Catherine Decaens
email: catherine.decaens@u-psud.fr

Marjorie Durand

Brigitte Grosse

Doris Cassio

Which in vitro models could be best used to study hepatocyte polarity?

Keywords: hepatocyte, polarity, cytoskeleton, IP3R, in vitro models

The correct functioning of the liver is ensured by the setting and the maintenance of hepatocyte polarity. The complex polarity of hepatocyte is characterized by the existence of several basolateral and apical poles per cell. Many in vitro models are available for studying hepatocyte polarity. But which are the more suitable? To answer this question, we wished to identify criteria which determine the typical hepatocyte polarity. Therefore, we compiled a battery of protein markers of membrane domains in rat hepatocytes and looked for their involvement in hepatocytic functions.

Then, we focused on the relationship between hepatic functions and the cytoskeleton, Golgi apparatus and endoplasmic reticulum. Subsequently, we compared different cell lines expressing hepatocyte polarity. Finally, to demonstrate the usefulness of some of these lines, we presented new data on reticulum organization in relation to polarity.

INTRODUCTION

Although the Ancient Greeks already knew that the liver is able to regenerate, nowadays many people are still working hard to understand how this main organ functions. The liver is made up of 60% hepatocytes that produce 90% of the whole hepatic proteins and which are responsible for most of the main liver functions such as bile production, detoxification… As all epithelial cells, hepatocytes must be polarized to be functional. This implies the formation of specific membrane domains and special cytoskeletal and endoplasmic reticulum (ER) networks. Hepatocyte polarity is very peculiar and complex [START_REF] Hubbard | Hepatocyte surface polarity[END_REF] compared to the so-called « simple polarity » of most epithelial cells such as intestinal cells (Figure 1). « Simple » polarized cells have one apical pole located at the cell apex and one basolateral pole corresponding to the remaining domain of the plasma membrane. In contrast, hepatocytes that are organized in plates in the liver have several apical and basolateral poles. In contact with the external environment, the apical poles of frontfacing and adjacent hepatocytes form a continuous network of bile canaliculi (BC), into which bile is secreted. In contact with the blood, the basal membrane domain called sinusoidal pole, secretes various components -proteins, drugs-into the circulation and takes up recycled biliary salts. In both types of polarized cells, the tight junctions make the frontier between apical and lateral poles.

The first studies on hepatocytes were performed with isolated perfused organ and subcellular fractionations [START_REF] Bartles | Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation[END_REF]. Later, easier in vitro models were obtained by enzymatic digestion of the liver : freshly isolated hepatocytes induced to repolarize [START_REF] Musat | Reestablishment of cell polarity of rat hepatocytes in primary culture[END_REF] and hepatocytes isolated as couplets that keep a bile canalicular space between the two cells [START_REF] Graf | Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function[END_REF]. Both models have been widely used to study bile secretion [START_REF] Graf | The use of isolated rat hepatocyte couplets in hepatobiliary physiology[END_REF], protein trafficking [START_REF] Maurice | Formation of plasma membrane domains in rat hepatocytes and hepatoma cell lines in culture[END_REF] and calcium waves (Combettes et al., 1994). However, variations occur between different isolations and both models have a limited life span allowing only shortterm experiments. Primary hepatocyte repolarization is slow and variable.

To circumvent these disadvantages, hepatic cell lines were used. A few human lines exhibit polarity and some rat polarized lines were generated. The aim of this review is to make a survey of these cell lines. First, we will sum up data from literature concerning the relationship between liver functionality and hepatocyte polarity. Then, we will compare the cell lines. To exemplify their usefulness, some new data on inositol 1,4,5-trisphosphate receptors (IP 3 Rs) distribution in relation to polarity will be presented.

HEPATIC FUNCTIONALITY AND POLARITY

Which are the contributors in hepatic polarity? We will look at the membrane domain proteins, the cytoskeleton and two cytoplasmic organelles, focusing on their relationship with hepatic functionality.

MEMBRANE PROTEINS

Poles of polarized cells are characterized by a special plasma membrane protein distribution [START_REF] Simons | Cell surface polarity in epithelia[END_REF]. A list of membrane protein markers of polarity in rat hepatocyte is given in Supplementary Table 1. Among hepatocyte membrane markers, the bile salt export pump (Bsep/BSEP for rodent/HUMAN forms), the Na + /taurocholate cotransporting polypeptide (Ntcp/NTCP) and the asialoglycoprotein receptor are liver-specific. The other markers are also expressed in « simple » polarized cells of other tissues and are similarly localized in both types of polarized cells.

Biliary function is ensured by a set of transporters that need to be properly settled at their specific pole. At the apical pole of hepatocytes, several ATP-binding cassette (gene symbol Abc/ABC) transporters are localized, in particular, two multidrug resistance proteins Mdr1/MDR1 and Mdr2/MDR3, one multidrug resistance-associated protein Mrp2/MRP2 and Bsep/BSEP. Bsep/BSEP is the major bile salt transporter that ensures bile secretion at the BC [START_REF] Trauner | Bile salt transporters: molecular characterization, function, and regulation[END_REF]. Several clinical forms of cholestasis have been related to mutations in genes encoding for bile salt transporters [START_REF] Pauli-Magnus | Enterohepatic transport of bile salts and genetics of cholestasis[END_REF]. (Definition cholestasis : reduction or suppression of bile flow resulting from liver injury and diseases). For example, various BSEP mutations prevent the protein from trafficking to the apical membrane and account for the syndrome of progressive familial intrahepatic cholestasis of type 2 [START_REF] Strautnieks | A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis[END_REF]. Protein sorting is a prerequisite for polarized membrane domains. Most apical proteins of the hepatocyte are « indirectly » sorted, first addressed to the basolateral membrane prior to their subsequent transcytosis to the apical membrane [START_REF] Bartles | Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation[END_REF]. But apical Abc transporters such as Bsep are directly sorted to the apical pole through an endosomal compartment from which they can be released when increased bile secretion is needed [START_REF] Kipp | Trafficking of canalicular ABC transporters in hepatocytes[END_REF]. Myosin Vb and Rab11 participate in apical targeting of these recycling endosomes, thereby contributing to canalicular biogenesis [START_REF] Wakabayashi | Transporters on Demand: Intracellular Reservoirs and Cycling of Bile Canalicular ABC Transporters[END_REF].

At the sinusoidal pole of hepatocyte, Ntcp/NTCP and organic anion transporting polypeptide proteins are in charge of bile salt uptake from portal circulation. These transporters are essential for bile formation as 95% of the biliary secreted bile salts re-enter into portal circulation. Some members of the Mrp/MRP family, also localized at the sinusoidal pole, are responsible for the basolateral efflux of bile salts and organic anions. The Mrp3 up-regulation observed under cholestatic conditions facilitates clearance of anionic conjugates in the blood when canalicular secretion is impaired [START_REF] Donner | Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver[END_REF].

Among polarity markers, proteins of the apical junctional complex -the adherens and tight junctions- [START_REF] Nelson | Adaptation of core mechanisms to generate cell polarity[END_REF] and of the gap junctions [START_REF] Kojima | Regulation of the blood-biliary barrier: interaction between gap and tight junctions in hepatocytes[END_REF] are of special interest.

Cell junctions are essential for several hepatocyte-specific functions [START_REF] Vinken | Involvement of cell junctions in hepatocyte culture functionality[END_REF], including bile secretion [START_REF] Nathanson | Communication via gap junctions modulates bile secretion in the isolated perfused rat liver[END_REF], cytochrome P-450 isoenzyme expression [START_REF] Hamilton | Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell-cell interactions[END_REF] and glycogenolysis [START_REF] Nelles | Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice[END_REF]. According to their gate function in paracellular diffusion, tight junctions in hepatocytes can be called the blood-biliary barrier as they serve to keep bile in the BC away from blood circulation. These junctions are composed of transmembrane constitutive proteins and of various cytoplasmic proteins directly bound to the constitutive ones, such as the Zonula Occludens (ZO)-protein family. Recently, two of these proteins have been found to play a direct role in human hereditary liver diseases : claudin 1 in neonatal sclerosing cholangitis associated with ichthyosis syndrome [START_REF] Hadj-Rabia | Claudin-1 gene mutations in neonatal sclerosing cholangitis associated with ichthyosis: A tight junction disease[END_REF] and ZO-2 in familial hypercholanemia (Carlton et al., 2003). Tight and gap junctions are closely associated in hepatocytes. Connexin 32, specifically expressed in liver gap junctions, partly colocalizes with occludin and claudin 1 [START_REF] Kojima | Regulation of the blood-biliary barrier: interaction between gap and tight junctions in hepatocytes[END_REF]. These interactions could indicate some regulatory mechanism of the blood-biliary barrier by the gap junctions. In cholestasis, a loss of gap junctions is always observed in association with leaky tight junctions [START_REF] Anderson | Leaky junctions and cholestasis : A tight correlation[END_REF].

CYTOSKELETON

Polarity also means special cell shape and architecture. The cytoskeleton, widely responsible for this three dimensional organization, has a fundamental role in the generation of cell polarity [START_REF] Yeaman | New perspectives on mechanisms involved in generating epithelial cell polarity[END_REF]. The cytoskeletal components, microfilaments (MF), microtubules (MT) and intermediate filaments (IF), provide structural asymmetry in the plasma membrane; the three major cytoskeletal proteins -actin, tubulin and cytokeratin-are connected, directly or via protein scaffolds, to the plasma membrane especially at the intercellular junctions. How is the cytoskeletal network involved in hepatocyte polarity and functionality?

Studies on human isolated BC membrane preparations have provided clues on the organization of the BC cytoskeleton [START_REF] Tsukada | The structure and organization of the bile canalicular cytoskeleton with special reference to actin and actin-binding proteins[END_REF]. The BC is surrounded by two circumferential MF belts of actin and actin-binding proteins. One belt containing myosin II and associated with the adherens junctions is responsible for the contractility of BC implicated in the bile flow; the other belt regulates vesicle transport near the plasma membrane [START_REF] Tsukada | Bile canalicular contraction is coincident with reorganization of pericanalicular filaments and co-localization of actin and myosin-II[END_REF]. Experimental cholestasis models always show alterations in the pericanalicular MF. When cholestasis was induced in rats by treatment with phalloidin (which prevents F-actin depolymerization), bile flow and bile acid secretion decreased and tight junction permeability was altered. This demonstrates the role of MF on the hepatocyte junctional barrier and on bile production [START_REF] Elias | Phalloidin-induced cholestasis: A microfilament-mediated change in junctional complex permeability[END_REF]. Among the proteins associated with MF, the ezrin-radixin-moesin proteins are implicated in apical protein retention. Kikuchi et al. have shown that radixin-null mice have a loss of Mrp2 transporter at the BC membrane and hyperbilirubinemia [START_REF] Kikuchi | Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes[END_REF]. In human primary biliary cirrhosis, a large reduction in radixin expression is associated with a partial loss of canalicular MRP2 (Kojima, H. et al., 2003) .

MT are polarized filaments with the fast-growing minus-ends facing the apical pole of polarized cells (Figure 1). Highly concentrated near the BC [START_REF] Novikoff | Three-dimensional organization of rat hepatocyte cytoskeleton: relation to the asialoglycoprotein endocytosis pathway[END_REF], MT are involved in hepatic protein trafficking [START_REF] Pous | Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells[END_REF]. MT are also essential in BC formation : during epithelial polarity establishment, MT are reorganized according to an apico-basal polarity axis; this allows transport of apical proteins to the MT minus-ends which contributes to the apical pole formation (Cohen et al., 2004a). MT also organize signaling as demonstrated by the specific involvement of the dynamic MT sub-population in the growth hormone pathway [START_REF] Phung-Koskas | STAT5B-mediated Growth Hormone Signaling Is Organized by Highly Dynamic Microtubules in Hepatic Cells[END_REF].

IF are arranged as a pericanalicular sheath, inserted into desmosomes, around the BC that they stabilize. IF network perturbation by nickel leads to abnormal canalicular function [START_REF] Kawahara | Role of cytokeratin intermediate filaments in transhepatic transport and canalicular secretion[END_REF]. IF are also targets and modulators of toxic stress and apoptosis in hepatocyte [START_REF] Zatloukal | The keratin cytoskeleton in liver diseases[END_REF]. In hepatocyte, the keratin expression pattern is rather simple, exclusively composed of cytokeratins 8 and 18. In cytokeratin 8-null mice, the apical HA4 protein is not only found in BC as usual but also at the basolateral pole [START_REF] Ameen | Anomalous apical plasma membrane phenotype in CK8-deficient mice indicates a novel role for intermediate filaments in the polarization of simple epithelia[END_REF]. Mutations in cytokeratin 8 and 18 genes were shown to be risk factors in patients for developing liver cirrhosis [START_REF] Ku | Keratin 8 and 18 mutations are risk factors for developing liver disease of multiple etiologies[END_REF].

GOLGI APPARATUS AND ENDOPLASMIC RETICULUM

In addition to the cytoskeleton components, organelles are not randomly distributed in the cytoplasm. The Golgi apparatus, involved in protein selective targeting to the poles, is localized between nucleus and the apical pole in polarized cells (Figure 1), namely between the nucleus and BC in hepatocytes [START_REF] Musat | Reestablishment of cell polarity of rat hepatocytes in primary culture[END_REF]. During cell polarization, Golgi apparatus undergoes a complex rearrangement, moving along the new apico-basal polarity axis of MT towards their minus-ends in the apical region [START_REF] Bacallao | The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium[END_REF].

The ER is a continuous and dynamic membrane system, connected to the cytoskeleton and closely associated with plasma membrane and organelles such as Golgi apparatus [START_REF] Voeltz | Structural organization of the endoplasmic reticulum[END_REF]. It exhibits structural and functional distinct sub-regions [START_REF] Baumann | Endoplasmic reticulum of animal cells and its organization into structural and functional domains[END_REF] which fulfil specialized biological functions, such as calcium (Ca 2+ ) signalling. Stored in the smooth ER, Ca 2+ is released in response to specific signals through two types of Ca 2+ channels, the IP 3 Rs and the ryanodine receptors, organized in specific domains of the ER [START_REF] Meldolesi | The Heterogeneity of ER Ca2+ Stores Has a Key Role in Nonmuscle Cell Signaling and Function[END_REF]. The subcellular distribution of the three IP 3 R isoforms depends on cell types, physiological status [START_REF] Vermassen | Subcellular distribution of the inositol 1,4,5trisphosphate receptors: functional relevance and molecular determinants[END_REF]) and even correlates with polarity state in Madin-Darby canine kidney (MDCK) cells (Colosetti et al., 2003).

Hepatocytes are exceptionally active in protein and lipid synthesis. Accordingly, their ultrastructural examination reveals quantities of rough and smooth ER, occupying 15% of the total cell volume. Ca 2+ signalling regulates diverse hepatocyte functions, in particular bile secretion [START_REF] Leite | Calcium signaling in the hepatocyte[END_REF]) and a predominant role of IP 3 Rs was evidenced. Hepatocytes express only the IP 3 R1 and IP 3 R2 isoforms [START_REF] Dufour | Expression of inositol 1,4,5trisphosphate receptor isoforms in rat cirrhosis[END_REF]. The preponderant IP 3 R2 isoform is concentrated at the BC level, precisely where calcium signals originate [START_REF] Nathanson | Multistep mechanism of polarized Ca2+ wave patterns in hepatocytes[END_REF]. This enrichment might be essential for the formation of Ca 2+ waves in hepatocytes in vivo and could also play a role in the formation of intercellular Ca 2+ waves because some gap junctions, necessary for the cell-to-cell wave propagation, are also located in that region [START_REF] Hernandez | The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves[END_REF]. So, although still poorly understood, ER could take part in polarity.

IN VITRO HEPATOCYTE MODELS : MAIN USES, INTERESTS AND LIMITS

A comparison of these various lines is given in Supplementary Table 2.

HUMAN LINES

Huh7 cells are poorly polarized. They make few BC structures according to the localization of apical aminopeptidase N (immunohistochemistry and immunoelectron microscopy) but the protein is also present in numerous cytoplasmic vesicles and on the remainder of the cell membrane (Chiu et al., 1990). ZO-1 protein does not localize at the BC structures but makes belts at the cell apex as in « simple » polarized cells [START_REF] Harada | Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes[END_REF]. Huh7 cells have been used in the study of hepatitis C virus [START_REF] Lohmann | Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line[END_REF] and of copper excretion in Wilson's disease [START_REF] Harada | Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes[END_REF].

HepG2 cells have polarity features. BC like-spaces, microvilli-lined and sealed by tight junctions are present. Apical markers, dipeptidyl peptidase IV (DPP IV), B442 (BC antigen) and ABC transporters, especially BSEP, are properly addressed [START_REF] Zegers | Sphingolipid Transport to the Apical Plasma Membrane Domain in Human Hepatoma Cells Is Controlled by PKC and PKA Activity: A Correlation with Cell Polarity in HepG2 Cells[END_REF][START_REF] Kubitz | Trafficking of the bile salt export pump from the Golgi to the canalicular membrane is regulated by the p38 MAP kinase[END_REF]. Various cytoskeletal components (actin, villin, myosin II and b-tubulin) localize as in hepatocytes [START_REF] Sormunen | Bile canaliculus formation in cultured HEPG2 cells[END_REF]. HepG2 cells express some efflux transporters (Cantz et al., 2000;[START_REF] Vee | Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line[END_REF] and accumulate cholylglycylamidofluorescein (a fluorescent bile acidderivative) in more than 70% of their BC structures (Figure 2). However, only 20-40% of HepG2 cells are polarized [START_REF] Theard | Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions[END_REF]; their BC structures are hard to detect in phase contrast and mislocalization of apical markers and microvilli may occur [START_REF] Zegers | Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells[END_REF]. Recently, HepG2 cell polarity was improved by growth in multilayering clusters on their predeposited extracellular matrix; this led to the formation of elongated canalicular lumens that span several cells and appear as large acinar structures [START_REF] Herrema | Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells[END_REF]. Since HepG2 cells express numerous metabolizing enzymes, they are an excellent tool for genotoxic studies [START_REF] Knasmuller | Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge[END_REF]. They are currently used for polarity development and lipid and protein trafficking studies [START_REF] Theard | Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions[END_REF].

The HepaRG cell line displays an heterogeneous morphology with hepatocyte-like and biliary-like epithelial phenotypes [START_REF] Gripon | Infection of a human hepatoma cell line by hepatitis B virus[END_REF]. Hepatocyte-like cells have BC-like structures (phase contrast and electron microscopy), that are functional as evidenced by fluorescein excretion (Cerec et al., 2007). They correctly localize DPP IV, E-cadherin, ZO-1 and cytokeratin 18 [START_REF] Parent | Origin and characterization of a human bipotent liver progenitor cell line[END_REF]. More polarity markers have to be looked for to better define the phenotype. The HepaRG cells express drug transporters and various active P450 cytochromes at levels comparable to those found in cultured human hepatocytes. Thus, the HepaRG cell line is a good tool for xenotoxic and genotoxic studies [START_REF] Guillouzo | The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics[END_REF]. As bipotent progenitors, HepaRG cells are of great interest for differentiation studies. However, no more than 55% of the cells display the hepatocytic phenotype, favoured by growth with 2% DMSO and epidermal growth factor.

CELL LINES DERIVED FROM THE Fao RAT HEPATOMA

Several polarized lines have been obtained by hybridization and various treatments.

WIF-B and WIF-B9

were derived from hybrid cells obtained by fusion of Fao with human fibroblast (Cassio et al., 1991;[START_REF] Ihrke | WIF-B cells: an in vitro model for studies of hepatocyte polarity[END_REF]. More than 90% of the cells create BC structures, well visible in phase contrast microscopy (Figure 2), microvilli-lined and sealed by typical tight junction continuous strands (freeze-fracture replica) [START_REF] Decaens | Establishment of hepatic cell polarity in the rat hepatoma-human fibroblast hybrid WIF-B9. A biphasic phenomenon going from a simple epithelial polarized phenotype to an hepatic polarized one[END_REF]. These BC are surrounded by sheats of actin, myosin IIA, ezrin, a-tubulin and cytokeratins 8 and 18, as in hepatocytes in situ [START_REF] Decaens | Spatiotemporal expression of catenins, ZO-1, and occludin during early polarization of hepatic WIF-B9 cells[END_REF]. WIF-B and WIF-B9 cell lines express and correctly settle a large pool of the polarity markers listed in supplementary Table 1 [START_REF] Decaens | Establishment of hepatic cell polarity in the rat hepatoma-human fibroblast hybrid WIF-B9. A biphasic phenomenon going from a simple epithelial polarized phenotype to an hepatic polarized one[END_REF][START_REF] Bender | Expression and localization of hepatocyte domainspecific plasma membrane proteins in hepatoma x fibroblast hybrids and in hepatoma dedifferentiated variants[END_REF]. These cells communicate via connexin 32-constituted gap junctions (Chaumontet et al., 1998). Their Golgi apparatus is located between the BC region and the nucleus [START_REF] Pous | Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells[END_REF]. They make vectorial transport of bile acids (Figure 2) [START_REF] Bravo | Efficient in vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells[END_REF] and express several rat and even human hepatobiliary transporters [START_REF] Briz | Ca(2+)mobilizing hormones induce sequentially ordered Ca2+ signals in multicellular systems of rat hepatocytes[END_REF]. WIF-B and WIF-B9 cell lines are now used as in vitro models for many hepatocytic trait studies. The respective role of stable and dynamic MT classes in specialized transport steps was evidenced using WIF-B9 cells [START_REF] Pous | Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells[END_REF]. These cells permit hepatocytic traffic studies because they sort most of their apical proteins just as hepatocytes do [START_REF] Ihrke | Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes[END_REF][START_REF] Sai | Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells[END_REF][START_REF] Nyasae | Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells[END_REF][START_REF] Wakabayashi | Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes[END_REF][START_REF] Guo | NH2-terminal signals in ATP7B Cu-ATPase mediate its Cu-dependent anterograde traffic in polarized hepatic cells[END_REF][START_REF] Ramnarayanan | Exogenous MAL Reroutes Selected Hepatic Apical Proteins into the Direct Pathway in WIF-B Cells[END_REF]. Overcoming drug resistance [START_REF] Briz | Usefulness of liposomes loaded with cytostatic bile acid derivatives to circumvent chemotherapy resistance of enterohepatic tumors[END_REF] and the hepatotoxicity profile of chemicals [START_REF] Biagini | Investigation of the hepatotoxicity profile of chemical entities using Liverbeads and WIF-B9 in vitro models[END_REF] were evaluated using WIF-B9 cells. Bile acid metabolism [START_REF] Monte | Predominance of human versus rat phenotype in the metabolic pathways for bile acid synthesis by hybrid WIF-B9 cells[END_REF] and biliary cholesterol transport [START_REF] Harder | SR-BI Undergoes Cholesterol-stimulated Transcytosis to the Bile Canaliculus in Polarized WIF-B Cells[END_REF] have been studied, since WIF-B and WIF-B9 cells correctly localize transporters for bile secretion and aquaporin water channels [START_REF] Nies | Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF-B[END_REF][START_REF] Gradilone | Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B[END_REF]. Recently, alcohol-induced hepatotoxicity was investigated using WIF-B cells which efficiently metabolize ethanol into acetaldehyde as hepatocytes in situ [START_REF] Kannarkat | Microtubules are more stable and more highly acetylated in ethanol-treated hepatic cells[END_REF].

WIF-B9 cells can also be used to study both types of polarity (Figure 1) since they express the « simple » polarized phenotype before expressing the typical hepatocytic phenotype [START_REF] Decaens | Establishment of hepatic cell polarity in the rat hepatoma-human fibroblast hybrid WIF-B9. A biphasic phenomenon going from a simple epithelial polarized phenotype to an hepatic polarized one[END_REF]Cohen et al., 2004a).

11-3 is a mouse monochromosomal-Fao rat hepatoma hybrid. More than 90% of the cells form functional BC-like structures and have an hepatocyte polarized phenotype according to the localization of many membrane polarity markers. Before expressing the hepatocyte phenotype, 11-3 cells express the « simple » polarized phenotype. Two subclones, 11-3-5 which only displays the simple polarized phenotype, and 11-3-1 which is unpolarized [START_REF] Peng | How to induce non-polarized cells of hepatic origin to express typical hepatocyte polarity: generation of new highly polarized cell models with developed and functional bile canaliculi[END_REF]) might be useful to compare protein expression profile with polarity state. 11-3 cells were used to study the regulation of alcohol dehydrogenase [START_REF] Majewski | The class I alcohol dehydrogenase gene is glucocorticoidresponsive in the rat hepatoma microcell hybrid cell line, 11-3[END_REF].

Fao-BA1 and C2rev7-BA1 were generated by Fao or C2rev7 induction with chenodeoxycholic acid [START_REF] Ng | Reversible induction of rat hepatoma cell polarity with bile acids[END_REF]. They form functional BC-like structures easily detected in phase contrast, microvilli-lined, and delimited by tight junctions (electron microscopy). Apical protein markers (gglutamyl transferase, aminopeptidase N, Bsep and Mrp2, but not DPP IV), 1-18 basolateral marker and ZO-1 protein are correctly localized. However, some polarity traits are missing: Ntcp and organic anion transporting polypeptide 1 are localized in the cytoplasm rather than at the basolateral membrane ; apical markers are not present in all the BC. As hepatocytic phenotype is maintained only in the presence of bile acids, Fao-BA1 and C2rev7-BA1 cells may be suitable for studies on hepatocyte polarization and bile salt trafficking.

Can 3-1 and Can 10 were generated by Fao culture in spheroids, which favours contacts between cells, followed by enrichment in BC-forming zones in monolayers.

More than 95% of Can 3-1 cells form BC structures, easily detectable in phase contrast. Numerous apical, basolateral and tight junction protein markers are correctly localized. Can 3-1 cells make vectorial transport of bile acids but the expression of their hepatobiliary transporters is not bile-acid inducible [START_REF] Briz | Ca(2+)mobilizing hormones induce sequentially ordered Ca2+ signals in multicellular systems of rat hepatocytes[END_REF]. They polarize only when plated at high density, which makes them particularly suitable for studies on hepatocyte polarization.

Can 10 cells form long and branched BC structures made by union of the apical poles of tens of adjacent cells (Figure 3). These long BC, hardly detectable in phase contrast, are delimited by a complex network of tight junctions (freeze-fracture replica) (unpublished data) and surrounded by actin, radixin, ezrin, a-tubulin and g-tubulin (Figures 34). According to the correct localization of many polarity markers, more than 90% of Can 10 cells are polarized and vectorially transport bile acids. Their transporters are correctly localized (as illustrated for multidrug resistance protein in Figure 3) and are inducible by bile-acids [START_REF] Briz | Ca(2+)mobilizing hormones induce sequentially ordered Ca2+ signals in multicellular systems of rat hepatocytes[END_REF]. Therefore, Can 10 is the first model with functional highly developed BC, reminiscent of those found in vivo. According to their protein repertoire close to that of the hepatocyte, especially for claudins (unpublished data), Can 10 cells can be a useful tool in tight junction studies. They may contribute to understanding the way that neighbouring cells are able to form long BC.

CELL LINES DERIVED FROM LIVER OF TRANSGENIC MICE

mhAT3F is derived from the liver of a transgenic mouse expressing SV40. It forms organoids in a Matrigel-based 3D culture with intercellular lumens having polarized traits: they are microvillilined with tight junction complexes clearly visible in electron microscopy; ZO-1, g-catenin, actin and radixin are localized at membranes delimiting the lumens. However, these apical lumens are more related to acini than to BC-structures. mhAT3F cells have been used to study apoptotic signalling [START_REF] Haouzi | Threedimensional polarization sensitizes hepatocytes to Fas/CD95 apoptotic signalling[END_REF].

AML12 and AML14 are derived from the livers of transgenic mice overexpressing transforming growth factor a. Adjacent cells form BC-like structures with microvilli and tight junctions (electron microscopy). They express the liver-specific connexins 32 and 26. The stable AML12 line expresses numerous hepatocyte secretory proteins and enzymes. This line is used for studying the role of transforming growth factor b1 [START_REF] Yang | Transforming growth factor-[beta]1 induces epithelial-to-mesenchymal transition and apoptosis via a cell cycle-dependent mechanism[END_REF] and of cytokines (Brooling et al., 2005) on various cellular processes and lipogenesis in steatotic hepatocytes [START_REF] Schadinger | PPAR{gamma}2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes[END_REF].

EMK1 OVEREXPRESSING MDCK CELLS

Finally, we want to point to MDCK cells, of kidney origin but able to express the typical hepatocyte polarity by overexpression of EMK1. EMK1 is the mammalian homologue of PAR-1, an essential kinase for the establishment of polarity in Caenorhabditis elegans, regulating MT organization.

Increased expression of EMK1 promotes hepatocytic-type lumen formation between more than 90% of neighbouring MDCK cells during their polarization (Cohen et al., 2004a). These lumens are microvilli-lined and express the MDCK apical markers gp135 and gp114, ZO-1 and actin. Ecadherin localized on the plasma membrane is excluded from these lumens. MT are oriented with minus-end towards the lumens. According to these criteria, these lumens look like BC structures.

The transcytotic trafficking route for apical markers, typical of hepatocyte, is promoted in those cells (Cohen et al., 2004b). So EMK1-overexpressing MDCK cells constitute an excellent model to study the regulation of epithelial lumen morphogenesis and the candidates involved in the branching decision between simple or hepatocytic-like phenotypes.

PERSPECTIVES

All the cell lines described above have interesting features and deserve to be further characterized. Some aspects of cell organization have not been yet approached, for example Ca 2+ signalling. Very little information is available on their ER organization and IP 3 R distribution. We have used two of these cell lines to study the distribution of the three IP 3 R isoforms. In WIF-B9 and Can10 cells, the three isoform mRNA were detected (K. Schmidt, J.F. Dufour, personal communication). In WIF-B9 cells, IP 3 R2 and IP 3 R3 have an enriched pericanalicular localization whereas IP 3 R1 is homogeneously distributed in the cytoplasm (Figure 4). The expression of IP 3 R3, an isoform absent from hepatocytes but present in cholangiocytes, was noticed in several cells in culture [START_REF] Blondel | Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues[END_REF]. Sehgal et al. have also found the expression of the three IP 3 Rs in WIF-B cells but they did not observe a polarized distribution for either IP 3 R2, or for IP 3 R3 [START_REF] Sehgal | Protein 4.1N does not interact with the inositol 1,4,5-trisphosphate receptor in an epithelial cell line[END_REF]. As we both used the same commercial antibodies for IP 3 R3, this discrepancy could be due to a difference in the polarity state of the cells; we have previously shown that several days in culture are needed before WIF-B9 cells express a fully polarized hepatocyte phenotype. Can 10 cells express the three IP 3 Rs with a pericanalicular enrichment for IP 3 R2 (not shown) and IP 3 R3 (Figure 4). Thus, in WIF-B9 and Can 10 cells, two IP 3 Rs exhibit a polarized distribution as observed in hepatocytes. These first results indicate that polarized cell models coud be very useful to study the relationship between ER organization, Ca 2+ signalling and polarity.

Numerous studies have been carried out to find a way to keep the hepatocytes isolated from normal liver in well differentiated and polarized status. For example, hepatocyte culture on and in various matrices preserves more or less their polarized functional features [START_REF] Moghe | Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function[END_REF]. However polarity establishment study with normal hepatocytes is limited because these cells do not divide.

An in vivo model with chimeric human livers in mice was recently developed [START_REF] Kneteman | Mice with chimeric human livers: who says supermodels have to be tall?[END_REF] in which the human hepatocytes repopulated the diseased mouse liver and formed functional BC. These chimeric human-mouse livers were used to study hepatitis B and C virus infection. This model of great interest has also limits as pointed out by users who stated : « While the scope of utility of the chimeric mouse model is vast, the model is not without its limitations. Investigators should continue to work towards developing stable cell lines that maintain a differentiated hepatocyte phenotype. » [START_REF] Kneteman | Mice with chimeric human livers: who says supermodels have to be tall?[END_REF].

The development of new cell lines -a fully polarized human line would be necessary-can lead to useful tools in hepatocyte polarity and functionality studies, as shown with the models described in this review. Brooling, J.T., Campbell, J.S., Mitchell, C., Yeoh, G.C. and Fausto, N. (2005). Differential regulation of rodent hepatocyte and oval cell proliferation by interferon gamma. Hepatology 41, 906-915 Cantz, T., Nies, A.T., Brom, M., Hofmann, A.F. and Keppler, D. (2000). MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells. Am. J.

Online supplementary material

  Physiol. Gastrointest. Liver Physiol. 278, G522-531 Carlton, V.E., Harris, B.Z., Puffenberger, E.G., Batta, A.K., Knisely, A.S., Robinson, D.L.,Strauss, K.A., Shneider, B.L., Lim, W.A., Salen, G. et al. (2003). Complex inheritance of familial hypercholanemia with associated mutations in TJP2 andBAAT. Nat. Genet. 34, 91-96 Cassio, D., Hamon-Benais, C., Guerin, M. and Lecoq, O. (1991). Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatomaderived hybrid cells able to form functional bile canaliculi in vitro. J. Cell Biol. 115, 1397-1408 Cassio, D., Macias, R.I., Grosse, B., Marin, J.J. and Monte, M.J. (2007). Expression, localization, and inducibility by bile acids of hepatobiliary transporters in the new polarized rat hepatic cell lines, Can 3-1 and Can 10. Cell Tissue Res. 330, 447-460 Cerec, V., Glaise, D., Garnier, D., Morosan, S., Turlin, B., Drenou, B., Gripon, P., Kremsdorf, D., Guguen-Guillouzo, C. and Corlu, A. (2007). Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor. Hepatology 45, 957-967 Chaumontet, C., Mazzoleni, G., Decaens, C., Bex, V., Cassio, D. and Martel, P. (1998). The polarized hepatic human/rat hybrid WIF 12-1 and WIF-B cells communicate efficiently in vitro via connexin 32-constituted gap junctions. Hepatology 28, 164-172 Chiu, J.H., Hu, C.P., Lui, W.Y., Lo, S.C. and Chang, C.M. (1990). The formation of bile canaliculi in human hepatoma cell lines. Hepatology 11, 834-842 Cohen, D., Brennwald, P.J., Rodriguez-Boulan, E. and Musch, A. (2004a). Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton. J. Cell Biol. 164, 717-727 Cohen, D., Rodriguez-Boulan, E. and Musch, A. (2004b). Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells. Proc.Natl.Acad.Sci. USA 101, 13792-13797 Colosetti, P., Tunwell, R.E., Cruttwell, C., Arsanto, J.P., Mauger, J.P. and Cassio, D. (2003). The type 3 inositol 1,4,5-trisphosphate receptor is concentrated at the tight junction level in polarized MDCK cells. J. Cell Sci. 116, 2791-2803 apical poles of two WIF-B9 cells. Confocal microscopy : compilations of 3-8 xy sections taken at 0.4 mm steps and, for Can 10, xz sections (g, i, k) taken as indicated (long arrows) on f, h, j images. Scale bar 10 mm.

Figure 5 :

 5 Figure 5 : possible cover figure

Table 1 :

 1 MEMBRANE PROTEIN MARKERS OF POLARITY IN RAT HEPATOCYTE

Table 2 :

 2 COMPARISON OF HEPATIC CELL LINES enhanced expression of canalicular ABC transporters involved in phase III of hepatic detoxification. Toxicology 232, 24-36

Acknowledgments : We greatly thank B.R. Stevenson and D. Haouzi for kindly providing antibodies, J.G. Marin for cholylglycylamidofluorescein and K. Schmidt and J.F. Dufour for unpublished results on IP 3 R mRNA levels. We also thank V. Nicolas (IFR141-ITFM, Chatenay Malabry, France) for help with confocal analysis and L. Mery for critical reading of the manuscript. This work has been funded by institutional grants from INSERM and supported by a grant from Egide (PAI Picasso) and the Acción Integrada Hispano-Francesa (HF2003-0089).

LEGENDS

Figure 1 : Diagrams of a simple epithelial cell and of an hepatocyte.

The apical pole is separated from the lateral pole by tight junctions .

The Golgi apparatus is always facing the apical pole.

Microtubules are oriented with minus-end at the apical pole .

BC : bile canaliculus.