-PCR, mRNA-seq, Western and immunofluorescence analyses. Hippo signaling was modulated using pathway agonists and antagonists. Results: In both Pkd1 mutant cell lines expression of Ctgf and Cyr61, two target genes for hippo activity, is dramatically reduced in response to FBS/LPA stimulation. The normal signaling of Pkd1KO cells is restored by inhibiting the critical signaling mediator Lats1. This was due to cilia-dependent signaling as 3T3-Ift88KO cells lacking proper cilia also show an 80% reduction in Hippo signaling after stimulation. Pathway analysis demonstrates that Pkd1KO cells have normal expression of the component of the hippo signaling pathway yet increased active RhoA and phospho-Yap levels. Most strikingly, is an almost 50% reduction of LPA receptor 1 (Lpar1) in the cilia in Pkd1KO cells compared to wild type, without significant changes in total protein or RNA levels. Conclusion: Our findings support the hypothesis that PC1 is required for adequate translocation and signaling of Lpar1 to the cilia to mediate a Hippo signaling. The fact that inactivating Lats1 restores normal Hippo signaling in Pkd1KO cells suggests a potential therapeutic target for clinical intervention in ADPKD. Funding: NIDDK Support