Centrosomal Abnormalities Characterize Human and Rodent Cystic Cholangiocytes and Are Associated with Cdc25A Overexpression

Hepatic cystogenesis in polycystic liver diseases is associated with abnormalities of cholangiocyte cilia. Given the crucial association between cilia and centrosomes, we tested the hypothesis that centrosomal defects occur in cystic cholangiocytes of rodents (Pkd2^WS25/− mice and PCK rats) and of patients with polycystic liver diseases, contributing to disturbed ciliogenesis and cyst formation. We examined centrosomal cytoarchitecture in control and cystic cholangiocytes, the effects of centrosomal abnormalities on ciliogenesis, and the role of the cell-cycle regulator Cdc25A in centrosomal defects by depleting cholangiocytes of Cdc25A in vitro and in vivo and evaluating centrosome morphology, cell-cycle progression, proliferation, ciliogenesis, and cystogenesis. The cystic cholangiocytes had atypical centrosome positioning, supernumerary centrosomes, multipolar spindles, and extra cilia. Structurally aberrant cilia were present in cystic cholangiocytes during ciliogenesis. Depletion of Cdc25A resulted in i) a decreased number of centrosomes and multiciliated cholangiocytes, ii) an increased fraction of ciliated cholangiocytes with longer cilia, iii) a decreased proportion of cholangiocytes in G1/G0 and S phases of the cell cycle, iv) decreased cell proliferation, and v) reduced cyst growth in vitro and in vivo. Our data support the hypothesis that centrosomal abnormalities in cholangiocytes are associated with aberrant ciliogenesis and that accelerated cystogenesis is likely due to overexpression of Cdc25A, providing additional evidence that pharmacological targeting of Cdc25A has therapeutic potential in polycystic liver diseases.Polycystic liver diseases (PLDs) are inherited genetic disorders that include but are not limited to i) autosomal dominant polycystic kidney disease (ADPKD), the most common of these disorders; ii) autosomal recessive PKD (ARPKD), less common but generally more severe; and iii) autosomal dominant polycystic liver disease (ADPLD), a rare condition in which cyst formation is limited to the liver (unlike the other two conditions, in which cysts occur in both liver and kidneys).^1–4 Defects in ciliary morphology and impaired ciliary-mediated intracellular signaling trigger atypical cell cycle and benign cell hyperproliferation, which are the major contributors to cyst formation and expansion. Indeed, we and others have shown that abnormalities in ciliary length (both elongation and shortening) and disturbed expression of ciliary-associated proteins are sufficient to generate the pathological cystic phenotype.^5–11Primary cilia have an intimate relationship with another cell organelle, the centrosome. Centrosomes play an important role in microtubule nucleation and organization, in cell-cycle progression, migration, ubiquitin-proteasome degradation, cell polarity and, importantly, in cilia formation.^12,13 Centrosomes are composed of two centrioles linked by interconnecting fibers and surrounded by pericentriolar material. The older centriole (the mother centriole) is structurally distinguished from the daughter centriole by the presence of fibrous appendages. In the G1/G0 phase of the cell cycle, after centriole duplication, the mother centriole is recruited to the apical cell membrane to become the basal body, providing a template for nucleation of the ciliary axoneme.^13–17Both ciliary and centrosomal defects have been detected in nearly all human cancers. Many molecules have been implicated in centrosomal abnormalities, but most experimental evidence defines the tumor suppressor p53 (levels of which are abnormal in most tumors) as a major regulator of centrosome number, size, and appearance in cancer.^{13,14,18–23} Dysfunctional links between centrosomes and primary cilia have also been observed in polycystic kidney. Indeed, in several animal models of PKD and in patients with ADPKD, renal epithelial and endothelial cells have supernumerary centrosomes, defects in mitotic spindle formation, abnormal cell division, and malformed primary cilia.^24–29Our research group previously showed that primary cilia in cholangiocytes lining liver cysts of animal models of PLD (PCK rat and Pkd2^WS25/− and Pkhd1^del2/del2 mice) are morphologically abnormal.^7,30,31 However, the structural and functional relationships between the centrosome and primary cilia in cystic cholangiocytes have not been addressed previously. Thus, to test the hypothesis that centrosomal defects contribute to ciliogenesis and cyst formation, we analyzed i) the centrosome cytoarchitecture (ie, their number, size, and cellular position), ii) the ability of supernumerary centrosomes to nucleate extra cilia, iii) the effects of centrosomal abnormalities on temporal ciliary growth (ie, ciliogenesis), and iv) the potential mechanisms responsible for centrosomal aberrations in PLD. Our results show that i) cystic cholangiocytes of animal models and human patients with PLD have improper centrosome positioning, overduplicated centrosomes, multipolar spindles, and extra cilia; ii) these defects are associated with impaired ciliogenesis in cystic cholangiocytes; iii) Cdc25A, a cell-cycle phosphatase overexpressed in PLD,⁸ is involved in regulation of the observed centrosomal abnormalities; and iv) genetic and pharmacological suppression of Cdc25A reduces the abnormal number of centrosomes per cell, reduces the proportion of cholangiocytes with multiple centrosomes and extra cilia, and alters the cell cycle, leading to decreased cholangiocyte proliferation and attenuated cyst growth. Taken together, our data confirm the importance of centrosomal abnormalities in hepatic cystogenesis and provide further support for Cdc25A as a potential therapeutic target in PLD.