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Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia.

Tokuda S, Kim YH, Matsumoto H, Muro S, Hirai T, Mishima M, Furuse M - PLoS ONE (2015)

Bottom Line: The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability.The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification.The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

ABSTRACT
The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

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Localization of claudin-3, E-cadherin, occludin and Na+/K+ ATPase under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells and barrier function of TJs around the cavities within the multi-layered epithelia.(A and B) Immunofluorescence microscopy for claudin-3 and E-cadherin in z-axis plane under the ‘Apical’ (A) and ‘Basal’ (B) conditions in MDCK I cells. Signals of claudin-3 were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (C and D) Immunofluorescence microscopy for occludin and Na+/K+ ATPase in z-axis plane under the ‘Apical’ (C) and ‘Basal’ (D) conditions in MDCK I cells. Signals of occludin were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (E and F) A tracer experiment using a primary amine-reactive biotinylation reagent was performed in MDCK I cells cultured under the ‘Apical’ (E) and ‘Basal’ (F) conditions. The biotinylation reagent was administered in the basal side for 10 min, and the bound biotin was detected by streptavidin. The biotinylation reagent appeared to stop at TJs around the cavities within the multi-layered epithelia under the ‘Basal’ condition (arrows). Scale bars = 10 μm.
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pone.0145522.g006: Localization of claudin-3, E-cadherin, occludin and Na+/K+ ATPase under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells and barrier function of TJs around the cavities within the multi-layered epithelia.(A and B) Immunofluorescence microscopy for claudin-3 and E-cadherin in z-axis plane under the ‘Apical’ (A) and ‘Basal’ (B) conditions in MDCK I cells. Signals of claudin-3 were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (C and D) Immunofluorescence microscopy for occludin and Na+/K+ ATPase in z-axis plane under the ‘Apical’ (C) and ‘Basal’ (D) conditions in MDCK I cells. Signals of occludin were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (E and F) A tracer experiment using a primary amine-reactive biotinylation reagent was performed in MDCK I cells cultured under the ‘Apical’ (E) and ‘Basal’ (F) conditions. The biotinylation reagent was administered in the basal side for 10 min, and the bound biotin was detected by streptavidin. The biotinylation reagent appeared to stop at TJs around the cavities within the multi-layered epithelia under the ‘Basal’ condition (arrows). Scale bars = 10 μm.

Mentions: Currently many proteins have been identified as TJ components such as a scaffold protein zonula occludens-1 (ZO-1) [15] and integral membrane proteins occludin [16] and claudins [17]. To confirm the localization of TJ proteins at cell-cell contacts around the cavities present within the multi-layered epithelia, we examined the localization of ZO-1 in MDCK I cells at four days after the culture under the ‘Apical’ and ‘Basal’ conditions by immunofluorescence microscopy (Fig 5). Under the ‘Apical’ condition, signals of ZO-1 were concentrated at the most apical regions of cell-cell contacts with the faint signals along the lateral membranes in MDCK I cells (Fig 5A), which was consistent with previous studies [18–20]. In contrast, signals of ZO-1 were found within the multi-layered epithelia as well as at cell-cell contacts of the outermost cell layer (Fig 5B). In three-dimensional images constructed from an integration of confocal scanning images, closed lines of ZO-1 signals were found within the multi-layered epithelia under the ‘Basal’ condition (Fig 5E and S1 Movie). Spherical staining of F-actin was also observed within the multi-layered epithelia under the ‘Basal’ condition (Fig 5D), which was thought to represent the signals of F-actin of microvilli observed in the electron microscopy. We also examined the localization of claudin-3 and occludin in MDCK I cells (Fig 6A–6D). Signals of claudin-3 and occludin were also observed within the multi-layered epithelia under the ‘Basal’ condition. These results indicate that TJ proteins including ZO-1, claudin(s) and occludin localize at the TJs in the MDCK I cells surrounding the cavities.


Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia.

Tokuda S, Kim YH, Matsumoto H, Muro S, Hirai T, Mishima M, Furuse M - PLoS ONE (2015)

Localization of claudin-3, E-cadherin, occludin and Na+/K+ ATPase under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells and barrier function of TJs around the cavities within the multi-layered epithelia.(A and B) Immunofluorescence microscopy for claudin-3 and E-cadherin in z-axis plane under the ‘Apical’ (A) and ‘Basal’ (B) conditions in MDCK I cells. Signals of claudin-3 were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (C and D) Immunofluorescence microscopy for occludin and Na+/K+ ATPase in z-axis plane under the ‘Apical’ (C) and ‘Basal’ (D) conditions in MDCK I cells. Signals of occludin were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (E and F) A tracer experiment using a primary amine-reactive biotinylation reagent was performed in MDCK I cells cultured under the ‘Apical’ (E) and ‘Basal’ (F) conditions. The biotinylation reagent was administered in the basal side for 10 min, and the bound biotin was detected by streptavidin. The biotinylation reagent appeared to stop at TJs around the cavities within the multi-layered epithelia under the ‘Basal’ condition (arrows). Scale bars = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696811&req=5

pone.0145522.g006: Localization of claudin-3, E-cadherin, occludin and Na+/K+ ATPase under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells and barrier function of TJs around the cavities within the multi-layered epithelia.(A and B) Immunofluorescence microscopy for claudin-3 and E-cadherin in z-axis plane under the ‘Apical’ (A) and ‘Basal’ (B) conditions in MDCK I cells. Signals of claudin-3 were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (C and D) Immunofluorescence microscopy for occludin and Na+/K+ ATPase in z-axis plane under the ‘Apical’ (C) and ‘Basal’ (D) conditions in MDCK I cells. Signals of occludin were observed within the multi-layered MDCK I cells under the ‘Basal’ condition (arrows). (E and F) A tracer experiment using a primary amine-reactive biotinylation reagent was performed in MDCK I cells cultured under the ‘Apical’ (E) and ‘Basal’ (F) conditions. The biotinylation reagent was administered in the basal side for 10 min, and the bound biotin was detected by streptavidin. The biotinylation reagent appeared to stop at TJs around the cavities within the multi-layered epithelia under the ‘Basal’ condition (arrows). Scale bars = 10 μm.
Mentions: Currently many proteins have been identified as TJ components such as a scaffold protein zonula occludens-1 (ZO-1) [15] and integral membrane proteins occludin [16] and claudins [17]. To confirm the localization of TJ proteins at cell-cell contacts around the cavities present within the multi-layered epithelia, we examined the localization of ZO-1 in MDCK I cells at four days after the culture under the ‘Apical’ and ‘Basal’ conditions by immunofluorescence microscopy (Fig 5). Under the ‘Apical’ condition, signals of ZO-1 were concentrated at the most apical regions of cell-cell contacts with the faint signals along the lateral membranes in MDCK I cells (Fig 5A), which was consistent with previous studies [18–20]. In contrast, signals of ZO-1 were found within the multi-layered epithelia as well as at cell-cell contacts of the outermost cell layer (Fig 5B). In three-dimensional images constructed from an integration of confocal scanning images, closed lines of ZO-1 signals were found within the multi-layered epithelia under the ‘Basal’ condition (Fig 5E and S1 Movie). Spherical staining of F-actin was also observed within the multi-layered epithelia under the ‘Basal’ condition (Fig 5D), which was thought to represent the signals of F-actin of microvilli observed in the electron microscopy. We also examined the localization of claudin-3 and occludin in MDCK I cells (Fig 6A–6D). Signals of claudin-3 and occludin were also observed within the multi-layered epithelia under the ‘Basal’ condition. These results indicate that TJ proteins including ZO-1, claudin(s) and occludin localize at the TJs in the MDCK I cells surrounding the cavities.

Bottom Line: The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability.The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification.The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

ABSTRACT
The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

Show MeSH
Related in: MedlinePlus