Banales Jesús M, Masyuk Tatyana V, Gradilone Sergio A, Masyuk Anatoliy I, Medina Juan F, LaRusso Nicholas F
Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Hepatology. 2009 Jan;49(1):160-74. doi: 10.1002/hep.22636.
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.
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.
PCK大鼠是常染色体隐性多囊肾病（ARPKD）的动物模型，会形成与细胞内3',5'-环磷酸腺苷（cAMP）增加相关的胆管细胞源性肝囊肿，抑制cAMP可抑制囊肿生长。我们假设升高的cAMP通过两种下游效应器，即cAMP激活的交换蛋白（Epac1和Epac2亚型）和蛋白激酶A（PKA）刺激胆管细胞增殖，并且细胞内钙也参与此过程。在信使RNA和蛋白质水平对Epac亚型和PKA调节亚基的评估表明，培养的正常大鼠胆管细胞表达Epac1、Epac2和所有PKA调节亚基。Epac亚型和PKA RIβ亚基在培养的PCK胆管细胞中过表达。对Epac和PKA激活的增殖分析表明，正常和PCK胆管细胞在Epac特异性刺激后均增加其生长，而PKA特异性刺激则产生不同的效果，抑制正常胆管细胞的增殖，但加速PCK胆管细胞中的这一过程。另一方面，当在三维条件下培养时，正常和PCK胆管细胞产生的囊性结构的PKA和Epac激活均导致囊肿生长增加，特别是在PCK胆管细胞衍生的囊肿中。药理抑制剂和小干扰RNA介导的基因沉默证明了每种效应器激活的特异性，以及MEK-ERK1/2信号传导参与所有观察到的效应器相关增殖变化。发现PCK胆管细胞对PKA刺激而非Epac刺激的过度增殖与细胞内钙减少有关，钙水平的恢复通过PI3K/AKT途径阻断了PKA依赖性增殖。
我们的数据提供了强有力的证据，表明cAMP效应器Epac和PKA以及细胞内钙水平均参与ARPKD的肝囊肿形成。
