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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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Comparison of Pa-4-vec (1) and Pa-4ΔRaf-1:ER (2) cells based on phenotype and occludin expression. Stable Pa-4 cell clones were selected after transfection with a control vector or an oncogenic Raf-1, ΔRaf-1:ER (Samuels et al. 1993). (A) Activation of ERK1 and ERK2 in Pa-4ΔRaf-1:ER cells. Protein lysates were immunoblotted with an antibody to phosphorylated ERK1 and ERK2. (B) Pa-4-vec and Pa-4ΔRaf-1:ER cells grown on plastic displayed differences in plating phenotype. (C) Rhodamine-phalloidin labeling of actin showed the loss of circumferential actin rings and appearance of stress fibers in Pa-4ΔRaf-1:ER cells. (D) Pa-4-vec cells grew as epithelium monosheets on semipermeable supports with high TEER, whereas Pa-4ΔRaf-1:ER cells acquired a stratified phenotype and lost functional TJs. Average TEER values of both cell types are shown. (E) Immunofluorescence staining (Cy5) of occludin in Pa-4-vec and Pa-4ΔRaf-1:ER cells. (F) Western blots with an antibody to occludin. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. Actin was used as loading control. (G) Northern blots using a probe derived from human occludin cDNA sequence (Furuse et al. 1993). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA levels were shown as loading control. Bars, 10 μm.
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Figure 1: Comparison of Pa-4-vec (1) and Pa-4ΔRaf-1:ER (2) cells based on phenotype and occludin expression. Stable Pa-4 cell clones were selected after transfection with a control vector or an oncogenic Raf-1, ΔRaf-1:ER (Samuels et al. 1993). (A) Activation of ERK1 and ERK2 in Pa-4ΔRaf-1:ER cells. Protein lysates were immunoblotted with an antibody to phosphorylated ERK1 and ERK2. (B) Pa-4-vec and Pa-4ΔRaf-1:ER cells grown on plastic displayed differences in plating phenotype. (C) Rhodamine-phalloidin labeling of actin showed the loss of circumferential actin rings and appearance of stress fibers in Pa-4ΔRaf-1:ER cells. (D) Pa-4-vec cells grew as epithelium monosheets on semipermeable supports with high TEER, whereas Pa-4ΔRaf-1:ER cells acquired a stratified phenotype and lost functional TJs. Average TEER values of both cell types are shown. (E) Immunofluorescence staining (Cy5) of occludin in Pa-4-vec and Pa-4ΔRaf-1:ER cells. (F) Western blots with an antibody to occludin. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. Actin was used as loading control. (G) Northern blots using a probe derived from human occludin cDNA sequence (Furuse et al. 1993). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA levels were shown as loading control. Bars, 10 μm.

Mentions: Raf-1 is a serine/threonine kinase composed of two regulatory and one catalytic domain (Morrison and Cutler 1997). Deletion of the regulatory domains of Raf-1 results in a constitutively active form capable of driving MEK-ERK pathway kinase activities. Pa-4 is an immortalized epithelial cell line derived from rat parotid gland, which grows as polarized monosheet in vitro with high transepithelial resistance. Stable transfections of Pa-4 cells were generated with a constitutively active construct of Raf-1 (Li et al. 1997). The expression of ΔRaf-1:ER protein and activation of the Raf-MEK-ERK kinase pathway in the stably transfected cells, Pa-4ΔRaf-1:ER, have been characterized previously (Li et al. 1997). Here, we verified an increased Raf-1 activity in Pa-4ΔRaf-1:ER cells by measuring the phosphorylation levels of ERK1 and ERK2 (Fig. 1 A). When grown on plastic, Pa-4ΔRaf-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-4ΔRaf-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-4ΔRaf-1:ER cells (Fig. 1 E). Western analyses revealed that Pa-4-vec cells had high levels of occludin protein in both Triton X-100–soluble and –insoluble lysates (Fig. 1 F). In the Triton X-100–insoluble fraction, where cytoskeleton-associated proteins are enriched (Wong 1997), a large percentage of occludin was hyperphosphorylated and probably represented a functional component of the TJ (Fig. 1 F). By comparison, Pa-4ΔRaf-1:ER cells completely lost their expression of occludin (Fig. 1 F, arrow). Northern analysis demonstrated that the downregulation of occludin protein in Pa-4ΔRaf-1:ER cells correlated with a complete loss of occludin mRNA (Fig. 1 G).


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

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

Comparison of Pa-4-vec (1) and Pa-4ΔRaf-1:ER (2) cells based on phenotype and occludin expression. Stable Pa-4 cell clones were selected after transfection with a control vector or an oncogenic Raf-1, ΔRaf-1:ER (Samuels et al. 1993). (A) Activation of ERK1 and ERK2 in Pa-4ΔRaf-1:ER cells. Protein lysates were immunoblotted with an antibody to phosphorylated ERK1 and ERK2. (B) Pa-4-vec and Pa-4ΔRaf-1:ER cells grown on plastic displayed differences in plating phenotype. (C) Rhodamine-phalloidin labeling of actin showed the loss of circumferential actin rings and appearance of stress fibers in Pa-4ΔRaf-1:ER cells. (D) Pa-4-vec cells grew as epithelium monosheets on semipermeable supports with high TEER, whereas Pa-4ΔRaf-1:ER cells acquired a stratified phenotype and lost functional TJs. Average TEER values of both cell types are shown. (E) Immunofluorescence staining (Cy5) of occludin in Pa-4-vec and Pa-4ΔRaf-1:ER cells. (F) Western blots with an antibody to occludin. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. Actin was used as loading control. (G) Northern blots using a probe derived from human occludin cDNA sequence (Furuse et al. 1993). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA levels were shown as loading control. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169369&req=5

Figure 1: Comparison of Pa-4-vec (1) and Pa-4ΔRaf-1:ER (2) cells based on phenotype and occludin expression. Stable Pa-4 cell clones were selected after transfection with a control vector or an oncogenic Raf-1, ΔRaf-1:ER (Samuels et al. 1993). (A) Activation of ERK1 and ERK2 in Pa-4ΔRaf-1:ER cells. Protein lysates were immunoblotted with an antibody to phosphorylated ERK1 and ERK2. (B) Pa-4-vec and Pa-4ΔRaf-1:ER cells grown on plastic displayed differences in plating phenotype. (C) Rhodamine-phalloidin labeling of actin showed the loss of circumferential actin rings and appearance of stress fibers in Pa-4ΔRaf-1:ER cells. (D) Pa-4-vec cells grew as epithelium monosheets on semipermeable supports with high TEER, whereas Pa-4ΔRaf-1:ER cells acquired a stratified phenotype and lost functional TJs. Average TEER values of both cell types are shown. (E) Immunofluorescence staining (Cy5) of occludin in Pa-4-vec and Pa-4ΔRaf-1:ER cells. (F) Western blots with an antibody to occludin. S, Triton X-100–soluble; I, Triton X-100–insoluble. Arrow indicates hyperphosphorylated occludin. Actin was used as loading control. (G) Northern blots using a probe derived from human occludin cDNA sequence (Furuse et al. 1993). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA levels were shown as loading control. Bars, 10 μm.
Mentions: Raf-1 is a serine/threonine kinase composed of two regulatory and one catalytic domain (Morrison and Cutler 1997). Deletion of the regulatory domains of Raf-1 results in a constitutively active form capable of driving MEK-ERK pathway kinase activities. Pa-4 is an immortalized epithelial cell line derived from rat parotid gland, which grows as polarized monosheet in vitro with high transepithelial resistance. Stable transfections of Pa-4 cells were generated with a constitutively active construct of Raf-1 (Li et al. 1997). The expression of ΔRaf-1:ER protein and activation of the Raf-MEK-ERK kinase pathway in the stably transfected cells, Pa-4ΔRaf-1:ER, have been characterized previously (Li et al. 1997). Here, we verified an increased Raf-1 activity in Pa-4ΔRaf-1:ER cells by measuring the phosphorylation levels of ERK1 and ERK2 (Fig. 1 A). When grown on plastic, Pa-4ΔRaf-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-4ΔRaf-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-4ΔRaf-1:ER cells (Fig. 1 E). Western analyses revealed that Pa-4-vec cells had high levels of occludin protein in both Triton X-100–soluble and –insoluble lysates (Fig. 1 F). In the Triton X-100–insoluble fraction, where cytoskeleton-associated proteins are enriched (Wong 1997), a large percentage of occludin was hyperphosphorylated and probably represented a functional component of the TJ (Fig. 1 F). By comparison, Pa-4ΔRaf-1:ER cells completely lost their expression of occludin (Fig. 1 F, arrow). Northern analysis demonstrated that the downregulation of occludin protein in Pa-4ΔRaf-1:ER cells correlated with a complete loss of occludin mRNA (Fig. 1 G).

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