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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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AMPK is activated in the epithelium-lymphocyte co-culture and by TNF-α.(a) Western blot results showing increased level of AMPK phosphorylation in MDCK-lymphocyte co-culture and Calu-3-lymphocyte co-culture as compared to MDCK and Calu-3 cultured alone, respectively. Total AMPK remained unchanged, indicating that AMPK is activated in these co-cultures, with beta-actin used as loading control. The corresponding statistical analysis is on the right panel (*, p<0.05; ***, p<0.001). (b) ATP assay showing no significant changes of cellular ATP levels in MDCK-lymphocyte co-culture vs. MDCK cells alone (‘ns’ stands for no significance). Cell lysates from MDCK cells with and without lymphocytes were subjected to an ATP assay. The ATP contents were normalized to the total protein amount. Data were from three experiments. (c) Lysates prepared from MDCK cells maintained in α-MEM in the absence or presence of TNF-α were blotted with the indicated antibodies. Treatment with AICAR served as the positive control for AMPK activation. (d) The activated AMPK was represented as a relative ratio to total AMPK. The phosphorylation of AMPK is significantly increased by exposure for 2 hours to TNF-α treated, as assessed by Student's test (**, p<0.01).
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pone-0012343-g002: AMPK is activated in the epithelium-lymphocyte co-culture and by TNF-α.(a) Western blot results showing increased level of AMPK phosphorylation in MDCK-lymphocyte co-culture and Calu-3-lymphocyte co-culture as compared to MDCK and Calu-3 cultured alone, respectively. Total AMPK remained unchanged, indicating that AMPK is activated in these co-cultures, with beta-actin used as loading control. The corresponding statistical analysis is on the right panel (*, p<0.05; ***, p<0.001). (b) ATP assay showing no significant changes of cellular ATP levels in MDCK-lymphocyte co-culture vs. MDCK cells alone (‘ns’ stands for no significance). Cell lysates from MDCK cells with and without lymphocytes were subjected to an ATP assay. The ATP contents were normalized to the total protein amount. Data were from three experiments. (c) Lysates prepared from MDCK cells maintained in α-MEM in the absence or presence of TNF-α were blotted with the indicated antibodies. Treatment with AICAR served as the positive control for AMPK activation. (d) The activated AMPK was represented as a relative ratio to total AMPK. The phosphorylation of AMPK is significantly increased by exposure for 2 hours to TNF-α treated, as assessed by Student's test (**, p<0.01).

Mentions: AMPK was reported to be involved in TJ assembly [9], [10] and it has also been implicated in inflammatory responses [16]. We decided, therefore, to examine whether AMPK is involved in the accelerating effect of lymphocytes on TJ assembly. As a first step, we investigated whether AMPK is activated in MDCK cells by Western blot. As shown in Fig. 2a, phosphorylation of AMPK at the Thr172 residue on the alpha-subunit was drastically increased in co-culture compared with MDCK alone whereas total AMPK remained unchanged as shown in a blot probed with AMPK (pan-α) antibody that recognizes both phosphorylated and unphosphorylated AMPK. This result indicates that lymphocyte co-culture creates the potential for involvement of AMPK in TJ assembly of MDCK cells. To determine whether AMPK activation is a general phenomenon that occurs when epithelial cells are co-cultured with lymphocytes, we also examined the level of AMPK activation in another epithelial cell line of different origin, the human airway epithelial cell line, Calu-3. Similar to what has been observed in MDCK cells,, the level of AMPK Thr-172 phosphorylation increased in Calu-3-lymphocyte co-culture, as shown in Fig. 2a, suggesting that AMPK activation may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia.


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)

AMPK is activated in the epithelium-lymphocyte co-culture and by TNF-α.(a) Western blot results showing increased level of AMPK phosphorylation in MDCK-lymphocyte co-culture and Calu-3-lymphocyte co-culture as compared to MDCK and Calu-3 cultured alone, respectively. Total AMPK remained unchanged, indicating that AMPK is activated in these co-cultures, with beta-actin used as loading control. The corresponding statistical analysis is on the right panel (*, p<0.05; ***, p<0.001). (b) ATP assay showing no significant changes of cellular ATP levels in MDCK-lymphocyte co-culture vs. MDCK cells alone (‘ns’ stands for no significance). Cell lysates from MDCK cells with and without lymphocytes were subjected to an ATP assay. The ATP contents were normalized to the total protein amount. Data were from three experiments. (c) Lysates prepared from MDCK cells maintained in α-MEM in the absence or presence of TNF-α were blotted with the indicated antibodies. Treatment with AICAR served as the positive control for AMPK activation. (d) The activated AMPK was represented as a relative ratio to total AMPK. The phosphorylation of AMPK is significantly increased by exposure for 2 hours to TNF-α treated, as assessed by Student's test (**, p<0.01).
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Related In: Results  -  Collection

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pone-0012343-g002: AMPK is activated in the epithelium-lymphocyte co-culture and by TNF-α.(a) Western blot results showing increased level of AMPK phosphorylation in MDCK-lymphocyte co-culture and Calu-3-lymphocyte co-culture as compared to MDCK and Calu-3 cultured alone, respectively. Total AMPK remained unchanged, indicating that AMPK is activated in these co-cultures, with beta-actin used as loading control. The corresponding statistical analysis is on the right panel (*, p<0.05; ***, p<0.001). (b) ATP assay showing no significant changes of cellular ATP levels in MDCK-lymphocyte co-culture vs. MDCK cells alone (‘ns’ stands for no significance). Cell lysates from MDCK cells with and without lymphocytes were subjected to an ATP assay. The ATP contents were normalized to the total protein amount. Data were from three experiments. (c) Lysates prepared from MDCK cells maintained in α-MEM in the absence or presence of TNF-α were blotted with the indicated antibodies. Treatment with AICAR served as the positive control for AMPK activation. (d) The activated AMPK was represented as a relative ratio to total AMPK. The phosphorylation of AMPK is significantly increased by exposure for 2 hours to TNF-α treated, as assessed by Student's test (**, p<0.01).
Mentions: AMPK was reported to be involved in TJ assembly [9], [10] and it has also been implicated in inflammatory responses [16]. We decided, therefore, to examine whether AMPK is involved in the accelerating effect of lymphocytes on TJ assembly. As a first step, we investigated whether AMPK is activated in MDCK cells by Western blot. As shown in Fig. 2a, phosphorylation of AMPK at the Thr172 residue on the alpha-subunit was drastically increased in co-culture compared with MDCK alone whereas total AMPK remained unchanged as shown in a blot probed with AMPK (pan-α) antibody that recognizes both phosphorylated and unphosphorylated AMPK. This result indicates that lymphocyte co-culture creates the potential for involvement of AMPK in TJ assembly of MDCK cells. To determine whether AMPK activation is a general phenomenon that occurs when epithelial cells are co-cultured with lymphocytes, we also examined the level of AMPK activation in another epithelial cell line of different origin, the human airway epithelial cell line, Calu-3. Similar to what has been observed in MDCK cells,, the level of AMPK Thr-172 phosphorylation increased in Calu-3-lymphocyte co-culture, as shown in Fig. 2a, suggesting that AMPK activation may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia.

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