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Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

Lejeune M, Moreau F, Chadee K - PLoS ONE (2014)

Bottom Line: Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells.However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4.We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

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The expression of tight junction mRNA and proteins in Caco-2 EP2 receptor transfectants.(A) The mRNA expression for tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in Caco-2 EP2S and EP2A cells was measured by Q-PCR. The expression of gene of interest (GOI) was normalized with the internal control (Actin) and expressed as fold changes with that of the vector control. (B) A representative immunoblot showing constitutive expression of the important tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in EP2S, EP2A and vector control cells. Actin used as internal control. (C) Densitometry analysis for claudin-4 expression in EP2S, EP2A and vector control cells. EP2A compared statistically with the vector control. ★★ p<0.01.
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pone-0113270-g003: The expression of tight junction mRNA and proteins in Caco-2 EP2 receptor transfectants.(A) The mRNA expression for tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in Caco-2 EP2S and EP2A cells was measured by Q-PCR. The expression of gene of interest (GOI) was normalized with the internal control (Actin) and expressed as fold changes with that of the vector control. (B) A representative immunoblot showing constitutive expression of the important tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in EP2S, EP2A and vector control cells. Actin used as internal control. (C) Densitometry analysis for claudin-4 expression in EP2S, EP2A and vector control cells. EP2A compared statistically with the vector control. ★★ p<0.01.

Mentions: Having identified that EP2A monolayer had extremely low TER compared to the vector control or EP2S cells, we correlated the baseline TER of these cell lines (as shown in Fig. 1B) with that of basal expression of TJ proteins. As TER is a measure of TJ integrity and that the presence or absence of TJ proteins is a major determinant of junctional integrity, we screened for expression of the important TJ proteins such as ZO-1, occludin, claudin-1, claudin-2 and claudin-4. Among the TJ proteins the presence of ZO-1, occludin, claudin-1 and claudin-4 can increase TER. In contrast, claudin-2 is a pore forming TJ protein whose presence can decrease TER. Accordingly, the basal mRNA expression of the TJ proteins were analyzed by Q-PCR in EP2A/S cells and compared with that of the vector control (Fig. 3A). As shown, the transcript for occludin was significantly decreased in both EP2A and EP2S cells whereas alterations in the expression for the other TJ proteins remained unchanged. The alterations observed in occludin in both EP2A/S cells did not correlate with differences in TER as depicted in Figure 1B. However, analysis of protein expression by Western blot (Figure 3B) revealed that the vector control express all the TJ proteins except claudin-2 which correlated well with high TER. EP2S cells normally expressed claudin-4 but moderately expressed ZO-1 and claudin-1. Occludin expression was very low but claudin-2 was moderately expressed and correlated with the TER as compared to the vector control. Similar to EP2S, EP2A cells moderately expressed ZO-1 and claudin-1 and had low expression of occludin. Claudin-2 expression was very low. Interestingly, EP2A cells significantly lacked claudin-4 as compared to the vector control or EP2S cells. Densitometry analysis clearly showed a significant decrease in claudin-4 expression (71% decrease; p<0.01) in EP2A cells as compared to the vector control (Fig. 3C). As EP2A monolayer had very low TER and decreased expression of claudin-4 was exclusively observed in these cells, we conclude that loss of EP2 receptor constitutively is related to the decreased expression of claudin-4 in Caco-2 epithelial monolayer.


Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

Lejeune M, Moreau F, Chadee K - PLoS ONE (2014)

The expression of tight junction mRNA and proteins in Caco-2 EP2 receptor transfectants.(A) The mRNA expression for tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in Caco-2 EP2S and EP2A cells was measured by Q-PCR. The expression of gene of interest (GOI) was normalized with the internal control (Actin) and expressed as fold changes with that of the vector control. (B) A representative immunoblot showing constitutive expression of the important tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in EP2S, EP2A and vector control cells. Actin used as internal control. (C) Densitometry analysis for claudin-4 expression in EP2S, EP2A and vector control cells. EP2A compared statistically with the vector control. ★★ p<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232557&req=5

pone-0113270-g003: The expression of tight junction mRNA and proteins in Caco-2 EP2 receptor transfectants.(A) The mRNA expression for tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in Caco-2 EP2S and EP2A cells was measured by Q-PCR. The expression of gene of interest (GOI) was normalized with the internal control (Actin) and expressed as fold changes with that of the vector control. (B) A representative immunoblot showing constitutive expression of the important tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in EP2S, EP2A and vector control cells. Actin used as internal control. (C) Densitometry analysis for claudin-4 expression in EP2S, EP2A and vector control cells. EP2A compared statistically with the vector control. ★★ p<0.01.
Mentions: Having identified that EP2A monolayer had extremely low TER compared to the vector control or EP2S cells, we correlated the baseline TER of these cell lines (as shown in Fig. 1B) with that of basal expression of TJ proteins. As TER is a measure of TJ integrity and that the presence or absence of TJ proteins is a major determinant of junctional integrity, we screened for expression of the important TJ proteins such as ZO-1, occludin, claudin-1, claudin-2 and claudin-4. Among the TJ proteins the presence of ZO-1, occludin, claudin-1 and claudin-4 can increase TER. In contrast, claudin-2 is a pore forming TJ protein whose presence can decrease TER. Accordingly, the basal mRNA expression of the TJ proteins were analyzed by Q-PCR in EP2A/S cells and compared with that of the vector control (Fig. 3A). As shown, the transcript for occludin was significantly decreased in both EP2A and EP2S cells whereas alterations in the expression for the other TJ proteins remained unchanged. The alterations observed in occludin in both EP2A/S cells did not correlate with differences in TER as depicted in Figure 1B. However, analysis of protein expression by Western blot (Figure 3B) revealed that the vector control express all the TJ proteins except claudin-2 which correlated well with high TER. EP2S cells normally expressed claudin-4 but moderately expressed ZO-1 and claudin-1. Occludin expression was very low but claudin-2 was moderately expressed and correlated with the TER as compared to the vector control. Similar to EP2S, EP2A cells moderately expressed ZO-1 and claudin-1 and had low expression of occludin. Claudin-2 expression was very low. Interestingly, EP2A cells significantly lacked claudin-4 as compared to the vector control or EP2S cells. Densitometry analysis clearly showed a significant decrease in claudin-4 expression (71% decrease; p<0.01) in EP2A cells as compared to the vector control (Fig. 3C). As EP2A monolayer had very low TER and decreased expression of claudin-4 was exclusively observed in these cells, we conclude that loss of EP2 receptor constitutively is related to the decreased expression of claudin-4 in Caco-2 epithelial monolayer.

Bottom Line: Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells.However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4.We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

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