Polarity is characterized by a specific organization of plasma membrane proteins and defines the shape and architecture of a cell. Hepatocyte polarity requires the coordinated establishment and maintenance of TJs and apical membrane domains (6
). We demonstrate that HepG2 polarization limits HCV entry (Fig. ) and that treating cells with PKA agonist(s), OSM, and Rho kinase inhibitor Y-27632, which stimulate polarization via distinct modes of action (25
), specifically reduce HCVpp infection (Fig. and ). HepG2 develops BC-like spaces that form elongated canalicular lumens spanning several cells (Fig. ) (25
), and the resistance of HepG2-CD81 cells to HCVpp infection following Rho kinase inhibition suggests that membrane remodeling associated with canalicular development restricts viral entry (Fig. ). In contrast, canalicular structures were infrequently detected in cultured PHH (<5%), consistent with their rapid dedifferentiation in culture (reviewed in reference 53
). We and others have reported that PHH support low-level HCVcc replication; however, it has been difficult to quantify the frequency of infected cells within the population and to study the role of polarity in HCV entry (reviewed in reference 17
To ascertain whether the reduced permissivity of polarized HepG2 cells to HCV infection was due to a reorganization of the viral receptors, we imaged endogenous and fluorescent-tagged versions of CD81 and CLDN1. Both receptors localized to areas of cell-cell contact in nonpolarized cells, and the majority of CLDN1 redistributed to TJs upon polarization (Fig. and ). In contrast, ZO-1 and OCLN appeared to localize exclusively to TJs surrounding the BC (Fig. and ). CD81, SR-BI, CLDN1, and OCLN localization in polarized HepG2 cells was comparable to that observed in healthy liver tissue (Fig. ), providing support for the use of HepG2 cells as a model system to study HCV entry into polarized hepatocytes.
TJs are dynamically regulated (49
), and the major pool(s) of OCLN and ZO-1 in the T84 epithelial cell line is reported to reside in Triton X-100-insoluble raft-like membrane microdomains (39
). Similarly, in polarized HepG2 cells we found that the basal and lateral pools of CLDN1 and JAM-A are Triton X-100 soluble, whereas TJ-associated CLDN1 and JAM-A are insoluble to detergent extraction (data not shown). The presence of TJ and nonjunctional pools of CLDNs in the liver is consistent with earlier reports (44
). We hypothesize that nonjunctional CLDN1 may be more accessible to interact with the viral glycoproteins during entry. Earlier studies showing that CLDN1 expression in nonpolarized human embryonal 293T kidney cells allow HCVpp entry support a model where entry is not dependent on the formation of functionally active TJs (15
). Indeed, the observation that CLDN1 lacking the C-terminal region that is required for transport to the TJs (46
) confers viral entry into 293T cells (15
) lends further support to a model where nonjunctional CLDN1 has a role in HCV entry.
The expression levels of tagged CLDN1 and CD81 at the basal and lateral membranes of polarized cells were comparable to that of the plasma membrane of nonpolarized HepG2 (Fig. ). We (24
) and others (11
) reported that CLDN1 and CD81 associate at the plasma membrane, suggesting that these complexes play a role in HCV entry. We demonstrate that FRET occurred between membrane-expressed pools of CLDN1 and CD81 at comparable efficiencies in polarized and nonpolarized HepG2 cells to those previously reported in Huh-7 and 293T cells (24
), suggesting that receptor complex formation does not limit HCV infection of polarized cells. Interestingly, there was no detectable FRET between TJ-associated pools of CLDN1 and CD81, suggesting that the conformation, density, or stoichiometry of the proteins at the TJ is not compatible with coreceptor association. These data are consistent with an earlier report by Kovalenko and colleagues, who demonstrated an association between CLDN1 and the tetraspanin proteins CD81 and CD9 in nonpolarized A431 and A549 epithelial cells (29
), leading them to suggest that tetraspanin-enriched microdomains regulate the trafficking of nonjunctional CLDNs (reviewed in reference 21
We recently demonstrated PKA-dependent localization of CLDN1 in Huh-7 cells, such that inhibiting PKAII reduced CLDN1 membrane expression and abrogated HCV entry (16
). To ascertain whether PKA regulates CLDN1 localization at basal and lateral membranes and at TJ domains, HepG2 cells expressing AcGFP.CLDN1 and DsRED.CD81 were treated with the PKA antagonist, Rp-8-Br-cAMPs. Treatment reduced CLDN1 expression at the basal membrane and increased lateral- and TJ-associated forms with no detectable effect on CD81 localization (Fig. ). The reduced CLDN1 expression at the basal membrane abrogated FRET association with CD81 at this site. Importantly, Rp-8-Br-cAMPs treatment induced a significant reduction in HCVpp entry. One consequence of HepG2 polarization in vitro, in particular the development of multicellular structures with cells sharing a BC, is the reduced availability of the basal membrane. Quantification of the relative amount of basal and lateral membranes and TJ domains in polarized HepG2 cells demonstrates that as more cells share a BC, the TJ and basal surfaces reduce and the area of lateral membrane increases, supporting the model that basal membrane-expressed pools of receptors have a role in viral entry.
Cytokine-mediated changes in cell permeability contribute to a wide range of pathological conditions, including inflammatory bowel disease, cystic fibrosis, and perturbation of the blood-brain barrier (reviewed in reference 8
). While the molecular mechanisms regulating these processes are incompletely understood, model cell culture systems of epithelial/endothelial barrier function have highlighted the important role of cytokines in regulating TJs. Treatment of HepG2-CD81 cells with TNF-α and IFN-γ reduced TJ integrity and yet had a minimal effect on cell polarization or HCVpp entry (Fig. ). TNF-α and IFN-γ have been reported to modulate actin- myosin contractility and to promote the endocytosis of TJ proteins (reviewed in reference 8
). However, the majority of reports have utilized epithelial and endothelial cells with simple polarity, and the effect of cytokines on hepatic permeability is poorly understood. Phorbol ester activation of PKC abrogated TJ integrity and significantly reduced HepG2 polarity, with a concomitant increase in HCVpp infection (Fig. ). PMA induced a relocalization of CLDN1, ZO-1, and OCLN from TJs, which was not apparent with either cytokine treatment (data not shown). Several reports demonstrate that PMA activation of PKC can signal a redistribution of TJ proteins from the cell borders to the cytoplasm (9
). The signaling events downstream of PKC that regulate cell permeability are incompletely defined; however, multiple components of the TJ complex may be directly phosphorylated by PKC (reviewed in reference 23
). These data suggest that functionally intact TJs per se do not limit HCV entry and highlight the differential effect(s) of cytokine and PMA modulation of TJ integrity and protein localization on HCV entry that is worthy of further investigation.
The establishment and maintenance of a fully polarized state requires the appropriate extracellular matrix, cell-cell contacts, and correct geometrical orientation, with many epithelial cells adopting a partially polarized morphology in monolayer culture. Huh-7 hepatoma and clonal derivatives such as Huh-7.5 (5
) support high-level HCV RNA replication and are used widely to study HCV infection and entry. We noted heterogeneous expression and localization of CLDN1, OCLN, and JAM-A in Huh-7-derived hepatomas, with frequent breaks in their lateral staining pattern(s). Furthermore, actin staining was heterogeneous, with limited evidence for circumferential bands colocalizing with ZO-1 and JAM-A (unpublished data), as would be expected in a polarized monolayer (33
). All treatments to regulate HepG2 polarity had minimal effects on HCVpp or HCVcc infection of Huh-7 or Huh-7.5 cells, in contrast to an earlier report demonstrating that TNF-α induced a redistribution of CLDN1 from the plasma membrane to intracellular sites in Huh-7.5.1 cells (55
). Huh-7 cells are heterogeneous with respect to morphology, CD81 expression, and viral RNA replication, and this can change with in vitro passage and culture conditions (1
), making data comparison between laboratories difficult. Cumulatively, our data suggest that Huh-7 cells fail to develop mature TJs, explaining the relatively low transepithelial resistance values reported (4
) compared to those of more differentiated epithelial cell lines. Recent reports of CLDN1 and OCLN colocalization with ZO-1 at the apex of Huh-7 cells (7
) provide insufficient evidence to demonstrate functional TJs, since both ZO-1 and OCLN have been shown to be expressed in cells that lack TJs (14
). In contrast, HepG2 cells that develop and maintain complex polarity have been used extensively to define TJ formation and regulation in the development of hepatic polarity (reviewed in references 13
). On the assumption that HepG2 cells mimic hepatocyte polarity within the liver, our data suggest that HCV entry may be suboptimal and that agents which disrupt hepatocyte polarity may promote HCV infection and transmission in the liver.