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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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Subcellular localization and expression levels of ZO-1 and E-cadherin. Cells grown on filters were fixed and labeled with antibodies for (A) occludin and ZO-1 (yz single focal planes), or (B) occludin and E-cadherin (yz single focal planes). Occludin, Cy5 labeled (red); ZO-1 and E-cadherin, FITC labeled (green). The lateral membrane staining of occludin is due to secondary antibody effects. Bar, 10 μm. (C) Protein immunoblots probed with anti–ZO-1, anti–E-cadherin, or antiactin antibodies. Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1:ER-occludin, respectively. S, Triton X-100–soluble; I, Triton X-100–insoluble. Blots shown are representatives from three independent experiments.
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Figure 4: Subcellular localization and expression levels of ZO-1 and E-cadherin. Cells grown on filters were fixed and labeled with antibodies for (A) occludin and ZO-1 (yz single focal planes), or (B) occludin and E-cadherin (yz single focal planes). Occludin, Cy5 labeled (red); ZO-1 and E-cadherin, FITC labeled (green). The lateral membrane staining of occludin is due to secondary antibody effects. Bar, 10 μm. (C) Protein immunoblots probed with anti–ZO-1, anti–E-cadherin, or antiactin antibodies. Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1:ER-occludin, respectively. S, Triton X-100–soluble; I, Triton X-100–insoluble. Blots shown are representatives from three independent experiments.

Mentions: 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-4ΔRaf-1:ER cells. Although ZO-1 protein was no longer exclusively located at the cell–cell contact points in Pa-4ΔRaf-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-100–insoluble fractions. The level of Triton X-100–insoluble 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-4ΔRaf-1:ER cells, and a reconcentration at the TJs in Pa-4ΔRaf-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 (Guilford 1999). Our observation implies that occludin may play a role in the localization of these junctional proteins.


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

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

Subcellular localization and expression levels of ZO-1 and E-cadherin. Cells grown on filters were fixed and labeled with antibodies for (A) occludin and ZO-1 (yz single focal planes), or (B) occludin and E-cadherin (yz single focal planes). Occludin, Cy5 labeled (red); ZO-1 and E-cadherin, FITC labeled (green). The lateral membrane staining of occludin is due to secondary antibody effects. Bar, 10 μm. (C) Protein immunoblots probed with anti–ZO-1, anti–E-cadherin, or antiactin antibodies. Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1:ER-occludin, respectively. S, Triton X-100–soluble; I, Triton X-100–insoluble. Blots shown are representatives from three independent experiments.
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Figure 4: Subcellular localization and expression levels of ZO-1 and E-cadherin. Cells grown on filters were fixed and labeled with antibodies for (A) occludin and ZO-1 (yz single focal planes), or (B) occludin and E-cadherin (yz single focal planes). Occludin, Cy5 labeled (red); ZO-1 and E-cadherin, FITC labeled (green). The lateral membrane staining of occludin is due to secondary antibody effects. Bar, 10 μm. (C) Protein immunoblots probed with anti–ZO-1, anti–E-cadherin, or antiactin antibodies. Lanes 1, 2, and 3 represent Pa-4-vec, Pa-4ΔRaf-1:ER, Pa-4ΔRaf-1:ER-occludin, respectively. S, Triton X-100–soluble; I, Triton X-100–insoluble. Blots shown are representatives from three independent experiments.
Mentions: 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-4ΔRaf-1:ER cells. Although ZO-1 protein was no longer exclusively located at the cell–cell contact points in Pa-4ΔRaf-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-100–insoluble fractions. The level of Triton X-100–insoluble 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-4ΔRaf-1:ER cells, and a reconcentration at the TJs in Pa-4ΔRaf-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 (Guilford 1999). Our observation implies that occludin may play a role in the localization of these junctional proteins.

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