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Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK).

Tang XX, Chen H, Yu S, Zhang L, Caplan MJ, Chan HC - PLoS ONE (2010)

Bottom Line: In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone.This accelaration was found to be mediated by AMP-activated protein kinase (AMPK).AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Cell Biology Research Center, School of Biomedial Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Shatin, Hong Kong.

ABSTRACT
The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.

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shRNA-mediated AMPK knockdown inhibits the lymphocyte-facilitation of tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in (A) Vector control cells; (B) Co-cultured vector control cells; (C) shRNA AMPK cells and (D) Co-cultured shRNA AMPK cells at various time points (0h, 1h, 2h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in culture conditions as indicated in (a) at various time points (0h, 1h, 2h, 3h) after calcium switch. The asterisks denote significant differences detected in (B) co-cultured vector control cells vs. (D) co-cultured shRNA AMPK cells by Student's t test (*, p<0.05; **, p<0.01).
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pone-0012343-g004: shRNA-mediated AMPK knockdown inhibits the lymphocyte-facilitation of tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in (A) Vector control cells; (B) Co-cultured vector control cells; (C) shRNA AMPK cells and (D) Co-cultured shRNA AMPK cells at various time points (0h, 1h, 2h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in culture conditions as indicated in (a) at various time points (0h, 1h, 2h, 3h) after calcium switch. The asterisks denote significant differences detected in (B) co-cultured vector control cells vs. (D) co-cultured shRNA AMPK cells by Student's t test (*, p<0.05; **, p<0.01).

Mentions: To further investigate whether the lymphocyte-accelerated TJ assembly was due to the activation of AMPK observed in the co-culture, we used the AMPK inhibitor, Compound C, as well as MDCK cells subjected to shRNA-mediated AMPK knockdown in the calcium switch model. Compound C abolished the lymphocyte-accelerated TJ assembly in the co-culture as measured by the extent of ZO-1 localization to the TJ after calcium switch (Fig. 3a, b), indicating that AMPK activation is critical to the lymphocyte-acceleration of TJ assembly. To further confirm the important role of AMPK in this process, MDCK cells expressing shRNA directed against the AMPK α1 subunit protein and a vector control cell line were tested for their rate of TJ assembly. The α1 isoform is the predominant AMPK α isoform expressed in MDCK cells [9]. As illustrated in Fig. 4a, b, the TJ assembly rate in the co-cultured vector control cells was significantly higher than that in the vector control cells alone, suggesting that the transfected control vector does not impair the lymphocyte-accelerated TJ assembly process in the MDCK cells. However, the TJ assembly rates in AMPK shRNA cells failed to elicit a lymphocyte-accelerated TJ assembly in the co-cultured condition, confirming the requirement for AMPK in this process. In short, forced suppression of AMPK activity, either through a chemical inhibitor or through shRNA-mediated expression knockdown, inhibited the lymphocyte-accelerated TJ formation.


Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK).

Tang XX, Chen H, Yu S, Zhang L, Caplan MJ, Chan HC - PLoS ONE (2010)

shRNA-mediated AMPK knockdown inhibits the lymphocyte-facilitation of tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in (A) Vector control cells; (B) Co-cultured vector control cells; (C) shRNA AMPK cells and (D) Co-cultured shRNA AMPK cells at various time points (0h, 1h, 2h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in culture conditions as indicated in (a) at various time points (0h, 1h, 2h, 3h) after calcium switch. The asterisks denote significant differences detected in (B) co-cultured vector control cells vs. (D) co-cultured shRNA AMPK cells by Student's t test (*, p<0.05; **, p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925955&req=5

pone-0012343-g004: shRNA-mediated AMPK knockdown inhibits the lymphocyte-facilitation of tight junction assembly.(a) Representative pictures of ZO-1 relocation to cell–cell junctions in (A) Vector control cells; (B) Co-cultured vector control cells; (C) shRNA AMPK cells and (D) Co-cultured shRNA AMPK cells at various time points (0h, 1h, 2h, 3h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in culture conditions as indicated in (a) at various time points (0h, 1h, 2h, 3h) after calcium switch. The asterisks denote significant differences detected in (B) co-cultured vector control cells vs. (D) co-cultured shRNA AMPK cells by Student's t test (*, p<0.05; **, p<0.01).
Mentions: To further investigate whether the lymphocyte-accelerated TJ assembly was due to the activation of AMPK observed in the co-culture, we used the AMPK inhibitor, Compound C, as well as MDCK cells subjected to shRNA-mediated AMPK knockdown in the calcium switch model. Compound C abolished the lymphocyte-accelerated TJ assembly in the co-culture as measured by the extent of ZO-1 localization to the TJ after calcium switch (Fig. 3a, b), indicating that AMPK activation is critical to the lymphocyte-acceleration of TJ assembly. To further confirm the important role of AMPK in this process, MDCK cells expressing shRNA directed against the AMPK α1 subunit protein and a vector control cell line were tested for their rate of TJ assembly. The α1 isoform is the predominant AMPK α isoform expressed in MDCK cells [9]. As illustrated in Fig. 4a, b, the TJ assembly rate in the co-cultured vector control cells was significantly higher than that in the vector control cells alone, suggesting that the transfected control vector does not impair the lymphocyte-accelerated TJ assembly process in the MDCK cells. However, the TJ assembly rates in AMPK shRNA cells failed to elicit a lymphocyte-accelerated TJ assembly in the co-cultured condition, confirming the requirement for AMPK in this process. In short, forced suppression of AMPK activity, either through a chemical inhibitor or through shRNA-mediated expression knockdown, inhibited the lymphocyte-accelerated TJ formation.

Bottom Line: In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone.This accelaration was found to be mediated by AMP-activated protein kinase (AMPK).AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Cell Biology Research Center, School of Biomedial Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Shatin, Hong Kong.

ABSTRACT
The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.

Show MeSH
Related in: MedlinePlus