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Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin.

Li D, Mrsny RJ - J. Cell Biol. (2000)

Bottom Line: 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 cell-cell contact growth control.Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs.Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Research and Development, Genentech Inc., South San Francisco, California 94080, USA.

ABSTRACT
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 cell-cell 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-1-activated 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-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.

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Related in: MedlinePlus

Expression of exogenous occludin induced the formation of functional epithelial cell TJs in Pa-4ΔRaf-1:ER cells. (A) Western blots using antibodies to human estrogen receptor (top), phosphorylated ERK1 and ERK2 (middle), or actin (bottom). Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1: ER-occludin, respectively. Similar results were seen in all eight Pa-4ΔRaf-1:ER-occludin clones. (B) Pa-4ΔRaf-1:ER-occludin lysates were immunoblotted with an antioccludin antibody. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. (C) Immunofluorescence staining (Cy5) showing occludin was concentrated at the cell borders. (D) Total RNA was isolated from Pa-4-vec (lane 1), Pa-4ΔRaf-1:ER (lane 2), and Pa-4ΔRaf-1:ER-occludin (lane 3) cells. Reverse transcription–PCR was performed using primer sets specific for rat occludin, human occludin, or glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (as control). (E) Pa-4ΔRaf-1:ER-occludin cells grown on plastic displayed similar phenotype to that of Pa-4-vec cells. (F) Rhodamine-phalloidin labeling of actin showed the reappearance of pericellular actin rings in Pa-4ΔRaf-1:ER-occludin cells. (G) Pa-4ΔRaf-1:ER-occludin cells cultured on semipermeable filters grew as monolayers. (H) Formation of functional epithelial TJs in Pa-4ΔRaf-1:ER-occludin cells. TEER of Pa-4-vec (square), Pa-4ΔRaf-1:ER (triangle), and Pa-4ΔRaf-1:ER-occludin (circle) cells grown on filters were measured using a chopstick voltmeter. Data represent the mean ± SEM of six filters from each cell type. Bars, 10 μm.
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Figure 3: Expression of exogenous occludin induced the formation of functional epithelial cell TJs in Pa-4ΔRaf-1:ER cells. (A) Western blots using antibodies to human estrogen receptor (top), phosphorylated ERK1 and ERK2 (middle), or actin (bottom). Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1: ER-occludin, respectively. Similar results were seen in all eight Pa-4ΔRaf-1:ER-occludin clones. (B) Pa-4ΔRaf-1:ER-occludin lysates were immunoblotted with an antioccludin antibody. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. (C) Immunofluorescence staining (Cy5) showing occludin was concentrated at the cell borders. (D) Total RNA was isolated from Pa-4-vec (lane 1), Pa-4ΔRaf-1:ER (lane 2), and Pa-4ΔRaf-1:ER-occludin (lane 3) cells. Reverse transcription–PCR was performed using primer sets specific for rat occludin, human occludin, or glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (as control). (E) Pa-4ΔRaf-1:ER-occludin cells grown on plastic displayed similar phenotype to that of Pa-4-vec cells. (F) Rhodamine-phalloidin labeling of actin showed the reappearance of pericellular actin rings in Pa-4ΔRaf-1:ER-occludin cells. (G) Pa-4ΔRaf-1:ER-occludin cells cultured on semipermeable filters grew as monolayers. (H) Formation of functional epithelial TJs in Pa-4ΔRaf-1:ER-occludin cells. TEER of Pa-4-vec (square), Pa-4ΔRaf-1:ER (triangle), and Pa-4ΔRaf-1:ER-occludin (circle) cells grown on filters were measured using a chopstick voltmeter. Data represent the mean ± SEM of six filters from each cell type. Bars, 10 μm.

Mentions: Although we made the observation that occludin was downregulated in Raf-1–activated 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-4ΔRaf-1:ER cells, which no longer express endogenous occludin. A total of eight Pa-4ΔRaf-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-4ΔRaf-1:ER cells. Occludin-transfected Pa-4ΔRaf-1:ER cells were verified to have similar levels of ΔRaf-1:ER protein and phosphorylated ERK1 and ERK2 compared with Pa-4ΔRaf-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-4ΔRaf-1:ER-occludin cells confirmed the presence of occludin in both Triton X-100–soluble and –insoluble fractions (Fig. 3 B), and hyperphosphorylated occludin in the Triton X-100–insoluble fraction only (Fig. 3 B, arrow). Immunostaining of Pa-4ΔRaf-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 transcription–PCR results revealed that rat occludin mRNA was only present in Pa-4-vec cells, whereas human occludin mRNA was only detectable in Pa-4ΔRaf-1:ER-occludin cells (Fig. 3 D). When cultured on plastic, Pa-4ΔRaf-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-4ΔRaf-1:ER-occludin cells formed monolayers (Fig. 3 G) with TEER values of ∼900 Ω·cm2 (Fig. 3 H), demonstrating the assembly of functional TJs. It is not surprising that the TEER of Pa-4ΔRaf-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-1–induced disruption of epithelial TJs.


Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin.

Li D, Mrsny RJ - J. Cell Biol. (2000)

Expression of exogenous occludin induced the formation of functional epithelial cell TJs in Pa-4ΔRaf-1:ER cells. (A) Western blots using antibodies to human estrogen receptor (top), phosphorylated ERK1 and ERK2 (middle), or actin (bottom). Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1: ER-occludin, respectively. Similar results were seen in all eight Pa-4ΔRaf-1:ER-occludin clones. (B) Pa-4ΔRaf-1:ER-occludin lysates were immunoblotted with an antioccludin antibody. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. (C) Immunofluorescence staining (Cy5) showing occludin was concentrated at the cell borders. (D) Total RNA was isolated from Pa-4-vec (lane 1), Pa-4ΔRaf-1:ER (lane 2), and Pa-4ΔRaf-1:ER-occludin (lane 3) cells. Reverse transcription–PCR was performed using primer sets specific for rat occludin, human occludin, or glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (as control). (E) Pa-4ΔRaf-1:ER-occludin cells grown on plastic displayed similar phenotype to that of Pa-4-vec cells. (F) Rhodamine-phalloidin labeling of actin showed the reappearance of pericellular actin rings in Pa-4ΔRaf-1:ER-occludin cells. (G) Pa-4ΔRaf-1:ER-occludin cells cultured on semipermeable filters grew as monolayers. (H) Formation of functional epithelial TJs in Pa-4ΔRaf-1:ER-occludin cells. TEER of Pa-4-vec (square), Pa-4ΔRaf-1:ER (triangle), and Pa-4ΔRaf-1:ER-occludin (circle) cells grown on filters were measured using a chopstick voltmeter. Data represent the mean ± SEM of six filters from each cell type. Bars, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169369&req=5

Figure 3: Expression of exogenous occludin induced the formation of functional epithelial cell TJs in Pa-4ΔRaf-1:ER cells. (A) Western blots using antibodies to human estrogen receptor (top), phosphorylated ERK1 and ERK2 (middle), or actin (bottom). Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1: ER-occludin, respectively. Similar results were seen in all eight Pa-4ΔRaf-1:ER-occludin clones. (B) Pa-4ΔRaf-1:ER-occludin lysates were immunoblotted with an antioccludin antibody. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. (C) Immunofluorescence staining (Cy5) showing occludin was concentrated at the cell borders. (D) Total RNA was isolated from Pa-4-vec (lane 1), Pa-4ΔRaf-1:ER (lane 2), and Pa-4ΔRaf-1:ER-occludin (lane 3) cells. Reverse transcription–PCR was performed using primer sets specific for rat occludin, human occludin, or glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (as control). (E) Pa-4ΔRaf-1:ER-occludin cells grown on plastic displayed similar phenotype to that of Pa-4-vec cells. (F) Rhodamine-phalloidin labeling of actin showed the reappearance of pericellular actin rings in Pa-4ΔRaf-1:ER-occludin cells. (G) Pa-4ΔRaf-1:ER-occludin cells cultured on semipermeable filters grew as monolayers. (H) Formation of functional epithelial TJs in Pa-4ΔRaf-1:ER-occludin cells. TEER of Pa-4-vec (square), Pa-4ΔRaf-1:ER (triangle), and Pa-4ΔRaf-1:ER-occludin (circle) cells grown on filters were measured using a chopstick voltmeter. Data represent the mean ± SEM of six filters from each cell type. Bars, 10 μm.
Mentions: Although we made the observation that occludin was downregulated in Raf-1–activated 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-4ΔRaf-1:ER cells, which no longer express endogenous occludin. A total of eight Pa-4ΔRaf-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-4ΔRaf-1:ER cells. Occludin-transfected Pa-4ΔRaf-1:ER cells were verified to have similar levels of ΔRaf-1:ER protein and phosphorylated ERK1 and ERK2 compared with Pa-4ΔRaf-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-4ΔRaf-1:ER-occludin cells confirmed the presence of occludin in both Triton X-100–soluble and –insoluble fractions (Fig. 3 B), and hyperphosphorylated occludin in the Triton X-100–insoluble fraction only (Fig. 3 B, arrow). Immunostaining of Pa-4ΔRaf-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 transcription–PCR results revealed that rat occludin mRNA was only present in Pa-4-vec cells, whereas human occludin mRNA was only detectable in Pa-4ΔRaf-1:ER-occludin cells (Fig. 3 D). When cultured on plastic, Pa-4ΔRaf-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-4ΔRaf-1:ER-occludin cells formed monolayers (Fig. 3 G) with TEER values of ∼900 Ω·cm2 (Fig. 3 H), demonstrating the assembly of functional TJs. It is not surprising that the TEER of Pa-4ΔRaf-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-1–induced disruption of epithelial TJs.

Bottom Line: 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 cell-cell contact growth control.Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs.Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Research and Development, Genentech Inc., South San Francisco, California 94080, USA.

ABSTRACT
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 cell-cell 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-1-activated 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-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.

Show MeSH
Related in: MedlinePlus