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.

Ciliary subcellular localization of TGR5 determines the cholangiocyte functional response to bile acid signaling.

TGR5, the G protein-coupled bile acid receptor that transmits bile acid signaling into a cell functional response via the intracellular cAMP signaling pathway, is expressed in human and rodent cholangiocytes. However, detailed information on the localization and function of cholangiocyte TGR5 is limited. We demonstrated that in human (H69 cells) and rat cholangiocytes, TGR5 is localized to multiple, diverse subcellular compartments, with its strongest expression on the apical plasma, ciliary, and nuclear membranes. To evaluate the relationship between ciliary TGR5 and the cholangiocyte functional response to bile acid signaling, we used a model of ciliated and nonciliated H69 cells and demonstrated that TGR5 agonists induce opposite changes in cAMP and ERK levels in cells with and without primary cilia. The cAMP level was increased in nonciliated cholangiocytes but decreased in ciliated cells. In contrast, ERK signaling was induced in ciliated cholangiocytes but suppressed in cells without cilia. TGR5 agonists inhibited proliferation of ciliated cholangiocytes but activated proliferation of nonciliated cells. The observed differential effects of TGR5 agonists were associated with the coupling of TGR5 to Gαi protein in ciliated cells and Gαs protein in nonciliated cholangiocytes. The functional responses of nonciliated and ciliated cholangiocytes to TGR5-mediated bile acid signaling may have important pathophysiological significance in cilia-related liver disorders (i.e., cholangiociliopathies), such as polycystic liver disease. In summary, TGR5 is expressed on diverse cholangiocyte compartments, including a primary cilium, and its ciliary localization determines the cholangiocyte functional response to bile acid signaling.

HDAC6 inhibition restores ciliary expression and decreases tumor growth.

Primary cilia are multisensory organelles recently found to be absent in some tumor cells, but the mechanisms of deciliation and the role of cilia in tumor biology remain unclear. Cholangiocytes, the epithelial cells lining the biliary tree, normally express primary cilia and their interaction with bile components regulates multiple processes, including proliferation and transport. Using cholangiocarcinoma as a model, we found that primary cilia are reduced in cholangiocarcinoma by a mechanism involving histone deacetylase 6 (HDAC6). The experimental deciliation of normal cholangiocyte cells increased the proliferation rate and induced anchorage-independent growth. Furthermore, deciliation induced the activation of mitogen-activated protein kinase and Hedgehog signaling, two important pathways involved in cholangiocarcinoma development. We found that HDAC6 is overexpressed in cholangiocarcinoma and overexpression of HDAC6 in normal cholangiocytes induced deciliation and increased both proliferation and anchorage-independent growth. To evaluate the effect of cilia restoration on tumor cells, we targeted HDAC6 by short hairpin RNA (shRNA) or by the pharmacologic inhibitor, tubastatin-A. Both approaches restored the expression of primary cilia in cholangiocarcinoma cell lines and decreased cell proliferation and anchorage-independent growth. The effects of tubastatin-A were abolished when cholangiocarcinoma cells were rendered unable to regenerate cilia by stable transfection of IFT88-shRNA. Finally, inhibition of HDAC6 by tubastatin-A also induced a significant decrease in tumor growth in a cholangiocarcinoma animal model. Our data support a key role for primary cilia in malignant transformation, provide a plausible mechanism for their involvement, and suggest that restoration of primary cilia in tumor cells by HDAC6 targeting may be a potential therapeutic approach for cholangiocarcinoma.

Pasireotide is more effective than octreotide in reducing hepatorenal cystogenesis in rodents with polycystic kidney and liver diseases.

UNLABELLED: In polycystic liver (PLD) and kidney (PKD) diseases, increased cyclic adenosine monophosphate (cAMP) levels trigger hepatorenal cystogenesis. A reduction of the elevated cAMP by targeting somatostatin receptors (SSTRs) with octreotide (OCT; a somatostatin analog that preferentially binds to SSTR2) inhibits cyst growth. Here we compare the effects of OCT to pasireotide (PAS; a more potent somatostatin analog with broader receptor specificity) on: (1) cAMP levels, cell cycle, proliferation, and cyst expansion in vitro using cholangiocytes derived from control and PCK rats (a model of autosomal recessive PKD [ARPKD]), healthy human beings, and patients with autosomal dominant PKD (ADPKD); and (2) hepatorenal cystogenesis in vivo in PCK rats and Pkd2(WS25/-) mice (a model of ADPKD). Expression of SSTRs was assessed in control and cystic cholangiocytes of rodents and human beings. Concentrations of insulin-like growth factor 1 (IGF1) and vascular endothelial growth factor (VEGF) (both involved in indirect action of somatostatin analogs), and expression and localization of SSTRs after treatment were evaluated. We found that PAS was more potent (by 30%-45%) than OCT in reducing cAMP and cell proliferation, affecting cell cycle distribution, decreasing growth of cultured cysts in vitro, and inhibiting hepatorenal cystogenesis in vivo in PCK rats and Pkd2(WS25/-) mice. The levels of IGF1 (but not VEGF) were reduced only in response to PAS. Expression of SSTR1 and SSTR2 (but not SSTR3 and SSTR5) was decreased in cystic cholangiocytes compared to control. Although both OCT and PAS increased the immunoreactivity of SSTR2, only PAS up-regulated SSTR1; neither drug affected cellular localization of SSTRs. CONCLUSION: PAS is more effective than OCT in reducing hepatorenal cystogenesis in rodent models; therefore, it might be more beneficial for the treatment of PKD and PLD.

Inhibition of Cdc25A suppresses hepato-renal cystogenesis in rodent models of polycystic kidney and liver disease.

BACKGROUND & AIMS: In polycystic kidney disease and polycystic liver disease (PLD), the normally nonproliferative hepato-renal epithelia acquire a proliferative, cystic phenotype that is linked to overexpression of cell division cycle 25 (Cdc25)A phosphatase and cell-cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats via genetic and pharmacologic approaches. METHODS: Cdc25A(+/-) mice (which have reduced levels of Cdc25A) were cross-bred with polycystic kidney and hepatic disease 1 (Pkhd1(del2/del2)) mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and polycystic kidney (PCK) rats, control and polycystic kidney 2 (Pkd2(ws25/-)) mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2(ws25/-)mice; and expression of Cdc25A and the cell-cycle proteins regulated by Cdc25A. We also examined the effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2(ws25/-) mice. RESULTS: Liver weights and hepatic and fibrotic areas were decreased by 32%-52% in Cdc25A(+/-):Pkhd1(del2/del2) mice, compared with Pkhd1(del2/del2) mice. VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%-83% and decreased growth of cultured cysts by 23%-67%. In PCK rats and Pkd2(ws25/-) mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%-34%. PM-20 decreased hepato-renal cystogenesis in Pkd2(ws25/-) mice by 15%. CONCLUSIONS: Cdc25A inhibitors block cell-cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of polycystic kidney disease and PLD, and might be developed as therapeutics for these diseases.

Activation of Trpv4 reduces the hyperproliferative phenotype of cystic cholangiocytes from an animal model of ARPKD.

BACKGROUND & AIMS: In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes. METHODS: Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats. RESULTS: Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts. CONCLUSIONS: Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD.