There is convincing evidence that PLD are disorders of intracellular signaling; altered Ca2+
homeostasis, and increased production of cAMP being the main defects. Lower intracellular Ca2+
and higher cAMP levels have been consistently reported in cystic epithelia from ADPKD and ARPKD models10, 23
. The mechanistic relationship between defective polycystins/fibrocystin and the disorder of these second messengers is unclear. Here, we will provide a reasonable, but yet speculative, working model based on experimental evidence generated in ADPKD models (). It is important to emphasize that the fine molecular tuning of these interactions remains largely unknown.
Alterations of intracellular signaling in PLD. See text for details.
Several studies, including our own observations in cystic cholangiocytes, indicate that intracellular Ca2+
levels are lower in the cystic epithelium (below 100 nM)10,23
. PC2 functions as a membrane Ca2+
channel, in response to mechanical stimulations (in partnership with PC1) or to variations in osmolarity (in partnership with TRVP4)32
. PC-2 also interacts both physically and functionally with IP3
R and RyR, thereby regulating Ca2+
homeostasis in the ER9,10
, the largest controllable intracellular Ca2+
store in non-excitable cells. Thus, a defect in PC-2 may affect resting [Ca2+
] by reducing extracellular Ca2+
entry and by altering ER Ca2+
The amount of cAMP produced by a given cell is the result of the activity of several adenylyl cyclase (AC) isoforms, which respond to different stimuli and second messengers. At least seven of them are expressed in cholangiocytes25
. Among them, AC8 is regulated by Ca2+
/calmodulin, while AC5 and AC6 are inhibited at physiological Ca2+
concentrations (100 to 200 nM), but can be activated at the Ca2+
concentrations measured in polycystin-defective cells. Thus, at the [Ca2+
]i measured in cystic cholangiocytes, AC6 becomes more prone to activation than AC8. Of note, AC6 gene silencing abolishes shear stress-induced signaling in polarized cholangiocytes23
Thus, the increased cAMP level in cystic epithelia may be related to the activation of AC6, facilitated by the changes in intracellular Ca2+
homeostasis. Increased epithelial levels of cAMP stimulate fluid secretion and also the proliferative activity of cystic cholangiocytes. Alpini and coworkers have shown that cAMP stimulates proliferation in normal cholangiocytes via the PKA/Src/Raf/MEK/ERK1/2 cascade33
. In cystic cholangiocytes, the proliferative response and ERK1/2 activation in response to cAMP is significantly higher, suggesting that cAMP levels are not the only determinant of ERK1/2 activation. In fact, polycystin may have additional effects directly mediated by the proteolytic cleavage and nuclear translocation of its carboxy-terminal tail to the nucleus.
Activation ERK1/2 has a number of effects, including stimulation of the mTOR pathway20
. Both ERK1/2 and mTOR converge in stimulating cyclins and HIF1α. The list of HIF-1α-regulated genes is large, and includes genes coding for proteins involved in energy metabolism, erythropoiesis, and cell proliferation, in addition to VEGF. Mice deficient in PC2 show a severe liver phenotype, high proliferation rate of the cystic epithelium and high expression of VEGF and its receptor VEGFR-2. Expression of pERK1/2, p-mTOR and HIF1α, are also increased, suggesting that wild type PC2 acts as repressors of the cAMP/PKA/Raf/MEK/ERK/mTOR cascade20, 21
. The observation that changes in Ca2+
/cAMP signalling similar to the one described in PC-2 defective cells are common to cystic diseases characterized by different genetic defects and phenotypes suggests that PC2 is central to the pathogenesis of all PLD forms.
The pathophysiological relevance of this model is demonstrated by the reduction of cyst growth in vivo, after administration of SU5418 (inhibition of VEGFR2 signalling), or rapamycin (inhibition of mTOR and of VEGF production)20,21
. Inhibition of cAMP production was also exploited as a therapeutic strategy. Somatostatin represses AC function through its receptor SSTR2, which is expressed in the liver only by cholangiocyte. Octreotide, given in vivo
to PCK rats, reduced liver and kidney weight, hepatic and renal cyst volume and fibrosis, and diminished the rate of cell proliferation in hepatic and renal epithelia34
. A recent randomized controlled double blind clinical trial revealed a 5% reduction in liver cyst volume in patients with symptomatic ADPKD/PLCD treated with the long-acting somatostatin analogue lanreotide35
Several open questions remain. The mechanism leading to reduced cellular levels of Ca2+ are still obscure, and the impact of the lack of polycystins on ER Ca2+ homeostasis, and the role of AC6 need better understanding. In wild type animals, the result of increased cAMP-dependent proliferation is an expansion of the bile duct mass, rather than cystic transformation. Thus, the above working model addresses the mechanisms of progression of the established cysts, but does not explain how polycystins and fibrocystins actually interfere with the morphogenetic mechanisms in the biliary tree.