Elena Sáez

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)

Bicarbonate‐rich choleresis induced by secretin in normal rat is taurocholate‐dependent and involves AE2 anion exchanger

Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR)–dependent Cl− efflux and subsequent biliary HCO3− secretion, possibly via Cl−/HCO3− anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl−/HCO3− exchange in secretin‐stimulated HCO3− secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3− and Cl− excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl− channel inhibitor 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB) or the Cl−/HCO3− exchange inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3− and Cl−. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin‐induced increases in bile flow and HCO3− excretion but not the increased Cl− excretion, revealing a role of biliary Cl−/HCO3− exchange in secretin‐induced, bicarbonate‐rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+‐independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin‐stimulated Cl−/HCO3− exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate‐rich choleresis in the normal rat and that this occurs via a chloride–bicarbonate exchange process consistent with AE2 function. (HEPATOLOGY 2006;43:266–275.)

Anion exchanger 2 is essential for spermiogenesis in mice

Na+-independent anion exchangers (AE) mediate electroneutral exchange of Cl- for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 (Slc4a2) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. Male Ae2-/- mice (but not female Ae2-/- mice) are infertile. Histopathological analysis of Ae2-/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.

Ursodeoxycholic Acid Is Conjugated with Taurine to Promote Secretin-Stimulated Biliary Hydrocholeresis in the Normal Rat

Background & Aims Secretin induces bicarbonate-rich hydrocholeresis in healthy individuals, but not in untreated patients with primary biliary cirrhosis (PBC). Ursodeoxycholic acid (UDCA) – the first choice treatment for PBC – restores the secretin response. Compared with humans, secretin has poor effect in experimental normal-rat models with biliary drainage, although it may elicit hydrocholeresis when the bile-acid pool is maintained. In view of the benefits of UDCA in PBC, we used normal-rat models to unravel the acute contribution of UDCA (and/or taurine-conjugated TUDCA) for eliciting the biliary secretin response. Methods Intravascular and/or intrabiliary administration of agonists and inhibitors was performed in normal rats with biliary monitoring. Secretin/bile-acid interplay was analyzed in 3D cultured rat cholangiocytes that formed expansive cystic structures with intralumenal hydroionic secretion. Results In vivo, secretin stimulates hydrocholeresis upon UDCA/TUDCA infusion, but does not modify the intrinsic hypercholeretic effect of dehydrocholic acid (DHCA). The former effect is dependent on microtubule polymerization, and involves PKCα, PI3K and MEK pathways, as shown by colchicine (i.p.) and retrograde biliary inhibitors. In vitro, while secretin alone accelerates the spontaneous expansion of 3D-cystic structures, this effect is enhanced in the presence of TUDCA, but not UDCA or DHCA. Experiments with inhibitors and Ca2+-chelator confirmed that the synergistic effect of secretin plus TUDCA involves microtubules, intracellular Ca2+, PKCα, PI3K, PKA and MEK pathways. Gene silencing also demonstrated the involvement of the bicarbonate extruder Ae2. Conclusions UDCA is conjugated in order to promote secretin-stimulated hydrocholeresis in rats through Ae2, microtubules, intracellular Ca2+, PKCα, PI3K, PKA, and MEK.

Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

Background: Inositol 1,4,5-trisphosphate receptor (InsP3R3) is critical to secretion in a number of epithelia and its expression is lost in secretory disorders. Results: miR-506 down-regulates InsP3R3 expression and impairs Ca2+ signaling and secretion. Conclusion: Post-translational regulation of InsP3R3 expression by miR-506 might contribute to disease phenotype. Significance: Restoring InsP3R3 expression by use of anti-miR-506 therapy might be beneficial in a variety of secretory disorders. The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R3) is apically localized and triggers Ca2+ waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP3R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP3R3 in cholangiocyte Ca2+ signaling and secretion is well established and because loss of InsP3R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3′-UTR of human InsP3R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP3R3–3′UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP3R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP3R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP3-mediated Ca2+ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP3R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP3R3 expression and InsP3R3-mediated Ca2+ signaling and secretion.

Ursodeoxycholic acid inhibits hepatic cystogenesis in experimental models of polycystic liver disease

BACKGROUND & AIMS Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive biliary cystogenesis. Current therapies show short-term and/or modest beneficial effects. Cystic cholangiocytes hyperproliferate as a consequence of diminished intracellular calcium levels ([Ca(2+)]i). Here, the therapeutic value of ursodeoxycholic acid (UDCA) was investigated. METHODS Effect of UDCA was examined in vitro and in polycystic (PCK) rats. Hepatic cystogenesis and fibrosis, and the bile acid (BA) content were evaluated from the liver, bile, serum, and kidneys by HPLC-MS/MS. RESULTS Chronic treatment of PCK rats with UDCA inhibits hepatic cystogenesis and fibrosis, and improves their motor behaviour. As compared to wild-type animals, PCK rats show increased BA concentration ([BA]) in liver, similar hepatic Cyp7a1 mRNA levels, and diminished [BA] in bile. Likewise, [BA] is increased in cystic fluid of PLD patients compared to their matched serum levels. In PCK rats, UDCA decreases the intrahepatic accumulation of cytotoxic BA, normalizes their diminished [BA] in bile, increases the BA secretion in bile and diminishes the increased [BA] in kidneys. In vitro, UDCA inhibits the hyperproliferation of polycystic human cholangiocytes via a PI3K/AKT/MEK/ERK1/2-dependent mechanism without affecting apoptosis. Finally, the presence of glycodeoxycholic acid promotes the proliferation of polycystic human cholangiocytes, which is inhibited by both UDCA and tauro-UDCA. CONCLUSIONS UDCA was able to halt the liver disease of a rat model of PLD through inhibiting cystic cholangiocyte hyperproliferation and decreasing the levels of cytotoxic BA species in the liver, which suggests the use of UDCA as a potential therapeutic tool for PLD patients.

Anion exchanger 2 is critical for CD8(+) T cells to maintain pHi homeostasis and modulate immune responses.

Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi). Subsequent pHi regulation may involve HCO3− extrusion through Cl−/HCO3− exchangers and/or Na+‐HCO3− co‐transporters with acid‐loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8+ T‐cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na+‐independent Cl−/HCO3− anion‐exchange activity). Here we report that CD8+ T cells but not CD4+ T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4+ T cells) exhibit Ae1 expression and Na+‐HCO3− co‐transport with acidifying potential, CD8+ T cells lack these acid‐loading mechanisms. In Ae2‐KO mice, CD4+ but not CD8+ T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2‐KO CD8+ T cells exhibit alkalinized pHi, and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2‐deficient CD8+ T cells show enhanced intracellular production of IL‐2 and membrane expression of its receptor IL‐2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8+ T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8+ T‐cell responses.

CD8+ T cells undergo activation and programmed death-1 repression in the liver of aged Ae2a,b-/- mice favoring autoimmune cholangitis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl−/HCO3− anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b−/− mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b−/− mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8+ T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2′-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8+ T cells from aged knockouts. Conclusion: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.

MiRNA-506 promotes primary biliary cholangitis-like features in cholangiocytes and immune activation

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR‐506 is overexpressed in PBC cholangiocytes and directly targets both Cl–/ HCO3− anion exchanger 2 and type III inositol 1,4,5‐trisphosphate receptor, leading to cholestasis. Here, the regulation of miR‐506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin‐8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR‐506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR‐506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR‐506 (1) induced dedifferentiation with down‐regulation of biliary and epithelial markers together with up‐regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase‐3‐dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR‐506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. Conclusion: Different proinflammatory cytokines enhance the expression of miR‐506 in biliary epithelial cells; miR‐506 induces PBC‐like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (Hepatology 2018;67:1420‐1440)

Impact of Scaffold Exploration on Novel Dual-Acting Histone Deacetylases and Phosphodiesterase 5 Inhibitors for the Treatment of Alzheimer’s Disease

A novel systems therapeutics approach, involving simultaneous inhibition of phosphodiesterase 5 (PDE5) and histone deacetylase (HDAC), has been validated as a potentially novel therapeutic strategy for the treatment of Alzheimers disease (AD). First-in-class dual inhibitors bearing a sildenafil core have been very recently reported, and the lead molecule 7 has proven this strategy in AD animal models. Because scaffolds may play a critical role in primary activities and ADME-Tox profiling as well as on intellectual property, we have explored alternative scaffolds (vardenafil- and tadalafil-based cores) and evaluated their impact on critical parameters such as primary activities, permeability, toxicity, and in vivo (pharmacokinetics and functional response in hippocampus) to identify a potential alternative lead molecule bearing a different chemotype for in vivo testing.