Jesus M. Banales

Ae2a,b-Deficient Mice Develop Antimitochondrial Antibodies and Other Features Resembling Primary Biliary Cirrhosis

BACKGROUND & AIMS Cl(-)/HCO(3)(-) anion exchanger 2 (AE2) is involved in intracellular pH (pH(i)) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2(a.b)-deficient mice. METHODS In this murine model, splenocyte pH(i) and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. RESULTS Ae2(a,b)(-/-) mice exhibit splenomegaly, elevated pH(i) in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8(+) T-cell population, and under represented CD4(+)FoxP3(+)/regulatory T cells. Most Ae2(a,b)(-/-) mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2(a,b)(-/-) mice had extensive portal inflammation with CD8(+) and CD4(+) T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2(a,b)(-/-) mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. CONCLUSIONS Ae2 deficiency alters pH(i) homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC.

Cholangiocyte cilia express TRPV4 and detect changes in luminal tonicity inducing bicarbonate secretion

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.e., luminal fluid flow) by alterations in intracellular Ca2+ and cAMP. Because cholangiocyte cilia are also ideally positioned to detect changes in composition and tonicity of bile, we hypothesized that cilia also function as osmosensors. TRPV4, a Ca2+-permeable ion channel, has been implicated in signal transduction of osmotic stimuli. Using purified rat cholangiocytes and perfused intrahepatic bile duct units (IBDUs), we found that TRPV4 is expressed on cholangiocyte cilia, and that hypotonicity induces an increase in intracellular Ca2+ in a TRPV4-, ciliary-, and extracellular calcium-dependent manner. The osmosensation of luminal tonicity by ciliary TRPV4 induces bicarbonate secretion, the main determinant of ductal bile formation, by a mechanism involving apical ATP release. Furthermore, the activation of TRPV4 in vivo, by its specific agonist, 4αPDD, induces an increase in bile flow as well as ATP release and bicarbonate secretion. Our results suggest that cholangiocyte primary cilia play an important role in ductal bile formation by acting as osmosensors.

MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease

Hyperproliferation of bile duct epithelial cells due to cell-cycle dysregulation is a key feature of cystogenesis in polycystic liver diseases (PCLDs). Recent evidence suggests a regulatory role for microRNAs (miRNAs) in a variety of biological processes, including cell proliferation. We therefore hypothesized that miRNAs may be involved in the regulation of selected components of the cell cycle and might contribute to hepatic cystogenesis. We found that the cholangiocyte cell line PCK-CCL, which is derived from the PCK rat, a model of autosomal recessive polycystic kidney disease (ARPKD), displayed global changes in miRNA expression compared with normal rat cholangiocytes (NRCs). More specific analysis revealed decreased levels of 1 miRNA, miR15a, both in PCK-CCL cells and in liver tissue from PCK rats and patients with a PCLD. The decrease in miR15a expression was associated with upregulation of its target, the cell-cycle regulator cell division cycle 25A (Cdc25A). Overexpression of miR15a in PCK-CCL cells decreased Cdc25A levels, inhibited cell proliferation, and reduced cyst growth. In contrast, suppression of miR15a in NRCs accelerated cell proliferation, increased Cdc25A expression, and promoted cyst growth. Taken together, these results suggest that suppression of miR15a contributes to hepatic cystogenesis through dysregulation of Cdc25A.

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y(12) purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y(12) receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y(12) siRNAs, suggesting that cilia and ciliary P2Y(12) are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y(12) receptors and transduce corresponding signals into a cAMP response.

Expert consensus document: Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the “European Network for the Study of Cholangiocarcinoma” (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted.

Up‐regulation of microRNA 506 leads to decreased Cl−/HCO3− anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis

Cl−/HCO  3− anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin‐stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2‐deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR‐506) — predicted as candidate to target AE2 mRNA — for the decreased expression of AE2 in PBC. Real‐time quantitative polymerase chain reaction showed that miR‐506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR‐506 is up‐regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor‐mediated overexpression of miR‐506 in SV40‐immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR‐506 overexpression in three‐dimensional (3D)‐cultured H69 cholangiocytes blocked the secretin‐stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site‐directed mutagenesis demonstrated that miR‐506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR‐506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti‐miR‐506 was able to improve their AE2 activity. Conclusion: miR‐506 is up‐regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR‐506 may constitute a potential therapeutic target for this disease. (HEPATOLOGY 2012)

Mutations in GANAB, Encoding the Glucosidase IIα Subunit, Cause Autosomal-Dominant Polycystic Kidney and Liver Disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is a common, progressive, adult-onset disease that is an important cause of end-stage renal disease (ESRD), which requires transplantation or dialysis. Mutations in PKD1 or PKD2 (∼85% and ∼15% of resolved cases, respectively) are the known causes of ADPKD. Extrarenal manifestations include an increased level of intracranial aneurysms and polycystic liver disease (PLD), which can be severe and associated with significant morbidity. Autosomal-dominant PLD (ADPLD) with no or very few renal cysts is a separate disorder caused by PRKCSH, SEC63, or LRP5 mutations. After screening, 7%-10% of ADPKD-affected and ∼50% of ADPLD-affected families were genetically unresolved (GUR), suggesting further genetic heterogeneity of both disorders. Whole-exome sequencing of six GUR ADPKD-affected families identified one with a missense mutation in GANAB, encoding glucosidase II subunit α (GIIα). Because PRKCSH encodes GIIβ, GANAB is a strong ADPKD and ADPLD candidate gene. Sanger screening of 321 additional GUR families identified eight further likely mutations (six truncating), and a total of 20 affected individuals were identified in seven ADPKD- and two ADPLD-affected families. The phenotype was mild PKD and variable, including severe, PLD. Analysis of GANAB-null cells showed an absolute requirement of GIIα for maturation and surface and ciliary localization of the ADPKD proteins (PC1 and PC2), and reduced mature PC1 was seen in GANAB(+/-) cells. PC1 surface localization in GANAB(-/-) cells was rescued by wild-type, but not mutant, GIIα. Overall, we show that GANAB mutations cause ADPKD and ADPLD and that the cystogenesis is most likely driven by defects in PC1 maturation.

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 RIβ 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. Conclusion: 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. (HEPATOLOGY 2009;49:160‐174.)

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.

Expression of SLC22A1 variants may affect the response of hepatocellular carcinoma and cholangiocarcinoma to sorafenib

Reduced drug uptake is an important mechanism of chemoresistance. Down‐regulation of SLC22A1 encoding the organic cation transporter‐1 (OCT1) may affect the response of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CGC) to sorafenib, a cationic drug. Here we investigated whether SLC22A1 variants may contribute to sorafenib chemoresistance. Complete sequencing and selective variant identification were carried out to detect single nucleotide polymorphisms (SNPs) in SLC22A1 complementary DNA (cDNA). In HCC and CGC biopsies, in addition to previously described variants, two novel alternative spliced variants and three SNPs were identified. To study their functional consequences, these variants were mimicked by directed mutagenesis and expressed in HCC (Alexander and SK‐Hep‐1) and CGC (TFK1) cells. The two novel described variants, R61S fs*10 and C88A fs*16, encoded truncated proteins unable to reach the plasma membrane. Both variants abolished OCT1‐mediated uptake of tetraethylammonium, a typical OCT1 substrate, and were not able to induce sorafenib sensitivity. In cells expressing functional OCT1 variants, OCT1 inhibition with quinine prevented sorafenib‐induced toxicity. Expression of OCT1 variants in Xenopus laevis oocytes and determination of quinine‐sensitive sorafenib uptake by high‐performance liquid chromatography‐dual mass spectrometry confirmed that OCT1 is able to transport sorafenib and that R61S fs*10 and C88A fs*16 abolish this ability. Screening of these SNPs in 23 HCC and 15 CGC biopsies revealed that R61S fs*10 was present in both HCC (17%) and CGC (13%), whereas C88A fs*16 was only found in HCC (17%). Considering all SLC22A1 variants, at least one inactivating SNP was found in 48% HCC and 40% CGC. Conclusion: Development of HCC and CGC is accompanied by the appearance of aberrant OCT1 variants that, together with decreased OCT1 expression, may dramatically affect the ability of sorafenib to reach active intracellular concentrations in these tumors. (Hepatology 2013;53:1065–1073)