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).
PCK rats, an animal model of autosomal recessive polycystic kidney disease (ARPKD), develop cholangiocyte-derived liver cysts associated with increased intracellular adenosine 3',5'-cyclic adenosine monophosphate (cAMP), the inhibition of which suppresses cyst growth. We hypothesized that elevated cAMP stimulates cholangiocyte proliferation via two downstream effectors, exchange proteins activated by cAMP (Epac1 and Epac2 isoforms) and protein kinase A (PKA), and that intracellular calcium is also involved in this process. Assessment of Epac isoforms and PKA regulatory subunits at the messenger RNA and protein level showed that cultured normal rat cholangiocytes express Epac1, Epac2, and all regulatory PKA subunits. Epac isoforms and the PKA RIbeta subunit were overexpressed in cultured PCK cholangiocytes. Proliferation analysis in response to Epac and PKA activation indicated that both normal and PCK cholangiocytes increase their growth upon Epac-specific stimulation, while PKA-specific stimulation results in differential effects, suppressing proliferation in normal cholangiocytes but accelerating this process in PCK cholangiocytes. On the other hand, both PKA and Epac activation of cystic structures generated by normal and PCK cholangiocytes when cultured under three-dimensional conditions resulted in increased cyst growth, particularly in PCK-cholangiocyte derived cysts. Pharmacological inhibitors and small interfering RNA-mediated gene silencing demonstrated the specificity of each effector activation, as well as the involvement of MEK-ERK1/2 signaling in all the observed effector-associated proliferation changes. Hyperproliferation of PCK cholangiocytes in response to PKA stimulation, but not to Epac stimulation, was found to be associated with decreased intracellular calcium, and restoration of calcium levels blocked the PKA-dependent proliferation via the PI3K/AKT pathway.


CONCLUSIONS
Our data provide strong evidence that both cAMP effectors, Epac and PKA, and the levels of intracellular calcium are involved in the hepatic cystogenesis of ARPKD.
Banales
Jesús M
JM
Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Masyuk
Tatyana V
TV
Gradilone
Sergio A
SA
Masyuk
Anatoliy I
AI
Medina
Juan F
JF
LaRusso
Nicholas F
NF
D000818:Animals; D001652:Bile Ducts / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D003560:Cysts / Q000209:etiology*; D004195:Disease Models, Animal; D004789:Enzyme Activation / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D008099:Liver / Q000187:drug effects; D008107:Liver Diseases / Q000209:etiology*; D020930:MAP Kinase Kinase Kinases / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3 / Q000502:physiology; D008954:Models, Biological; D017044:Polycystic Kidney, Autosomal Recessive / Q000503:physiopathology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D020662:Guanine Nucleotide Exchange Factors; D012333:RNA, Messenger; C526973:Rapgef3 protein, rat; C531571:Rapgef4 protein, rat; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020930:MAP Kinase Kinase Kinases; D002118:Calcium