Correspondence to: Randall J. Mrsny, Genentech Inc., MS #6, 1 DNA Way, South San Francisco, CA 94080. Tel:(650) 225-2592 Fax:650-225-1418 E-mail:mrsny{at}gene.com.
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Abstract |
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Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cellcell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.
Key Words: occludin, Raf-MEK-ERK signaling, tight junction, claudin-1, epithelium transformation
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Introduction |
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Epithelia exhibit specialized structures involved in cellcell contacts known as tight junctions (TJs)1 and adherens junctions (AJs). AJ complexes are involved in maintaining cellcell adhesions between adjacent epithelial cells, whereas TJ structures provide the barrier to uncontrolled paracellular permeability. The positioning of these junctions is coordinated and stabilized through associations with a continuous band of bundled actin filaments known as an adhesion belt (
Loss of TJ and AJ structures is frequently observed in epithelium-derived cancers (-, ß-, and
-catenins (
Sequence mutations that result in a constitutively active state of the small GTP-binding protein Ras are commonly associated with epithelium oncogenesis (Raf-1:ER, which was constructed by fusing the kinase domain of Raf-1 with the hormone-binding domain of estrogen receptor (
Raf:ER cells. Based on these results, we suggest that occludin plays a critical role in the disruption of epithelial TJs induced by oncogenic Raf-1.
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Materials and Methods |
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Reagents and Antibodies
An antibody that specifically recognizes the phosphorylated form of ERK1 and ERK2 was purchased from Santa Cruz Biotechnologies, Inc. Antibodies to occludin, ZO-1, and claudin-1 were purchased from Zymed Laboratories, Inc. An antibody to E-cadherin was from Transduction Laboratories. PD98059, a selective inhibitor of MEK, was purchased from Calbiochem. Rhodamine-phalloidin was obtained from Molecular Probes, Inc.
Constructs and Plasmids
A constitutively active Raf-1 (Raf-1:ER), comprised of the catalytic domain of human Raf-1 fused with the hormone-binding domain of human estrogen receptor was constructed as described (
Cell Culture, Transfections, and Treatments
Pa-4Raf-1:ER cells were established as described previously by stably transfecting Pa-4 cells with the
Raf-1:ER construct (
Raf-1:ER cells were cultured in DMEM/F12 (1:1) medium supplemented with 2.5% FBS, insulin (5 µg/ml), transferrin (5 µg/ml), EGF (25 ng/ml), hydrocortisone (1.1 µM), glutamate (5 mM), G418 (600 µg/ml), and were maintained in a humidified atmosphere containing 5% CO2 and 95% air at 35°C. Pa-4
Raf-1:ER-occludin cells were raised by stably transfecting Pa-4
Raf-1:ER cells with pCB6-occludin and pTK-Hyg vector (Clontech) for hygromycin selection (100 µg/ml). Charcoal-stripped serum was used in maintaining Pa-4
Raf-1:ER and Pa-4
Raf-1:ER-occludin cells to minimize the estrogen level in the culture medium. Cells grown on semipermeable supports were plated at 106 cells/cm2 onto collagen-coated clear polyester membrane of Costar Transwell® (0.4-µm pore size, 1-cm2 surface area). The transepithelial resistance of confluent epithelial sheets was measured after 1 wk in culture with a chopstick Millicell-ERS® voltmeter (Millipore). Transient transfections were performed using Lipofectamine PlusTM (Life Technologies, Inc.) and following the manufacturer's protocol. Cells were collected and lysed 48 h after the start of transfection. Treatment of PD98059 in the transient transfection experiments was carried out by adding the inhibitor (20 µM final) 5 h after the start of transfection. Medium was replaced every 24 h with fresh PD98059.
Western and Northern Blottings
Western Blotting.
Cell lysates, prepared as Triton X-100soluble and insoluble fractions, were made as described previously (
Northern Blotting.
Total cellular RNA was prepared using TRIzolTM reagent according to the manufacturer's instructions (Life Technologies). Denatured samples were size fractionated on a formaldehyde (2.2 M)/agarose (1.5%) gel, blotted onto a ZetaProbe nylon membrane (Bio-Rad Laboratories) and hybridized with a 32P-labeled DNA probe prepared using the Ready-To-Go DNA labeling kit (Amersham Pharmacia Biotech). Northern analysis for claudin-1 message levels was carried out using a PCR-derived cDNA sequence probe (Furuse et al., 1998). Glyceraldehyde 3-phosphate dehydrogenase mRNA levels were monitored as loading controls.
Microscopy and Confocal Imaging
Phase-contrast microscopy of cells on plastic culture dishes was done using a Nikon Diaphot 300 inverted microscope attached to a Nikon N6006 camera. Cells grown on Transwell® filters were fixed in 10% normal buffered formalin. Thin sections cut from paraffin-embedded samples were stained with hematoxylin and eosin and viewed by light microscopy. Characterization of filamentous actin distribution was achieved using cells fixed in formalin, permeabilized with 0.2% (vol/vol) Triton X-100, and labeled with rhodamine-phalloidin as described previously (
Measurement of Cloning Efficiency in Soft Agarose
Pa-4-vec, Pa-4Raf-1:ER, and Pa-4
Raf-1:ER-occludin cells were plated at 10,000 cells per 35-mm culture dish in 1 ml of 0.35% (wt/vol) low melting temperature (LMT) agarose solution diluted with medium in the absence or presence of 1 µM estradiol. The dishes were coated with 1 ml of 0.7% (wt/vol) LMT agarose before cell plating, and 1 ml of overlay medium was added after cell plating. The overlay medium was changed every 3 d and fresh estradiol was added. After 15 d, the cells were stained with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; 0.05 mg/ml). The stained plates were photographed, and colonies >0.4 mm in diameter were counted and analyzed.
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Results |
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Activated Raf-1 Modulates Epithelial Cell Phenotype and Downregulates Occludin Expression
Raf-1 is a serine/threonine kinase composed of two regulatory and one catalytic domain (Raf-1:ER protein and activation of the Raf-MEK-ERK kinase pathway in the stably transfected cells, Pa-4
Raf-1:ER, have been characterized previously (
Raf-1:ER cells by measuring the phosphorylation levels of ERK1 and ERK2 (Fig 1 A). When grown on plastic, Pa-4
Raf-1:ER cells displayed significant morphological changes compared with vector-transfected control cells (Pa-4-vec) (Fig 1 B), and had prominent stress fibers instead of pericellular actin rings (Fig 1 C). When cultured on semipermeable filter supports, Pa-4
Raf-1:ER cells lost their ability to form high-resistance monolayers and acquired a stratified, low-resistance phenotype (Fig 1 D). Immunofluorescence staining of occludin showed normal peripheral distribution pattern in control cells, but there was only background staining of occludin in Pa-4
Raf-1:ER cells (Fig 1 E). Western analyses revealed that Pa-4-vec cells had high levels of occludin protein in both Triton X-100soluble and insoluble lysates (Fig 1 F). In the Triton X-100insoluble fraction, where cytoskeleton-associated proteins are enriched (
Raf-1:ER cells completely lost their expression of occludin (Fig 1 F, arrow). Northern analysis demonstrated that the downregulation of occludin protein in Pa-4
Raf-1:ER cells correlated with a complete loss of occludin mRNA (Fig 1 G).
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A recent study on the actions of vascular permeability factor on endothelial cell function has suggested that the loss of occludin at cell junctions occurs via the ERK-involved pathway (
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Introduction of Exogenous Occludin into Pa-4Raf-1:ER Cells Resulted in Reacquisition of Normal Epithelial Phenotype and Functionally Intact TJs
Although we made the observation that occludin was downregulated in Raf-1activated cells, this downregulation could be a side effect of Raf-1 activation and have no relevance to the disruption of epithelial TJs. To directly assess the potential role of occludin in stabilizing functional epithelial TJs, we introduced an exogenous occludin gene (human) driven by the cytomegalovirus promoter into Pa-4Raf-1:ER cells, which no longer express endogenous occludin. A total of eight Pa-4
Raf-1:ER-occludin cell clones were isolated and analyzed. Data from a representative clone, clone No. 2, have been presented for most studies. Control transfections with pCB6 vector alone did not yield any clones distinguishable from Pa-4
Raf-1:ER cells. Occludin-transfected Pa-4
Raf-1:ER cells were verified to have similar levels of
Raf-1:ER protein and phosphorylated ERK1 and ERK2 compared with Pa-4
Raf-1:ER cells (Fig 3 A), indicating that elevated activity of the Raf-MEK-ERK kinase pathway was maintained. Immunoblotting of protein lysates of Pa-4
Raf-1:ER-occludin cells confirmed the presence of occludin in both Triton X-100soluble and insoluble fractions (Fig 3 B), and hyperphosphorylated occludin in the Triton X-100insoluble fraction only (Fig 3 B, arrow). Immunostaining of Pa-4
Raf-1:ER-occludin cells verified the normal distribution of occludin at the periphery of cells (Fig 3 C). To demonstrate that the occludin protein detected was exogenous, PCR primers were designed using unique sequences in the 5'-untranslated regions of rat and human occludin cDNAs. Reverse transcriptionPCR results revealed that rat occludin mRNA was only present in Pa-4-vec cells, whereas human occludin mRNA was only detectable in Pa-4
Raf-1:ER-occludin cells (Fig 3 D). When cultured on plastic, Pa-4
Raf-1:ER-occludin cells displayed morphology indistinguishable from that of Pa-4-vec cells (Fig 3 E), and had similar annular rings of actin (Fig 3 F). When cultured on semipermeable filter supports, Pa-4
Raf-1:ER-occludin cells formed monolayers (Fig 3 G) with TEER values of ~900
·cm2 (Fig 3 H), demonstrating the assembly of functional TJs. It is not surprising that the TEER of Pa-4
Raf-1:ER-occludin cells did not recover fully to control levels, because these cells still have elevated Raf-1 activity, which is likely to affect other components responsible for the fine-tuning of epithelial TJs. Another possible reason is that human occludin protein may not work perfectly in a rat cell line. But our results clearly demonstrated that occludin played a crucial role in oncogenic Raf-1induced disruption of epithelial TJs.
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Occludin Protein Assists in the Membrane Localization of ZO-1 and E-Cadherin in Raf-1activated Pa-4 Cells
To investigate the potential role of occludin in coordinating other junctional proteins, we examined cellular distribution and expression of ZO-1, a TJ-associated protein, and E-cadherin, an AJ-associated protein. In Pa-4-vec cells, ZO-1 colocalized with occludin at the TJs (Fig 4 A), whereas E-cadherin localized next to occludin towards the basolateral side (Fig 4 B). However, the distribution patterns of ZO-1 and E-cadherin were disrupted in the occludin-absent Pa-4Raf-1:ER cells. Although ZO-1 protein was no longer exclusively located at the cellcell contact points in Pa-4
Raf-1:ER cells, there was still a substantial amount of ZO-1 appearing as plaques along the cell border, suggesting its membrane localization is independent of occludin expression and functional TJs. Introduction of exogenous occludin restored the distribution patterns of these two proteins to those observed in control cells (Fig 4A and Fig B). Raf-1 activation did not significantly affect the overall protein level of ZO-1, but slightly reduced the ZO-1 level in Triton X-100insoluble fractions. The level of Triton X-100insoluble ZO-1 recovered after introduction of occludin (Fig 4 C). This is consistent with our immunofluorescence results, where we observed a decrease in ZO-1 levels at the lateral membrane in Pa-4
Raf-1:ER cells, and a reconcentration at the TJs in Pa-4
Raf-1:ER-occludin cells. A similar scenario also occurred for E-cadherin distribution, although Raf-1 activation seemed to have decreased the total protein level of E-cadherin (Fig 4 C), consistent with other reports that E-cadherin is downregulated in transformed epithelial cells (
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Occludin Protein Stabilized Claudin-1 Protein in Raf-1activated Pa-4 Cells
Claudins have been implicated in the structure and function of TJ strands (·cm2, we examined claudin-1 as a representative of the claudin gene family in our system. Unlike occludin, the majority of claudin-1 protein was Triton X-100soluble in control Pa-4 cells and only a small percentage was detected in the Triton X-100insoluble fraction (Fig 5 A). This suggests that claudin-1 may not be as directly associated with the actin cytoskeleton as occludin. Claudin-1 protein was almost undetectable in Pa-4
Raf-1:ER cells, but recovered to the levels seen in Pa-4-vec cells after the reexpression of occludin (Fig 5 A). Although there was a decrease of claudin-1 mRNA level in Pa-4
Raf-1:ER cells, surprisingly, Pa-4
Raf-1:ER-occludin cells showed a level of claudin-1 mRNA comparable to that of Pa-4
Raf-1:ER cells (Fig 5 B) even with the apparent difference in protein levels. To further investigate this observation, we examined the protein levels of occludin and claudin-1 in eight different Pa-4
Raf-1:ER-occludin cell clones. The levels of occludin protein varied significantly among the clones (Fig 5 C). This is likely due to the location effects of gene insertion during stable transfection. We observed a general correlation between the protein levels of claudin-1 and those of occludin in the Pa-4
Raf-1:ER-occludin clones (Fig 5 C), but the levels of claudin-1 mRNA among the clones did not change significantly (Fig 5 D). Therefore, it seems possible that claudin-1 protein was stabilized by the presence of occludin protein in Raf-1activated Pa-4 cells. Costaining of claudin-1 and occludin revealed some colocalization between these two proteins in Pa-4-vec and Pa-4
Raf-1:ER-occludin cells (Fig 5 E). Thus, active Raf-1 can completely downregulate the expression of occludin (no detectable mRNA or protein) and decrease the expression of claudin-1, although to a lesser extent compared with occludin (50% decrease of mRNA, 95% decrease of protein). It is possible that the loss of claudin-1 protein in Pa-4
Raf-1:ER cells was due to accelerated protein degradation, implicating a role for occludin in the stabilization of claudin-1. The mechanism of this stabilization is unknown, but may result from proteinprotein interactions between occludin and claudin-1 at the TJs. We also noticed that the TEER among the Pa-4
Raf-1:ER-occludin clones did not correlate with the expression levels of occludin or claudin-1 (data not shown), suggesting that occludin and claudins are not the only players in regulating epithelial TJ function. A delicate balance between claudins and occludin levels and other regulatory components of TJs might be necessary to achieve the maximum barrier function.
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Occludin Expression Inhibited Clonal Formation of Pa-4Raf-1:ER Cells in Soft Agarose
Cancer cells grow aggressively and invasively. In a cell culture system, this is represented by their ability to grow anchorage-independently in soft agarose. Although the basal activity of Raf-1:ER fusion kinase was sufficient to disrupt TJ function and suppress occludin expression in Pa-4 cells, the kinase activity of
Raf-1:ER can be further induced in the presence of added estradiol. Further induction of Raf-1 activity can greatly increase the ability of Pa-4
Raf-1:ER cells to form colonies in soft agarose plates (
Raf-1:ER cells significantly decreased their ability to grow in soft agarose in the absence or presence of estradiol (Fig 6). Thus, there is a possibility that upregulation of occludin expression could be a potential approach to treat Raf-1induced epithelial cancers.
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Discussion |
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Mechanisms that control functional aspects of cellcell contacts are not yet fully understood. Events commonly associated with the oncogenic transformation of epithelial cells, however, include the loss of cellcell contacts and the acquisition of more migratory and invasive phenotypes (
Using the chimeric construct Raf-1:ER in a rat salivary epithelial cell system, we have shown that Raf-1 acts as a signal transducer capable of modulating TJ function. Regulation of occludin expression by Raf-1 may represent a previously unappreciated mechanism in epithelial cell transformation. Although the kinase activity of
Raf-1:ER can be further increased in the presence of added estradiol, the
Raf-1:ER fusion protein has a high level of basal activity (
Raf-1:ER-occludin clones obtained through stable transfection. Although these cells showed different levels of occludin and claudin-1 expression, many other properties of these clones that we have studied were similar. For instance, they all maintained high levels of
Raf-1:ER expression, comparable to that of the Pa-4
Raf-1:ER cells. When cultured on semipermeable filter supports, all eight clones grew as monolayers. Immunofluorescence staining also revealed normal distributions of occludin, claudin-1, ZO-1, and actin in these clones. We have also examined the potential effects of added estradiol on Pa-4
Raf-1:ER-occludin cells. Further activation of
Raf-1:ER by estradiol did not change the monolayer phenotype or TEER in any of the Pa-4
Raf-1:ER-occludin clones (data not shown), nor did it change the inability of these cells to grow in soft agarose (Fig 6). These results are consistent with our other observations, demonstrating that the downregulation of occludin is a downstream cellular event after Raf-1 activation, and reexpression of occludin can reverse some of the phenotypic changes induced by active Raf-1.
Although the MEK-ERK kinase pathway is likely involved in the Raf-1controlled occludin expression, the details of this regulation remain unresolved. Raf-1 signaling may downregulate occludin at the transcription level or through accelerated protein degradation. In Pa-4Raf-1:ER-occludin cells, exogenous (human) occludin protein did not appear to be degraded, even though these cells maintained elevated Raf-1 activity. Human occludin protein is highly homologous to rat occludin. If we assume that the occludin degradation machinery in rat cells can recognize human occludin, our data would suggest that the downregulation of occludin by activated Raf-1 was not through accelerated protein degradation. In addition, we have shown that constitutive activation of Raf-1 led to the complete loss of occludin mRNA in Pa-4 cells, clearly demonstrating that changes at the message level were involved. Furthermore, in A549 cells, which have an oncogenic K-ras mutation, inhibition of the Raf-MEK-ERK kinase pathway by a dominant negative Raf-1 mutant or by the MEK inhibitor PD98059 resulted in increased mRNA level of occludin (Li, D., and R.J. Mrsny, manuscript in preparation). Although we can not rule out the possibility that Raf-1 modulates the stability of occludin mRNA, we think it is more likely that Raf-1 regulates occludin expression at the transcription level rather than through degradation.
Raf-1 activation appeared to have broad effects on several other TJ-associated proteins, including claudin-1 and ZO-1. ZO-1 is known to directly interact with occludin (Raf-1:ER cells, the TJ localization of ZO-1 was disrupted and claudin-1 protein was downregulated. The introduction of exogenous occludin into these cells led to the reappearance of ZO-1 and claudin-1 proteins in the TJs. Although the mechanisms underlying these changes are unclear, our study suggests that the TJ targeting interaction among these proteins could be bidirectional. The changes of claudin-1 and ZO-1 in Pa-4
Raf-1:ER cells are likely to have contributed to the disruption of TJs induced by active Raf-1. However, the fact that reintroduction of occludin alone resulted in the return of normal epithelial phenotype and establishment of functional TJs implicates occludin as a more direct target in the signaling cascade originated from Raf-1. Expression of occludin seemed able to increase the protein level of claudin-1, possibly through stabilization, suggesting a direct interaction between occludin and claudins. The recovery of TEER seen in Pa-4
Raf-1:ER-occludin cells could be a direct result of the recovery in claudin-1 protein level, since there is evidence demonstrating TJ structures in the absence of occludin (
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Footnotes |
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1 Abbreviations used in this paper: AJ, adherens junction; ERK, extracellular-regulated kinase; MEK, mitogen-activated protein kinase; TEER, transepithelial electrical resistance; TJ, tight junction; ZO-1, zonula occludens 1.
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Acknowledgements |
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We thank David Ann for providing the Pa-4 and Pa-4Raf-1:ER cells, and Jeffrey Hooley, Robin Taylor, Wenlu Li, and Deborah Gonzalez for technical assistance.
Submitted: 26 October 1999
Revised: 2 December 1999
Accepted: 19 January 2000
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References |
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