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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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The presence of thymocytes do not change inter-TEC GJIC. Mouse TEC co-cultures (IT-76M1 cells) were maintained at 37°C for 2 hrs to establish an adherent confluent monolayer, and then simultaneously cultured with murine thymocytes at 1:5 or 1:10 (TEC:thymocytes) proportion for additional 5 hrs. After this incubation the thymocytes were discarded and epithelial cells were dissociated and analyzed by flow cytometry. The normalized dye coupling degree (gray columns) and the calcein mean fluorescence (white columns) of the double positive cells, clearly show the presence of thymocytes did not significantly modify the levels of dye coupling and dye transfer efficiency among mouse TEC. Data are expressed as mean SD, being derived from obtained from three independent experiments.
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Figure 6: The presence of thymocytes do not change inter-TEC GJIC. Mouse TEC co-cultures (IT-76M1 cells) were maintained at 37°C for 2 hrs to establish an adherent confluent monolayer, and then simultaneously cultured with murine thymocytes at 1:5 or 1:10 (TEC:thymocytes) proportion for additional 5 hrs. After this incubation the thymocytes were discarded and epithelial cells were dissociated and analyzed by flow cytometry. The normalized dye coupling degree (gray columns) and the calcein mean fluorescence (white columns) of the double positive cells, clearly show the presence of thymocytes did not significantly modify the levels of dye coupling and dye transfer efficiency among mouse TEC. Data are expressed as mean SD, being derived from obtained from three independent experiments.

Mentions: After the adhesion and establishment of mouse TEC co-cultures [containing calcein+DiIc18(3)- and calcein-DiIc18(3)+ cells], the thymocytes were added at 5 fold or 10 fold excess over TECs. After an additional 5 hours, the thymocytes were discarded and the TEC co-cultures were dissociated and analyzed by flow cytometry. In these conditions, the dye coupling between adjacent TEC was not significantly modulated by thymocytes, neither at 1:5 nor at 1:10 TEC:thymocytes proportions (Figure 6). The calcein fluorescence intensity of calcein+DiIc18(3)+ was not significantly modified as well (Figure 6).


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)

The presence of thymocytes do not change inter-TEC GJIC. Mouse TEC co-cultures (IT-76M1 cells) were maintained at 37°C for 2 hrs to establish an adherent confluent monolayer, and then simultaneously cultured with murine thymocytes at 1:5 or 1:10 (TEC:thymocytes) proportion for additional 5 hrs. After this incubation the thymocytes were discarded and epithelial cells were dissociated and analyzed by flow cytometry. The normalized dye coupling degree (gray columns) and the calcein mean fluorescence (white columns) of the double positive cells, clearly show the presence of thymocytes did not significantly modify the levels of dye coupling and dye transfer efficiency among mouse TEC. Data are expressed as mean SD, being derived from obtained from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: The presence of thymocytes do not change inter-TEC GJIC. Mouse TEC co-cultures (IT-76M1 cells) were maintained at 37°C for 2 hrs to establish an adherent confluent monolayer, and then simultaneously cultured with murine thymocytes at 1:5 or 1:10 (TEC:thymocytes) proportion for additional 5 hrs. After this incubation the thymocytes were discarded and epithelial cells were dissociated and analyzed by flow cytometry. The normalized dye coupling degree (gray columns) and the calcein mean fluorescence (white columns) of the double positive cells, clearly show the presence of thymocytes did not significantly modify the levels of dye coupling and dye transfer efficiency among mouse TEC. Data are expressed as mean SD, being derived from obtained from three independent experiments.
Mentions: After the adhesion and establishment of mouse TEC co-cultures [containing calcein+DiIc18(3)- and calcein-DiIc18(3)+ cells], the thymocytes were added at 5 fold or 10 fold excess over TECs. After an additional 5 hours, the thymocytes were discarded and the TEC co-cultures were dissociated and analyzed by flow cytometry. In these conditions, the dye coupling between adjacent TEC was not significantly modulated by thymocytes, neither at 1:5 nor at 1:10 TEC:thymocytes proportions (Figure 6). The calcein fluorescence intensity of calcein+DiIc18(3)+ was not significantly modified as well (Figure 6).

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