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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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Increase in cAMP enhances inter-TEC GJIC. Panels A and B depict untreated co-cultures of the mouse TEC line at zero and 6 hours time points, respectively. The percentage of double positive cells and the calcein geometric mean fluorescence intensity (MFI) of these populations are depicted at the upper right corner of each panel (%, above; MFI, below). Panels C and D show the inter-TEC coupling, following 6 hours of treatment with 1 mM 8-Br-cAMP or 10 μM forskolin. Both treatments enhanced the calcein mean fluorescence intensity of coupled cells (double positive cells). These data are representative of at least 4 separate experiments. Such enhancements can also be seen in Panels E to H, depicting TEC co-cultures treated for 6 hours with increasing concentrations of either 8-Br-cAMP (E-F) or forskolin (G-H). While percentages of coupled TEC was not significantly modified (E, G), the geometric mean fluorescence of calcein quantified from the double positive cells tripled after both treatments (F, H). The results are representative of 3 independent experiments (mean SD). Panel I shows that 8-Br-cAMP was also capable of enhancing inter-TEC GJIC, in primary cultures of human TNC-derived epithelial cells. Numbers of coupled cells were count in blind. * p < 0.05.
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Figure 2: Increase in cAMP enhances inter-TEC GJIC. Panels A and B depict untreated co-cultures of the mouse TEC line at zero and 6 hours time points, respectively. The percentage of double positive cells and the calcein geometric mean fluorescence intensity (MFI) of these populations are depicted at the upper right corner of each panel (%, above; MFI, below). Panels C and D show the inter-TEC coupling, following 6 hours of treatment with 1 mM 8-Br-cAMP or 10 μM forskolin. Both treatments enhanced the calcein mean fluorescence intensity of coupled cells (double positive cells). These data are representative of at least 4 separate experiments. Such enhancements can also be seen in Panels E to H, depicting TEC co-cultures treated for 6 hours with increasing concentrations of either 8-Br-cAMP (E-F) or forskolin (G-H). While percentages of coupled TEC was not significantly modified (E, G), the geometric mean fluorescence of calcein quantified from the double positive cells tripled after both treatments (F, H). The results are representative of 3 independent experiments (mean SD). Panel I shows that 8-Br-cAMP was also capable of enhancing inter-TEC GJIC, in primary cultures of human TNC-derived epithelial cells. Numbers of coupled cells were count in blind. * p < 0.05.

Mentions: To analyze the putative modulatory effects of cAMP on GJIC in TEC cultures, we stimulated the mouse TEC line with 8-Br-cAMP, a membrane permeable cAMP analog; or forskolin, which stimulates cAMP production through activation of adenylate cyclase isoforms [63]. For that, TEC co-cultures were either treated or not with 8-Br-cAMP (1 mM) or forskolin (10 μM) for 6 hrs and subsequently analyzed by flow cytometry. Two distinct quantitative parameters were calculated comparing the treated versus control double positive TEC: the extent of cell coupling (% double positive cells) and the cell coupling efficiency (based on calcein geometric mean fluorescence). Under these conditions, the extent of coupling was not significantly modified, ranging from 85.0 ± 8.2% (Mean ± SD) at control level to up to 96.7 ± 1.9% and 95.5 ± 2.2% with 8-Br-cAMP and forskolin, respectively, indicating that the TEC monolayer was functionally well coupled under control confluent culture conditions (Figure 2A-D). Nevertheless, treatment with 8-Br-cAMP or forskolin increased transfer efficiency, inducing respectively, 3.21 ± 0.58 and 3.18 ± 0.29 fold increase in geometric mean fluorescence of calcein labeling in double positive cells (Figure 2A-D). The half maximal effective concentration (EC50) for 8-Br-cAMP and forskolin was 98 μM and 0.470 μM, respectively (Figure 2E-H). Importantly, upregulation of inter-TEC GJIC triggered by 8-Br-cAMP was not restricted to the mouse TEC line, since similar results were seen when TNC-derived primary cultures of human TEC (Figure 2I) and IT76 M1 cells (data not shown) were treated with 8-Br-cAMP, as ascertained after lucifer yellow microinjection and blind evaluation of the numbers of coupled cells.


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)

Increase in cAMP enhances inter-TEC GJIC. Panels A and B depict untreated co-cultures of the mouse TEC line at zero and 6 hours time points, respectively. The percentage of double positive cells and the calcein geometric mean fluorescence intensity (MFI) of these populations are depicted at the upper right corner of each panel (%, above; MFI, below). Panels C and D show the inter-TEC coupling, following 6 hours of treatment with 1 mM 8-Br-cAMP or 10 μM forskolin. Both treatments enhanced the calcein mean fluorescence intensity of coupled cells (double positive cells). These data are representative of at least 4 separate experiments. Such enhancements can also be seen in Panels E to H, depicting TEC co-cultures treated for 6 hours with increasing concentrations of either 8-Br-cAMP (E-F) or forskolin (G-H). While percentages of coupled TEC was not significantly modified (E, G), the geometric mean fluorescence of calcein quantified from the double positive cells tripled after both treatments (F, H). The results are representative of 3 independent experiments (mean SD). Panel I shows that 8-Br-cAMP was also capable of enhancing inter-TEC GJIC, in primary cultures of human TNC-derived epithelial cells. Numbers of coupled cells were count in blind. * p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Increase in cAMP enhances inter-TEC GJIC. Panels A and B depict untreated co-cultures of the mouse TEC line at zero and 6 hours time points, respectively. The percentage of double positive cells and the calcein geometric mean fluorescence intensity (MFI) of these populations are depicted at the upper right corner of each panel (%, above; MFI, below). Panels C and D show the inter-TEC coupling, following 6 hours of treatment with 1 mM 8-Br-cAMP or 10 μM forskolin. Both treatments enhanced the calcein mean fluorescence intensity of coupled cells (double positive cells). These data are representative of at least 4 separate experiments. Such enhancements can also be seen in Panels E to H, depicting TEC co-cultures treated for 6 hours with increasing concentrations of either 8-Br-cAMP (E-F) or forskolin (G-H). While percentages of coupled TEC was not significantly modified (E, G), the geometric mean fluorescence of calcein quantified from the double positive cells tripled after both treatments (F, H). The results are representative of 3 independent experiments (mean SD). Panel I shows that 8-Br-cAMP was also capable of enhancing inter-TEC GJIC, in primary cultures of human TNC-derived epithelial cells. Numbers of coupled cells were count in blind. * p < 0.05.
Mentions: To analyze the putative modulatory effects of cAMP on GJIC in TEC cultures, we stimulated the mouse TEC line with 8-Br-cAMP, a membrane permeable cAMP analog; or forskolin, which stimulates cAMP production through activation of adenylate cyclase isoforms [63]. For that, TEC co-cultures were either treated or not with 8-Br-cAMP (1 mM) or forskolin (10 μM) for 6 hrs and subsequently analyzed by flow cytometry. Two distinct quantitative parameters were calculated comparing the treated versus control double positive TEC: the extent of cell coupling (% double positive cells) and the cell coupling efficiency (based on calcein geometric mean fluorescence). Under these conditions, the extent of coupling was not significantly modified, ranging from 85.0 ± 8.2% (Mean ± SD) at control level to up to 96.7 ± 1.9% and 95.5 ± 2.2% with 8-Br-cAMP and forskolin, respectively, indicating that the TEC monolayer was functionally well coupled under control confluent culture conditions (Figure 2A-D). Nevertheless, treatment with 8-Br-cAMP or forskolin increased transfer efficiency, inducing respectively, 3.21 ± 0.58 and 3.18 ± 0.29 fold increase in geometric mean fluorescence of calcein labeling in double positive cells (Figure 2A-D). The half maximal effective concentration (EC50) for 8-Br-cAMP and forskolin was 98 μM and 0.470 μM, respectively (Figure 2E-H). Importantly, upregulation of inter-TEC GJIC triggered by 8-Br-cAMP was not restricted to the mouse TEC line, since similar results were seen when TNC-derived primary cultures of human TEC (Figure 2I) and IT76 M1 cells (data not shown) were treated with 8-Br-cAMP, as ascertained after lucifer yellow microinjection and blind evaluation of the numbers of coupled cells.

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