The maintenance and generation of membrane polarity in hepatocytes (2004)

Hepatocytes, the major epithelial cells in the liver, are highly polarized. Their plasma membranes are separated by tight junctions into sinusoidal–basolateral and canalicular–apical domains, which contain distinct sets of proteins and lipids. A normal membrane polarity is vital for hepatocytes to perform many diverse functions, such as canalicular bile secretion and simultaneous sinusoidal secretion of large quantities of serum proteins into blood. Hepatocyte polarity is lost in many diseases like cholestasis. A complete understanding of the molecular mechanisms involved in hepatocyte polarity therefore is of considerable significance to both liver cell biology and the pathogenesis of liver diseases. To maintain the distinct distribution of proteins and lipids in the canalicular and sinusoidal membranes, hepatocytes have developed complex polarized trafficking and retention mechanisms. The establishment of membrane polarity in hepatocytes begins during liver embryogenesis. During hepatocyte differentiation, specific routes and mechanisms are defined for the delivery of plasma membrane proteins. How this polarity is maintained in adult hepatocytes and is generated during development are two major unanswered questions in this field. In this review, we first give an overview of the current knowledge about how membrane polarity is maintained in hepatocytes with the focus on recent developments in polarized protein trafficking. We then present a hypothetical model about how membrane polarity may be generated during liver development, and finally, we present an example of how membrane polarity is altered in cholestasis. Readers are directed to recent reviews for discussion of lipid trafficking in hepatocytes.1, 2 TGN, trans-Golgi network; DPP IV, dipeptidyl peptidase IV; pIgA-R, polymeric immunoglobulin A receptor; GPI, glycosyl-phosphatidylinositol; 5′NT, 5′-nucleotidase; MDCK, Madin-Darby canine kidney cells; ATP, adenosine triphosphate; Mdr1, multidrug resistance protein 1; Bsep, bile salt export pump; GFP, green fluorescent protein; Mrp2, multidrug resistant associated protein; LDL-R, low density lipoprotein receptor; AP, adaptor protein; SNARE, the soluble NSF attachment protein receptor; ERM, ezrin-radixin-moesin; TLCA, taurolithocholic acid. Three major intracellular trafficking pathways are involved in the polarized expression of plasma membrane proteins in hepatocytes. Through biogenesis, a plasma membrane protein is first synthesized in the endoplasmic reticulum, modified in Golgi, and finally sorted at the trans-Golgi network (TGN). From the TGN, they traffic along the post-Golgi biosynthetic pathway to the canalicular or sinusoidal membrane domain. After a protein reaches the plasma membrane, it can be either retained or internalized (endocytosed). After endocytosis, some proteins are transported to the opposite membrane domain of hepatocytes, a process known as transcytosis. During the past two decades, a key quest in liver cell biology has been to understand the trafficking routes and molecular mechanisms that are used by various canalicular and sinusoidal proteins as they journey along these pathways. Previous studies have demonstrated that proteins destined for the sinusoidal membrane in hepatocytes traffic from the Golgi complex to their destination directly (Fig. 1A).3 However, most canalicular proteins traffic by an indirect route, first to the sinusoidal membrane, followed by transcytosis through basolateral early endosomes and a subapical compartment, before final targeting to the canalicular domain (Fig. 1A). These canalicular proteins include single transmembrane proteins (such as dipeptidyl peptidase IV [DPP IV], aminopeptidase N, and polymeric immunoglobulin A receptor [pIgA-R]) and the glycosyl-phosphatidylinositol (GPI)-anchored proteins like 5′-nucleotidase (5′NT). Until recently, the indirect transcytotic pathway was thought to be the only route by which canalicular proteins reach their destination in hepatocytes. In contrast, most apical proteins traffic directly from TGN to the apical membrane in Madin-Darby canine kidney (MDCK) cells, a polarized kidney cell line, whereas both direct and indirect pathways operate in the retinal pigment and intestinal epithelial cells.4, 5 The post-Golgi biosynthetic trafficking and endocytic routes in hepatocytes. A hepatocyte couplet is shown with the basolateral–sinusoidal and apical–canalicular membranes separated by tight junctions (TJ). (A) Sinusoidal membrane proteins (blue circles) take a direct route (blue arrows) from the TGN to the basolateral membrane. Most canalicular proteins (green patches) are targeted initially to the basolateral membrane and then undergo transcytosis (green arrows) through the basolateral early endosome (BEE) and a subapical compartment (SAC) before reaching the canalicular membrane. (B) In contrast, Mdr1 (solid red patch) traffics directly (red arrow) from the TGN to the canalicular membrane. Bsep (hatched red patch) may traffic first to a subapical compartment (also termed as SAC) and then to the canalicular membrane. It remains unclear if Bsep and the transcytosed proteins traffic through the same SAC. (C) Some sinusoidal proteins recycle between the basolateral membrane, BEE, and SAC (dashed blue lines). The SAC functions as a common sorting station for both transcytosed and endocytosed proteins.65 Some sinusoidal proteins are targeted to late endosome (LE) and lysosomes (L) for degradation after endocytosis. At the canalicular membrane, most canalicular proteins are very slowly endocytosed with little recycling (dashed thin green lines). When an apical phosphoinositide 3 kinase is inhibited, some canalicular proteins (green patches) accumulate in lysosomes.46 (D) Mdr1 and Bsep both may recycle (dashed red lines) between the canalicular membranes and subapical compartment(s). The exact endocytic routes for Mdr1 and Bsep remain to be determined (indicated by ?). Possible intracellular compartments en route may include the apical early endosome (AEE), SAC, the apical recycling endosome (ARE), and lysosome for degradation. BC, bile canaliculi, N, nucleus. Recently, two canalicular adenosine triphosphate (ATP)–binding cassette transporters, the multidrug resistance protein 1 (Mdr1) and the bile salt export pump (Bsep), were shown to traffic directly from the Golgi to the canalicular domain (Fig. 1B). These findings were based on in vivo pulse-chase labeling in rats6 and expression of green fluorescent protein (GFP) fusion constructs in WIF-B cells, a polarized hepatic hybrid cell line.7 In addition to Mdr1 and Bsep, newly synthesized sphingolipids also reach the canalicular membrane directly from the TGN in hepatocytes.8 However, it remains unclear whether another major canalicular ATP-binding cassette transporter, the multidrug resistant-associated protein (Mrp2), which contains PDZ domains (a motif originally identified in postsynaptic protein PSD-95, Drosophila tumor suppressor Dlg and tight junction protein ZO-1; and used extensively for forming molecular scaffold as described below), also traffic directly from the Golgi to canalicular membrane. Indeed, a recent study demonstrated that Mrp2 resides in the same intracellular vesicles as pIgA-R, a canalicular protein that undergoes transcytosis.9 Much of our knowledge of the molecular mechanisms involved in polarized trafficking comes from work on epithelial cells other than hepatocytes.10 Direct basolateral targeting often involves cytoplasmic sorting signals, which can be either dileucine or tyrosine-based motifs.11 Two sinusoidal membrane proteins, the low-density lipoprotein receptor (LDL-R) and the sodium-taurocholate cotransporter, contain two tyrosine-based sorting signals.12, 13 Mutation of a critical glycine adjacent to the high-capacity tyrosine signal in LDL-R results in the mistargeting of LDL-R to the canalicular membrane in the WIF-B cells.14 Interestingly, β-turn structures, which are common to tyrosine-based basolateral sorting and endocytosis signals, are not present in the two tyrosine signals of sodium-taurocholate cotransporters.13 Basolateral sorting signals are believed to be recognized at the TGN by adaptor protein (AP) complexes, which are tetrameric protein complexes involved in both biosynthetic and endocytic sorting.15 Hepatocytes do not express μ1B, one important subunit of the AP-1B complex.16 μ1B is indispensable in the sorting of many basolateral proteins in kidney epithelial cells.17 It is not clear if hepatocytes use an unknown isoform of μ1 or a distinct AP complex to recognize the sinusoidal protein cargos. After they are bound to the AP complex, the sinusoidal proteins may be targeted and fused to the sinusoidal membrane, a process mediated in some epithelial cells by the soluble NSF (N-ethyl Maleimide-sensitive fusion protein) attachment protein receptor (SNARE) and an octomeric protein complex, which in yeast is called the exocyst-sec6/8 complex.18 The exact composition of the basolateral SNARE and exocyst complexes in hepatocytes and their roles in sinusoidal targeting have not been established. The canalicular transcytosing protein pIgA-R also takes a transcytotic route in MDCK and intestinal epithelial cells. Transcytosis of pIgA-R is stimulated by binding of immunoglobulin A and regulated by phosphorylation.19 pIgA-R contains a series of targeting signals within a cytoplasmic domain of 103 amino acids, which include a basolateral sorting signal and two tyrosine-based endocytosis signals.19 Notably, the basolateral sorting signal of pIgA-R is not dileucine or tyrosine based, and hence is different from the direct sinusoidal sorting signals discussed above. Different mechanisms other than the AP complex may determine the basolateral sorting of pIgA-R. The sorting signals in other single transmembrane proteins that undergo transcytosis are less well defined.20 Compared with direct basolateral sorting, the signals and machinery responsible for apical sorting are less well understood. Thus far, three types of apical sorting signals have been described: (1) GPI anchors, (2) transmembrane domains, and (3) N-glycans.10 In addition, the sphingolipid- and cholesterol-enriched domains, also known as lipid rafts, function as sorting platforms for apical cargos in the TGN.21 Lipid rafts are characterized by insolubility to detergent extraction at cold temperature. The enrichment of cholesterol and sphingolipid in canalicular membrane of hepatocytes has long been noted.22 Two recent independent studies demonstrate that lipid rafts mediate both transcytosis and direct apical trafficking of canalicular proteins in hepatocytes.23, 24 Slimane et al.23 showed that a GPI-anchored model protein, GPI-GFP, reaches the apical membrane of hepatic HepG2 cells by transcytosis and largely is present in lipid rafts, which are insoluble in both Triton X-100 and Lubrol WX at 4°C (Triton rafts). In contrast, two direct trafficking apical proteins, Mdr1-GFP and ATP7B (a P-type copper transporting ATPase), are contained in a distinct type of raft (Lubrol rafts), which are insoluble in Lubrol WX but are soluble in the stronger detergent, Triton X-100. Pulse-chase labeling indicated that both Triton and Lubrol rafts are formed at the TGN in HepG2 cells. Cholesterol depletion moderately increased the solubility of GPI-GFP to Triton X-100 without affecting its apical sorting. However, the same treatment led to missorting of Mdr1-GFP to the basolateral domain in HepG2 cells. Triton and Lubrol rafts also have been described in MDCK cells and segregate the GPI-anchored proteins from multispanning transmembrane proteins in the apical membrane.25 Nyasae et al.24 examined the solubility of a number of WIF-B cell apical proteins to Triton X-100 at 4°C. Two GPI-anchored proteins, 5′NT and CD59, are virtually insoluble to Triton X-100. In contrast, the single transmembrane proteins DDP IV and pIgA-R are completely soluble. Thus, not all of these canalicular proteins are contained in rafts, although they all use the indirect transcytotic pathway. Other mechanisms in addition to rafts must determine their indirect apical sorting. Moreover, the detergent-insoluble apical proteins seem to enter lipid rafts at different stages in the transcytotic pathway and with different kinetics.24 In this study, cholesterol depletion after 60 minutes severely impaired transcytosis of both raft-associated (CD59) and nonassociated proteins (DDP IV), presumably by blocking transcytotic flux from basolateral endosomes.24 Interestingly, cholesterol depletion did not affect the apical trafficking of GPI-GFP after 180 minutes in HepG2 cells in the study by Slimane et al.23 It is conceivable that the model protein GPI-GFP may not reflect faithfully the apical sorting of endogenous GPI-anchored canalicular proteins like CD59 and 5′NT. Despite this discrepancy, these two studies23, 24 together show that lipid rafts mediate the apical trafficking of some canalicular proteins in hepatocytes and that cholesterol is probably required for both Triton and Lubrol rafts. Furthermore, these two studies emphasize the complexity of membrane microdomains in the plasma membrane of hepatocytes. Judged by the sole criteria of detergent insolubility, the canalicular membrane consists of at least two types of rafts and a pool of single transmembrane proteins, some of which may be partly raft associated.23, 24 Lipid rafts also exist in the sinusoidal membrane of hepatocytes because some transcytosing proteins may have been incorporated into rafts before their transit to the sinusoidal membrane. In addition, caveolae, a special type of raft, have been detected at microvilli beneath the sinusoidal membranes of hepatocytes26 and contain raft-resident signaling proteins such as Ras and protein kinase C.27 Taken together, these studies suggest that both the canalicular and sinusoidal membranes can be viewed as a mosaic of raft-associated and nonassociated microdomains. A comprehensive analysis of detergent solubility of all canalicular and sinusoidal proteins will be required to appreciate fully the diversity of microdomains in the plasma membrane of hepatocytes and their functional implication. It also would be interesting to examine directly rafts and other microdomains in live hepatic cells using high-resolution fluorescence microscopy, which has been used successfully to examine raft-resident proteins in several cell types.28 Although it is not yet certain how raft mechanisms mediate the apical sorting of canalicular proteins, the work by Slimane et al.23 suggests that Lubrol rafts may segregate multispanning transmembrane proteins and may influence their direct apical sorting in HepG2 cells. It remains to be determined if other ATP-binding cassette proteins that traffic directly to the canalicular domain, such as Bsep, reside in the same type of lipid rafts. As discussed above, most GPI-anchored and single transmembrane proteins traffic directly from the TGN to the apical membrane in MDCK cells but indirectly via transcytosis to the canalicular membrane in hepatocytes. Why is there such a difference? Presumably, the plasma membrane and sorting mechanisms are organized differently between these two cell types. For of DPP IV and pIgA-R the transmembrane are the apical membrane in MDCK but the basolateral domain in hepatic WIF-B In hepatocytes, most proteins are the sinusoidal domain. proteins that undergo transcytosis seem to be sorted at the TGN as the sinusoidal proteins, that they are initially targeted to the sinusoidal domain. Basolateral endosomes also have been to function as a sorting station for proteins that undergo transcytosis to the apical membrane in In contrast, Mdr1 and Bsep must be sorted differently because they traffic directly to the canalicular domain. How these proteins are sorted through the TGN remains to be Transcytosis is not a to hepatocytes because a number of apical proteins are partly by transcytosis and partly by direct targeting to the apical membrane in intestinal epithelial Transcytosis also to be regulated during epithelial In cells, transcytosis functions as a major apical sorting for DPP IV in early epithelial differentiation, but is by direct apical targeting in epithelial It is that cells have protein secretion the basolateral membrane like hepatocytes. sinusoidal protein which is early in hepatocyte and in adult liver, may be the transcytosis is a major apical sorting for most canalicular proteins in adult hepatocytes. Interestingly, Bsep, a direct trafficking canalicular protein, is detected in liver only after with the that direct apical targeting may be in hepatocytes at a of In contrast, Mrp2 is detected at the bile before with the that Mrp2 may be sorted via the transcytotic than the direct pathway. To understand fully the in polarized trafficking between hepatocytes and other epithelial cells, a comprehensive of the and complexes involved in protein sorting will be For soluble NSF (N-ethyl fusion protein) attachment a key of the SNARE complex, is present at both the apical and basolateral membranes of many epithelial cells but is from the canalicular membrane of The apical SNARE complex the and fusion of Triton rafts GPI-anchored to the apical membrane in MDCK It remains to be determined if the of soluble NSF attachment in the canalicular membrane to the of transcytosis as a major apical delivery in hepatocytes. sinusoidal membrane proteins, such as LDL-R, the and the have a at the plasma membrane and undergo endocytosis (Fig. about canalicular et demonstrated that several indirect pathway proteins IV, aminopeptidase N, and as well as largely are retained at the apical membrane in hepatic WIF-B cells (Fig. However, these proteins recycle between plasma membrane and a intracellular compartment in less and cells, which contain only apical proteins. These suggest that apical retention mechanisms are during hepatocyte In contrast, Mdr1-GFP and presumably reflect Mdr1 and undergo endocytosis and recycle between the canalicular membrane and a subapical in WIF-B cells (Fig. Why is there such a in the of plasma membrane proteins in the is thought to be responsible for retention of like to both and single transmembrane proteins and with multispanning proteins like Mrp2 via their PDZ proteins mediate the of a molecular scaffold between apical membrane and which to the retention of these canalicular proteins. The of retention for single transmembrane proteins and Mrp2 may be different because of their differently in WIF-B In addition, of in led to a in the canalicular membrane expression of Mrp2 but not DDP IV, Mdr1, or At it is not known how or other retention mechanisms operate the sinusoidal membrane. membrane proteins like the receptor and raft-resident proteins are endocytosed by the and At it is not known which pathways mediate the apical endocytosis of Mdr1 and Bsep and the apical endocytosis of indirect pathway proteins and mechanisms may Mdr1 and Bsep recycle than retained in the canalicular membrane like (1) Mdr1 and Bsep reside in distinct lipid which may different and (2) Mdr1 and Bsep do not PDZ and presumably do not to proteins. Despite a tight apical retention endocytosis and targeting to lysosomes may function as a to and the indirect pathway proteins like aminopeptidase and Mrp2 from the canalicular membrane. of an apical phosphoinositide 3 kinase by or of led to of apical proteins like and in in WIF-B 3 kinase lipid from which in many of both biosynthetic and endocytic sorting. Thus far, we have how hepatic membrane polarity is How is such a complex polarity generated in hepatocytes during liver development and in how are the transcytotic delivery and apical retention mechanisms A number of studies have the in hepatocyte cells and hepatocytes at different stages of liver In the liver cells are not As the liver the the cells polarized at 5 are first the liver of bile canaliculi, and a canalicular network can be From this hepatocytes are but the bile do not a fully several after Interestingly, in hepatocytes, intracellular bile have been that may canalicular domains and may be to the intracellular apical compartment in cells described by et The development of membrane polarity is to the process of hepatocyte To understand fully the molecular involved in the of hepatocyte a analysis of the trafficking and signaling at different stages of hepatic are on recent work from other epithelial we that the of hepatic polarity involves the in In hepatocyte cells, sinusoidal and canalicular membrane proteins are sorted at the TGN into distinct vesicles by basolateral sorting signals and raft mechanisms (Fig. cells the these proteins may be into the but these microdomains are with other in the plasma membrane. Some intracellular compartments may be formed and may contain the canalicular proteins, an before of the canalicular membrane. As the liver the the of hepatocytes is by or (Fig. or the a process mediated by cell like may be to the of and may to the of or both a membrane or a that in may a number of (Fig. As by et and these include the establishment of apical junction complexes junction and the of signaling and polarized attachment of and which then to the of the in the canalicular and sinusoidal proteins distinct on the plasma membrane, followed by the of apical and basolateral the canalicular and basolateral domains are formed in hepatocytes (Fig. and also are in a polarized shown in A hypothetical model for the of membrane polarity during hepatocyte stages of during hepatocyte (A) hepatocyte cells are not polarized. The sinusoidal (blue circles) and canalicular proteins (green and red for indirect and direct pathway proteins, are sorted at the TGN and are in the plasma membrane. Some intracellular apical compartments may be formed that contain canalicular proteins. (B) As the liver the of hepatocytes is by exocyst complexes may be to the to the of (C) a membrane which the of apical The network to the complexes, to the and of the in hepatocytes. canalicular and sinusoidal proteins traffic to the plasma membrane (indicated by arrows) and the canalicular and sinusoidal (D) membrane polarity is fully in hepatocytes. The and of are The subapical network to tight junctions and the canalicular membrane, forming as a the canalicular domain. BC, bile tight During this specific that liver may influence the expression and distribution of specific membrane targeting soluble NSF attachment retention the and the of the subapical these the mechanisms for polarized protein For hepatocyte and the of tight junctions during and may function in liver of to of cells these cells to bile and to functional of cell Thus bile which are only at the of liver development, also may affect hepatocyte membrane polarity and the of bile canaliculi, by signaling through To these at the molecular will to in of the expression and signaling between the and at different during development of cell polarity may interesting about and how transcytosis and direct apical trafficking is in to hepatocyte of hepatocyte polarity is a of liver by or of to a of canalicular proteins such as pIgA-R and DPP IV to the domain and in subapical (Fig. and treatment also the of transcytotic with an in both the number and of transcytotic vesicles within the subapical intracellular in which may from fusion of transcytotic vesicles and may bile both and These intracellular seem to the apical compartments in less hepatic cells and liver, as discussed above. In liver, the tight junctions are also the between bile and Taken together, these suggest that transcytosis of canalicular proteins and to a or of membrane The mechanisms responsible for altered cell polarity in are understood. However, the of to the trafficking of Bsep to the canalicular domain is to be in hepatocytes in and can both transcytosis and the expression of Mrp2 in the canalicular The these of and the of and to the canalicular membrane, which may in affect the trafficking of It is conceivable that bile also may directly or indirectly affect the trafficking of other plasma membrane proteins in hepatocytes. Much work is in membrane polarity and polarized trafficking in hepatocytes. to the of canalicular proteins (green patches) to the sinusoidal and domains, of transcytotic vesicles red and the of intracellular junctions also are a between the and taurolithocholic the targeting and of transcytosed proteins and Mrp2 to the canalicular membrane, with the of to the canalicular membrane. the of and of to the canalicular membrane. studies described in this have into how sinusoidal and canalicular proteins traffic to and reside in the plasma membrane. However, the molecular mechanisms by which hepatocytes and maintain membrane polarity remain understood. of the polarized sorting machinery is by the of hepatocyte differentiation, we may into how hepatocyte polarity is generated and how the of hepatocytes to proteins to the apical canalicular membrane by transcytosis is established. studies of polarized trafficking in hepatocytes have been largely because of developments such as described in this the a understanding of this complex yet vital of hepatocytes to and maintain their polarity in and The and for during the of this and and for We to are not because of of