The cAMP effectors Epac and protein kinase a (PKA) are involved in the hepatic cystogenesis of an animal model of autosomal recessive polycystic kidney disease (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) is a genetic disorder in which affected infants often die at birth or shortly thereafter, primarily as the result of markedly enlarged kidney cysts and impaired lung function. In surviving patients, hepatic fibrosis, bile duct dilatation (Caroli’s disease) and/or cyst development becomes progressively more severe and may be the major cause of morbidity and mortality.1 ARPKD is linked to mutations in the PKHD1 gene which encodes fibrocystin, a large trans-membrane protein with unknown function,2, 3 located on cholangiocyte primary cilia;4 these non-motile long tubular organelles extend from the cholangiocyte apical membrane and function as mechano-,5 chemo-6 and osmo-sensors,7 detecting and transmitting luminal stimuli into intracellular signals. Although the pathophysiology of hepatic cystogenesis in ARPKD is unclear, abnormalities in cholangiocyte fluid secretion and proliferation likely contribute.1, 8 Indeed, in the PCK rat, a well-characterized animal model of ARPKD,9 liver cysts originate from intrahepatic bile ducts and their growth is associated with increased proliferative activity of cystic cholangiocytes.10 However, the mechanism underlying this benign hyperproliferative process is not fully understood. Accumulated evidence suggests that two intracellular signaling mediators, cAMP and Ca2+, regulate proliferation in different cell types,11, 12 including cholangiocytes.13 We recently demonstrated that cAMP levels are increased in cholangiocytes of the PCK rat and that octreotide, a somatostatin analog known to inhibit cAMP, decreases hepatic cyst volume, hepatic fibrotic scores and mitotic indices.10 cAMP has two downstream targets, cAMP-GEF/Epac (Epac) and protein kinase A (PKA).14 Epac proteins, a family of Rap guanine nucleotide exchange factors, regulate many cellular processes via PKA-independent mechanisms.15 There are two different Epac isoforms (Epac1 and Epac2, also known as RapGEF3 and RapGEF4, respectively) which are expressed from different genes in a variety of tissues.15–17 PKA is a heterotetrameric holoenzyme with two regulatory and two catalytic subunits, that responds to intracellular changes of cAMP regulating a wide range of intracellular processes.14 There exist several members of PKA regulatory (RIα, RIβ, RIIα and RIIβ) and catalytic (Cα, Cβ, Cγ and PrKX) subunits. The biochemical and functional features of PKA are largely determined by the structure and properties of the regulatory subunits, which are differentially expressed depending on the tissue and the cellular state.14 cAMP binds to the PKA regulatory subunits, leading to dissociation and activation of the catalytic subunits that may regulate the phosphorylation of a number of proteins and the expression of different genes.14 Both cAMP downstream effectors, Epac and PKA, may be involved in regulation of proliferation in different cell types. In polycystic diseases, however, only PKA has been shown to be responsible for hyperproliferation of epithelial cells lining renal cysts from patients with autosomal dominant polycystic kidney disease (ADPKD).18 But no data exist regarding the expression, intracellular localization and function of PKA regulatory subunits and Epac isoforms in cholangiocytes, and the involvement of these effectors in cystic cholangiocyte proliferation remains unknown. Intracellular Ca2+ [Ca2+]i may also participate in the control of cell growth.11, 12 In fact, renal cystic cells of patients with ARPKD have low calcium levels,11 and silencing of Pkhd1 in cultured normal kidney cells results in decreased intracellular calcium and hyperproliferation.19 The interconnection between cAMP and [Ca2+]i signaling pathways under normal and pathological conditions is currently attracting considerable attention. In particular, it has been shown that in renal cells from ADPKD patients, intracellular calcium modulates cAMP-dependent proliferation. Moreover, these cells were characterized by decreased [Ca2+]i and exhibited higher rates of cell proliferation in response to cAMP, while experimental restoration of [Ca2+]i inhibited their cAMP-stimulated growth.11 We recently demonstrated in normal rat cholangiocytes that elevated [Ca2+]i is able to terminate cAMP signaling (i.e., turn off regulation) initially activated by choleretic stimuli.5 However, it is unclear whether intracellular Ca2+ levels are altered in cystic cholangiocytes and what role, if any, intracellular Ca2+ plays in hepatic cystogenesis. Thus, we examined the: i) expression and intracellular localization of two cAMP downstream effectors, PKA and Epac, in cultured cholangiocytes from normal and PCK rats; ii) role of PKA and Epac activation in proliferation of normal and PCK cholangiocytes; iii) intracellular calcium levels in PCK cholangiocytes; and iv) relationships between cAMP-stimulated proliferation and calcium levels in PCK cholangiocytes.