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Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells.

Nihei OK, Fonseca PC, Rubim NM, Bonavita AG, Lyra JS, Neves-dos-Santos S, de Carvalho AC, Spray DC, Savino W, Alves LA - BMC Cell Biol. (2010)

Bottom Line: The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators.Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations.Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular Communication, Oswaldo Cruz Institute, The Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.

ABSTRACT

Background: We investigated the effects of the signaling molecules, cyclic AMP (cAMP) and protein-kinase C (PKC), on gap junctional intercellular communication (GJIC) between thymic epithelial cells (TEC).

Results: Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC.

Conclusions: Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

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Flow cytometric analysis of the mouse thymic epithelial cell line, showing inter-TEC gap junction intercellular communication. Calcein+Dilc18(3)- and calcein-DiIc18(3)+ IT-76M1 cells were co-cultured for 6 hr at 37°C. These cells were then dissociated and analyzed by flow cytometry to quantify the double positive [calcein+Dilc18(3)+] cells. Some calcein+Dilc18(3)- and calcein-DiIc18(3)+ cells were separately cultured and used to adjust the cytometry settings. These cells also were used to establish the control population (A). Data are presented in the form of dot plots (A, B, C), which depict two-dimensionally the labeling pattern of each cell population considering the fluorescence intensity (log scale) of calcein and DiIc18(3). In B, the 6 hr co-cultured cells are shown, where the presence of double positive cells is apparent, indicating the dye coupling. In C, cells co-cultured for 6 hours in the presence of 18-β-glycyrrhetinic acid (GRA; 100 μM) exhibited a complete inhibition of inter-TEC GJIC. These data are representative of at least 4 experiments.
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Figure 1: Flow cytometric analysis of the mouse thymic epithelial cell line, showing inter-TEC gap junction intercellular communication. Calcein+Dilc18(3)- and calcein-DiIc18(3)+ IT-76M1 cells were co-cultured for 6 hr at 37°C. These cells were then dissociated and analyzed by flow cytometry to quantify the double positive [calcein+Dilc18(3)+] cells. Some calcein+Dilc18(3)- and calcein-DiIc18(3)+ cells were separately cultured and used to adjust the cytometry settings. These cells also were used to establish the control population (A). Data are presented in the form of dot plots (A, B, C), which depict two-dimensionally the labeling pattern of each cell population considering the fluorescence intensity (log scale) of calcein and DiIc18(3). In B, the 6 hr co-cultured cells are shown, where the presence of double positive cells is apparent, indicating the dye coupling. In C, cells co-cultured for 6 hours in the presence of 18-β-glycyrrhetinic acid (GRA; 100 μM) exhibited a complete inhibition of inter-TEC GJIC. These data are representative of at least 4 experiments.

Mentions: Confirming previous results from our Laboratory [39,61,62] flow cytometry experiments revealed that after 6 hours of co-culture more than 65% of the initial single positive DiIc18(3)+ TEC acquired calcein (Figure 1B). To ascertain that gap junctions mediated this dye transfer we treated the cells for 6 hrs with 100 μM of 18-β-glycyrrhetinic acid (GRA), a gap junction inhibitor. Such procedure inhibited inter-TEC GJIC by >85% (Figure 1C). Similar results were also obtained when the epithelial were treated with carbenoxolone, another gap junction inhibitor (data not shown).


Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells.

Nihei OK, Fonseca PC, Rubim NM, Bonavita AG, Lyra JS, Neves-dos-Santos S, de Carvalho AC, Spray DC, Savino W, Alves LA - BMC Cell Biol. (2010)

Flow cytometric analysis of the mouse thymic epithelial cell line, showing inter-TEC gap junction intercellular communication. Calcein+Dilc18(3)- and calcein-DiIc18(3)+ IT-76M1 cells were co-cultured for 6 hr at 37°C. These cells were then dissociated and analyzed by flow cytometry to quantify the double positive [calcein+Dilc18(3)+] cells. Some calcein+Dilc18(3)- and calcein-DiIc18(3)+ cells were separately cultured and used to adjust the cytometry settings. These cells also were used to establish the control population (A). Data are presented in the form of dot plots (A, B, C), which depict two-dimensionally the labeling pattern of each cell population considering the fluorescence intensity (log scale) of calcein and DiIc18(3). In B, the 6 hr co-cultured cells are shown, where the presence of double positive cells is apparent, indicating the dye coupling. In C, cells co-cultured for 6 hours in the presence of 18-β-glycyrrhetinic acid (GRA; 100 μM) exhibited a complete inhibition of inter-TEC GJIC. These data are representative of at least 4 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823718&req=5

Figure 1: Flow cytometric analysis of the mouse thymic epithelial cell line, showing inter-TEC gap junction intercellular communication. Calcein+Dilc18(3)- and calcein-DiIc18(3)+ IT-76M1 cells were co-cultured for 6 hr at 37°C. These cells were then dissociated and analyzed by flow cytometry to quantify the double positive [calcein+Dilc18(3)+] cells. Some calcein+Dilc18(3)- and calcein-DiIc18(3)+ cells were separately cultured and used to adjust the cytometry settings. These cells also were used to establish the control population (A). Data are presented in the form of dot plots (A, B, C), which depict two-dimensionally the labeling pattern of each cell population considering the fluorescence intensity (log scale) of calcein and DiIc18(3). In B, the 6 hr co-cultured cells are shown, where the presence of double positive cells is apparent, indicating the dye coupling. In C, cells co-cultured for 6 hours in the presence of 18-β-glycyrrhetinic acid (GRA; 100 μM) exhibited a complete inhibition of inter-TEC GJIC. These data are representative of at least 4 experiments.
Mentions: Confirming previous results from our Laboratory [39,61,62] flow cytometry experiments revealed that after 6 hours of co-culture more than 65% of the initial single positive DiIc18(3)+ TEC acquired calcein (Figure 1B). To ascertain that gap junctions mediated this dye transfer we treated the cells for 6 hrs with 100 μM of 18-β-glycyrrhetinic acid (GRA), a gap junction inhibitor. Such procedure inhibited inter-TEC GJIC by >85% (Figure 1C). Similar results were also obtained when the epithelial were treated with carbenoxolone, another gap junction inhibitor (data not shown).

Bottom Line: The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators.Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations.Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular Communication, Oswaldo Cruz Institute, The Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.

ABSTRACT

Background: We investigated the effects of the signaling molecules, cyclic AMP (cAMP) and protein-kinase C (PKC), on gap junctional intercellular communication (GJIC) between thymic epithelial cells (TEC).

Results: Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC.

Conclusions: Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

Show MeSH