Polymeric IgA Receptor Research Articles

Specific IgA Enhances the Transcytosis and Excretion of Hepatitis A Virus.

Hepatitis A virus (HAV) replicates in the liver, and is excreted from the body in feces. However, the mechanisms of HAV transport from hepatocytes to the gastrointestinal tract are poorly understood, mainly due to lack of suitable in vitro models. Here, we use a polarized hepatic cell line and in vivo models to demonstrate vectorial transport of HAV from hepatocytes into bile via the apical cell membrane. Although this transport is specific for HAV, the rate of fecal excretion in inefficient, accounting for less than 1% of input virus from the bloodstream per hour. However, we also found that the rate of HAV excretion was enhanced in the presence of HAV-specific IgA. Using mice lacking the polymeric IgA receptor (pIgR−/−), we show that a proportion of HAV:IgA complexes are transported via the pIgR demonstrating a role for specific antibody in pathogen excretion.

IMMUNOGLOBULIN A (IgA) AND ITS RECEPTORS

Daily IgA production in human organism comprises 3 to 5 g, thus exceeding total synthesis of other Ig classes. IgA in human body is presented by 9 structural variants. Its molecules belong to two subclasses, IgA1 and IgA2, the latter represented by two allotypes. In human serum, IgA1 monomers predominate, that are produced by the bone marrow cells. Mucosa-associated lymphoid tissues produce dimeric IgA1 and IgA2 molecules containing an accessory polypeptide J-chain. When transported across epithelial layer to the mucosal surface, an extracellular segment of polymeric IgA receptor (pIgAR) is joining the dimeric IgA1, which becomes a ‘secretory’ component being a part cesretory IgA (sIgA) molecule. The main function of sIgA is to bind bacteria and viruses at the mucosal surfaces, thus preventing pathogens to invade the internal spaces of the organism (immune exclusion). If transferred across epithelium, IgA may neutralize the viruses penetrating the cells, like as bind and deliver proteins and other antigens to the mucosal surface. The leukocyte IgA receptor (FcαRI, CD89) is expressed on the neutrophils, eosinophiles, monocytes/macrophages, as well as dendritic and Kupffer cells. The cytoplasmic domain FcαRI is devoid of an activation ITAM motif. To transduce signal, an FcαRI-associated chain of Fcγ receptor is used. Due to this mechanism, IgA binding leads to activation of phagocytosis, endocytosis, antigen presentation, synthesis of proinflammatory mediator and other immune functions. Fcα/μR receptor is a structural homologue of pIgR, and it is able to bind IgA and IgM, being, however, expressed only at the surface of mature B lymphocytes and macrophages. Interaction of IgA with asialoglycoprotein and transferrin (CD71) receptors, like as with some other molecules, that have yet undetermined role in immune defense and development of pathological events.

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

MAL2 Selectively Regulates Polymeric IgA Receptor Delivery from the Golgi to the Plasma Membrane in WIF-B Cells

Myelin and lymphocyte protein 2 (MAL2) has been identified as a hepatic transcytotic regulator that mediates delivery from basolateral endosomes to the subapical compartment (SAC). However, overexpression of polymeric immunoglobulin A-receptor (pIgA-R) in polarized, hepatic WIF-B cells led to the dramatic redistribution of MAL2 into the Golgi and all the transcytotic intermediates occupied by the receptor. Although overexpressed hemagglutinin and dipeptidylpeptidase IV (DPPIV) distributed to the same compartments, MAL2 distributions did not change indicating the effect is selective. Cycloheximide treatment led to decreased pIgA-R and MAL2 intracellular staining, first in the Golgi then the SAC, suggesting they were apically delivered and that MAL2 was mediating the process. This was tested in Clone 9 cells (that lack endogenous MAL2). When expressed alone, pIgA-R was restricted to the Golgi whereas when coexpressed with MAL2, it distributed to the surface, was internalized and delivered to MAL2-positive puncta. In contrast, DPPIV distributions were independent of MAL2. Surface delivery of newly synthesized pIgA-R, but not DPPIV, was enhanced greater than ninefold by MAL2 coexpression. In WIF-B cells where MAL2 expression was knocked down, pIgA-R, but not DPPIV, was retained in the Golgi and its basolateral delivery was impaired. Thus, in addition to its role in transcytosis, MAL2 also regulates pIgA-R delivery from the Golgi to the plasma membrane.

Exogenous MAL Reroutes Selected Hepatic Apical Proteins into the Direct Pathway in WIF-B Cells

Unlike simple epithelial cells that directly target newly synthesized glycophosphatidylinositol (GPI)-anchored and single transmembrane domain (TMD) proteins from the trans-Golgi network to the apical membrane, hepatocytes use an indirect pathway: proteins are delivered to the basolateral domain and then selectively internalized and transcytosed to the apical plasma membrane. Myelin and lymphocyte protein (MAL) and MAL2 have been identified as regulators of direct and indirect apical delivery, respectively. Hepatocytes lack endogenous MAL consistent with the absence of direct apical targeting. Does MAL expression reroute hepatic apical residents into the direct pathway? We found that MAL expression in WIF-B cells induced the formation of cholesterol and glycosphingolipid-enriched Golgi domains that contained GPI-anchored and single TMD apical proteins; polymeric IgA receptor (pIgA-R), polytopic apical, and basolateral resident distributions were excluded. Basolateral delivery of newly synthesized apical residents was decreased in MAL-expressing cells concomitant with increased apical delivery; pIgA-R and basolateral resident delivery was unchanged. These data suggest that MAL rerouted selected hepatic apical proteins into the direct pathway.

Receptor Complexes Cotransported via Polarized Endocytic Pathways Form Clusters with Distinct Organizations

Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands. We show that pIgA-R and TFR complexes form increasingly organized clusters during cotransport from basolateral to perinuclear endosomes. The organization of these receptor clusters in basolateral versus perinuclear/apical endosomes is significantly different; the former showing a mixed random/clustered distribution while the latter highly organized clusters. Our results indicate that although both perinuclear and apical endosomes comprise pIgA-R and TFR clusters, their E% levels are significantly different suggesting that these receptors are packed into clusters in a distinct manner. The quantitative FRET-based assay presented here suggests that different receptor complexes form clusters, with diverse levels of organization, while being cotransported via the polarized endocytic pathways.

Tear Dynamics Measured with Real-Time Optical Coherence Tomography

Background & Aims This study demonstrates and characterizes apical (canalicular) endocytic pathways in hepatocytes in situ. Methods: Endocytic markers were administered by retrograde infusion through the common bile duct. Colocalization with proteins that are specific for various endocytic compartments was performed on stacks of deconvoluted confocal immunofluorescence images. The subcellular distribution of marker proteins was assessed by electron microscopy (EM). Results: Bulk-phase, as well as membrane-associated markers, were internalized readily at the apical cell pole. At the EM level, marker was found initially in 60–100-nm tubulovesicular structures and 150–200-nm cup-shaped vesicles, whereas multivesicular bodies and lysosomes became labeled after longer time intervals. Apical endocytosis involved clathrin and delivered marker to late endosomes (rab7+, cathepsin D+), as well as lysosomes (rab7−, cathepsin D+). Simultaneous labeling of the basolateral endocytic route resulted in overlap of both pathways in the late endosomal and lysosomal compartments. In addition, apical endocytosis involved a subapical compartment (endolyn-78+, rab11+, polymeric IgA receptor [pIgA-R+]) that is passed by the transcytotic route, thus constituting a crossroads. pIgA-R immunoreactivity, probably reflecting the cleaved receptor fragment, was associated with apical endocytic marker and colocalized with clathrin and later with cathepsin D. Conclusions: Apical endocytosis involves coated pits/vesicles, leads to a subapical compartment, and plays a role in the retrieval of canalicular plasma membrane components for lysosomal degradation.GASTROENTEROLOGY 2000;119:1692-1707

Diagnosis of Dry Eye with a Blue-Free Barrier Filter

Background & Aims This study demonstrates and characterizes apical (canalicular) endocytic pathways in hepatocytes in situ. Methods: Endocytic markers were administered by retrograde infusion through the common bile duct. Colocalization with proteins that are specific for various endocytic compartments was performed on stacks of deconvoluted confocal immunofluorescence images. The subcellular distribution of marker proteins was assessed by electron microscopy (EM). Results: Bulk-phase, as well as membrane-associated markers, were internalized readily at the apical cell pole. At the EM level, marker was found initially in 60–100-nm tubulovesicular structures and 150–200-nm cup-shaped vesicles, whereas multivesicular bodies and lysosomes became labeled after longer time intervals. Apical endocytosis involved clathrin and delivered marker to late endosomes (rab7+, cathepsin D+), as well as lysosomes (rab7−, cathepsin D+). Simultaneous labeling of the basolateral endocytic route resulted in overlap of both pathways in the late endosomal and lysosomal compartments. In addition, apical endocytosis involved a subapical compartment (endolyn-78+, rab11+, polymeric IgA receptor [pIgA-R+]) that is passed by the transcytotic route, thus constituting a crossroads. pIgA-R immunoreactivity, probably reflecting the cleaved receptor fragment, was associated with apical endocytic marker and colocalized with clathrin and later with cathepsin D. Conclusions: Apical endocytosis involves coated pits/vesicles, leads to a subapical compartment, and plays a role in the retrieval of canalicular plasma membrane components for lysosomal degradation.GASTROENTEROLOGY 2000;119:1692-1707

Intracellular accumulation of pIgA-R and regulators of transcytotic trafficking in cholestatic rat hepatocytes

Bile duct ligation (BDL) impairs basolateral-to-apical transcytosis in hepatocytes, causing accumulation of transcytotic carriers for the polymeric IgA receptor (pIgA-R) and redistribution of secretory component (SC) from bile to blood. To gain insight into the mechanisms regulating transcytosis and the pathophysiology of cholestasis, we investigated nascent protein trafficking in control and BDL livers using cell fractionation in the context of in vivo pulse-chase experiments and immunoblot analysis. Control and cholestatic hepatocytes trafficked [35S]-labeled serum proteins and the pIgA-R along the secretory pathway with identical kinetics. However, BDL impaired transcytosis, causing (1) accumulation of the pIgA-R, rab3D, rab11a, and other candidate regulators of apical-directed secretion in a crude vesicle carrier fraction (CVCF) enriched in transcytotic carriers; (2) slow delivery of [35S]-labeled SC to bile; and (3) paracellular reflux of SC from bile to blood. In conclusion, these data indicate that the secretory and transcytotic pathways remain polarized in cholestatic hepatocytes and suggest that the pIgA-R traffics through postendosomal rab3D-, rab11a-, and syntaxin 2-associated compartments, implicating these proteins in the regulation of transcytosis.

Role of passive and adaptive immunity in influencing enterocyte-specific gene expression.

Numerous genes expressed by intestinal epithelial cells are developmentally regulated, and the influence that adaptive (AI) and passive (PI) immunity have in controlling their expression has not been evaluated. In this study, we tested the hypothesis that both PI and AI influenced enterocyte gene expression by developing a breeding scheme that used T and B cell-deficient recombination-activating gene (RAG) mice. RNA was isolated from the liver and proximal/distal small intestine at various ages, and the steady-state levels of six different transcripts were evaluated by RNase protection assay. In wild-type (WT) pups, all transcripts [Fc receptor of the neonate (FcRn), polymeric IgA receptor (pIgR), GLUT5, lactase-phlorizin hydrolase (lactase), apical sodium-dependent bile acid transporter (ASBT), and Na+/glucose cotransporter (SGLT1)] studied were developmentally regulated at the time of weaning, and all transcripts except ASBT had the highest levels of expression in the proximal small intestine. In WT suckling pups reared in the absence of PI, pIgR mRNA levels were increased 100% during the early phase of development. In mice lacking AI, the expression of pIgR and lactase were significantly attenuated, whereas FcRn and GLUT5 levels were higher compared with WT mice. Finally, in the absence of both passive and active immunity, expression levels of pIgR and lactase were significantly lower than similarly aged WT mice. In summary, we report that the adaptive and passive immune status of mice influences steady-state mRNA levels of several important, developmentally regulated enterocyte genes during the suckling and weaning periods of life.

Dynamics of the apical plasma membrane recycling system during cell division.

The members of the family of Rab11 small GTPases are critical regulators of the plasma membrane vesicle recycling system. While previous studies have determined that the Golgi apparatus disperses during mitosis and reorganizes after cytokinesis, the fate of the recycling system during the cell cycle is more obscure. We have now studied in MDCK cells the fate during mitosis of an apical recycling system cargo, the polymeric IgA receptor (pIgAR), and regulators of the recycling system, Rab11a and its interacting proteins myosin Vb, Rab11-FIP1, Rab11-FIP2 and pp75/Rip11. Rab11a, pIgAR and myosin Vb containing vesicles dispersed into diffuse puncta in the cytosol during prophase and then became clustered near the spindle poles after metaphase, increasing in intensity throughout telophase. A similar pattern was observed for Rab11-FIP1 and Rab11-FIP2. However, Rab11-FIP1 lost colocalization with other recycling system markers during late prophase, relocating to the pericentriolar material. During telophase, Rab11-FIP1 returned to recycling system vesicles. Western blot analysis indicated that both Rab11a and pIgAR remained associated with membrane vesicles throughout the cell cycle. This behavior of the Rab11a-containing apical recycling endosome system during division was distinct from that of the Golgi apparatus. These results indicate that critical components of the apical recycling system remain associated on vesicles throughout the cell cycle and may provide a means for rapid re-establishment of plasma membrane components after mitosis.

IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component.

The bronchial epithelium is a source of both alpha and beta chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and alpha(2)-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene alpha, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC(50) approximately 0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC.

Myosin vb is associated with plasma membrane recycling systems.

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

Multiple transcription factors in the 5-flanking region of the human polymeric IgA receptor (pigR) controls its basal expression

Multiple transcription factors in the 5-flanking region of the human polymeric IgA receptor (pigR) controls its basal expression

Multiple transcription factors in the 5-flanking region of the human polymeric IgA receptor (pigR) controls its basal expression

Multiple transcription factors in the 5-flanking region of the human polymeric IgA receptor (pigR) controls its basal expression

Apical endocytosis in rat hepatocytes in situ involves clathrin, traverses a subapical compartment, and leads to lysosomes

Background & Aims This study demonstrates and characterizes apical (canalicular) endocytic pathways in hepatocytes in situ. Methods: Endocytic markers were administered by retrograde infusion through the common bile duct. Colocalization with proteins that are specific for various endocytic compartments was performed on stacks of deconvoluted confocal immunofluorescence images. The subcellular distribution of marker proteins was assessed by electron microscopy (EM). Results: Bulk-phase, as well as membrane-associated markers, were internalized readily at the apical cell pole. At the EM level, marker was found initially in 60–100-nm tubulovesicular structures and 150–200-nm cup-shaped vesicles, whereas multivesicular bodies and lysosomes became labeled after longer time intervals. Apical endocytosis involved clathrin and delivered marker to late endosomes (rab7+, cathepsin D+), as well as lysosomes (rab7−, cathepsin D+). Simultaneous labeling of the basolateral endocytic route resulted in overlap of both pathways in the late endosomal and lysosomal compartments. In addition, apical endocytosis involved a subapical compartment (endolyn-78+, rab11+, polymeric IgA receptor [pIgA-R+]) that is passed by the transcytotic route, thus constituting a crossroads. pIgA-R immunoreactivity, probably reflecting the cleaved receptor fragment, was associated with apical endocytic marker and colocalized with clathrin and later with cathepsin D. Conclusions: Apical endocytosis involves coated pits/vesicles, leads to a subapical compartment, and plays a role in the retrieval of canalicular plasma membrane components for lysosomal degradation.GASTROENTEROLOGY 2000;119:1692-1707

Rab3D, a small GTP–binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of ∼25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes. (Hepatology 2000;32:348-356.)

GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles.

Upon addition of GTPgammaS to in vitro budding reactions, COP I vesicles form but retain their coat, making them easy to isolate and analyze. We have developed an in vitro budding assay that reconstitutes the formation of COP I-derived vesicles under conditions where GTP hydrolysis can occur. Once formed, vesicles are uncoated and appear functional as they fuse readily with acceptor membranes. Electron microscopy shows a homogeneous population of uncoated vesicles that contain the medial/trans Golgi enzyme alpha1, 2-mannosidase II. Biochemical quantitation of vesicles reveals that resident Golgi enzymes are up to 10-fold more concentrated than in donor membranes, but vesicles formed in the presence of GTPgammaS show an average density of resident Golgi enzymes similar to that seen in donor membranes. We show that the sorting process is mediated by the small GTPase arf-1 as addition of a dominant, hydrolysis-deficient arf-1 (Q)71(L) mutant produced results similar to that of GTPgammaS. Strikingly, the average density of the anterograde cargo protein, polymeric IgA receptor, in COP I-derived vesicles was similar to that found in starting membranes and was independent of GTP hydrolysis. We conclude that hydrolysis of GTP bound to arf-1 promotes selective segregation and concentration of Golgi resident enzymes into COP I vesicles.

Transcriptional control of the murine polymeric IgA receptor promoter by glucocorticoids.

Glucocorticoids have been implicated as an important regulator of intestinal epithelial cell ontogeny. The polymeric IgA receptor (pIgR) is expressed in the intestinal epithelial layer and is regulated by several mediators, including glucocorticoids. The mechanism of how corticosteroids alter the transcriptional regulation of pIgR expression has not been defined. In this study, we demonstrated that glucocorticoids upregulate steady-state pIgR mRNA levels in the proximal intestine of suckling rats and in the IEC-6 intestinal cell line. We performed functional analysis of the 5'-flanking region in the presence of glucocorticoids and its receptor using the intestinal cell line Caco-2. We screened 4.7 kb of the upstream region of the murine gene and identified the most potent steroid response element to reside between nt -215 and -163 relative to the start of transcription. Substitution mutation analysis of this region identified the location of the putative steroid response element to be between nt -195 and -176. In vitro DNase I footprint analysis using the recombinant glucocorticoid receptor DNA binding domain confirmed a single area of protection that spans the nt identified by mutagenesis analysis. Electrophoretic mobility shift assays of the putative element confirmed the binding of both recombinant and cell synthesized glucocorticoid receptor in a specific manner. In summary, we report the identification and characterization of the glucocorticoid-DNA response element located in the immediate 5'-upstream region of the murine pIgR gene.

Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma × fibroblast hybrids and in hepatoma dedifferentiated variants

We have studied two aspects of the plasma membrane of hepatocytes, highly differentiated epithelial cells that exhibit a particular and complex polarity. Using a genetic approach, we have distinguished between the expression/regulation of proteins specific for all three hepatocyte membrane domains and their organization into discrete domains. For this analysis we used a panel of previously isolated cell clones, derived from the differentiated rat hepatoma line H4IIEC3, and that present different expression patterns for liver-specific genes. This panel was composed of (1) differentiated clones, (2) chromosomally reduced hepatoma-fibroblast hybrids characterized by a pleiotropic extinction/reexpression of liver-specific genes and (3) dedifferentiated variant and revertant clones. The expression of 16 hepatocyte membrane polarity markers was studied by western blotting and immunolocalization. Even though cells of differentiated clones express all of these polarity markers, they are not polarized, and are therefore suitable for studying the regulation of plasma membrane protein expression, and for identifying gene products implicated in the establishment of membrane polarity. In hepatoma-fibroblast hybrids the expression of four markers, three apical (dipeptidylpeptidase IV, alkaline phosphodiesterase B10 and polymeric IgA receptor) and one lateral (E-cadherin), is down-regulated in extinguished clones and restored in reexpressing subclones, as previously reported for liver-specific functions. The dipeptidylpeptidase IV mRNA was undetectable or strongly reduced in extinguished hybrids, but expressed at a robust level in some of the reexpressing clones. Concerning the dedifferentiated variants, each has its own pattern of membrane marker expression (loss of expression of three to six markers), that differs from that of extinguished hybrids. Revertant cells express all of the membrane markers examined. Among all of these hepatoma derivatives, only cells of reexpressing hybrids are polarized, and form bile canaliculi-like structures, with spherical and even, for one clone, long tubular and branched forms. All apical markers examined are confined in these canalicular structures, whereas the other markers are excluded from them, and present on the rest of the membrane (basolateral markers) or at the cell-cell contacts (lateral markers). Cells of reexpressing hybrids also express simple epithelial polarity. Thus the expression of only a few hepatocyte-domain-specific plasma membrane proteins is subject to down-regulation, as is the case for liver-specific genes so far studied, and the expression of polarity markers and the formation of poles are dissociable events.