Effects of Culture Conditions on Heterogeneity and the Apical Junctional Complex of the ARPE-19 Cell Line

Luo, Yan; Zhuo, Yehong; Fukuhara, Masayuki; Rizzolo, Lawrence J.

doi:10.1167/iovs.06-0166

Cited by 103 publications

(104 citation statements)

References 36 publications

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“…However, TER measurements are not necessarily fully reflective of functional tight junctions, nor do they correlate with adherens junction formation. A recent study found a discrepancy between TER, barrier function, and adherens junction formation in ARPE cells (25). Of note, we found that the ARPE cells displayed the lowest TER among the polarized cells studied yet were the most polarized with respect to susceptibility to HSV infection (Fig.…”

Section: Discussionmentioning

confidence: 48%

Access to Nectin Favors Herpes Simplex Virus Infection at the Apical Surface of Polarized Human Epithelial Cells

Galen

Cheshenko

Tuyama

et al. 2006

J Virol

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Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16-to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.

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Section: Discussionmentioning

confidence: 48%

Access to Nectin Favors Herpes Simplex Virus Infection at the Apical Surface of Polarized Human Epithelial Cells

Galen

Cheshenko

Tuyama

et al. 2006

J Virol

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“…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 (33,35). 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-␣ induced a redistribution of CLDN1 from the plasma membrane to intracellular sites in Huh-7.5.1 cells (55).…”

Section: Discussionmentioning

confidence: 84%

Polarization Restricts Hepatitis C Virus Entry into HepG2 Hepatoma Cells

Mee

Harris

Farquhar

et al. 2009

J Virol

117

141

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The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver.Hepatitis C virus (HCV) poses a global health problem, with over 170 million infected individuals worldwide. The principal reservoir for HCV replication is believed to be hepatocytes in the liver. Innate and adaptive cellular immune responses are thought to play a critical role in controlling HCV replication. However, in the majority of cases, the virus persists and the resulting hepatitis leads to progressive liver injury, frequently culminating in fibrosis and hepatocellular carcinoma. At the present time there is no vaccine and the only established treatment is alpha interferon (IFN-␣) in combination with ribavirin, which is only partially effective. Hence, there is an urgent need for the development of more effective therapies.The recent discovery that some strains of HCV can replicate in cell culture (HCVcc) and release infectious particles has allowed the complete viral life cycle to be studied (30,52,58). HCV initiates infection by attaching to molecules or receptors at the cell surface, and current evidence suggests that the tetraspanin CD81 (3, 10, 20, 26), scavenger receptor class B member I (SR-BI) (2, 22, 48), and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN) (15,37,43,55,57) are required for HCV entry (reviewed in reference 50). However, the exact role(s) played by each of the receptors is unclear.Many tissues in the body contain polarized cells,...

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“…Although this cell line has been widely used for experiments on RPE, several studies have shown that the properties of ARPE-19 cells vary according to culture conditions (Luo et al 2006). Therefore, our results should be considered provisional until they can be repeated in nontransformed human RPE cells.…”

Section: Discussionmentioning

confidence: 88%

Bevacizumab modulates epithelial‐to‐mesenchymal transition in the retinal pigment epithelial cells via connective tissue growth factor up‐regulation

Chen

Liang

Chen

et al. 2012

Acta Ophthalmologica

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ABSTRACT.Purpose: To investigate the effect of bevacizumab treatment on connective tissue growth factor (CTGF) expression and the induction of epithelial-to-mesenchymal transition in ARPE-19 cells and human donor retinal pigment epithelium (HRPE) cells in vitro. Methods: We quantitated the protein and gene expression level of CTGF by ELISA. The effect of Fc-Fc receptor (Fc-FcR) interaction on CTGF expression was evaluated by CD64 siRNA silencing. Expression of epithelial-to-mesenchymal transition markers, alpha-smooth muscle actin (a-SMA) and zona occludens protein (ZO-1) was evaluated by Western blot. Cell migration and collagen gel contraction assay were examined by light microscopy, and collagen production was measured by ELISA. Results: Bevacizumab stimulation increased CTGF expression in ARPE-19 and HRPE cells in a dose-dependent manner. CD64 gene silencing inhibited the effect of bevacizumab-induced CTGF up-regulation. Bevacizumab increased the expression of a-SMA and decreased the expression of ZO-1 in ARPE-19 cells. Bevacizumab also caused the release of type-1 collagen and increased cell migration and contraction of collagen. Conclusions: Bevacizumab exerts pro-fibrotic effects on human RPE cells at clinical doses by up-regulation of CTGF expression via an Fc-FcR interaction. This effect of bevacizumab may be one of the underlying mechanisms involved in age-related macular degeneration therapy or intravitreal bevacizumab-associated complications.

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Effects of Culture Conditions on Heterogeneity and the Apical Junctional Complex of the ARPE-19 Cell Line

Cited by 103 publications

References 36 publications

Access to Nectin Favors Herpes Simplex Virus Infection at the Apical Surface of Polarized Human Epithelial Cells

Access to Nectin Favors Herpes Simplex Virus Infection at the Apical Surface of Polarized Human Epithelial Cells

Polarization Restricts Hepatitis C Virus Entry into HepG2 Hepatoma Cells

Bevacizumab modulates epithelial‐to‐mesenchymal transition in the retinal pigment epithelial cells via connective tissue growth factor up‐regulation

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