In the present study we demonstrated, by various experimental approaches, that cAMP and PKC are involved in the modulation of inter-TEC GJIC: the cAMP agonist 8-Br-cAMP enhanced inter-TEC coupling whereas PMA-induced PKC activation triggered an opposite effect.
Using flow cytometry we first detected in a mouse TEC line, that up to 90% of DiIc18
cells co-cultured in 1:1 ratio with calcein+ cells became double positive, a phenomenon which was readily inhibited by the gap junction inhibitors 18-β-glycyrrhetinic acid and carbenoxolone. This result clearly demonstrates that the thymic epithelium spontaneously forms a GJIC-dependent functional syncytium in vitro
. Under these conditions, 8-Br-cAMP and forskolin did not significantly modify this spontaneous percentage of coupled cells. However, both compounds enhanced up to 3 fold the calcein fluorescence intensity of the double positive cells, indicating an increase in the rate of dye transfer among coupled cells. Importantly, 8-Br-cAMP also induced an increment in GJIC in TNC-derived primary cultures of human TEC. These data clearly show that elevation of cAMP upregulates inter-TEC GJIC, similar to what has been reported for effects on cell types in other systems [51
Experiments performed with the mouse TEC line revealed that 8-Br-cAMP and forskolin treatments induced an accumulation of Cx43 protein at cell-to-cell contact regions, seen as punctate clusters. Furthermore, 8-Br-cAMP induced an increase in Cx43 gene transcription, suggesting that the protein accumulation may be a consequence of altered Cx43 protein translation. Such an alteration in connexin synthesis and accumulation in the cell membrane is in accordance with previous description of modulation of gap junctions by cAMP elevating agonists after long-term evaluations (2-24 hrs, and 7 days) [51
]. However, short-term modulation also has been demonstrated, with description of changes in gap junctional conductance within minutes of treatment with 8-Br-cAMP [75
]; in some cell types, changes in dye coupling were also detected within 2-10 minutes after this treatment [55
]. In cases of both short- and long-term evaluations alteration in the degree of connexin phosphorylation has also been demonstrated [73
]. Interestingly, some of these reports have demonstrated that connexin isoforms other than Cx43 also may be regulated by cAMP analogs, such as Cx26, Cx40 and possibly Cx32 [49
]. Thus, we cannot exclude the possibility that other connexin isoforms, not yet characterized in TEC, might also be regulated by 8-Br-cAMP and forskolin in IT-76M1 cells.
We also investigated whether physiological stimuli, could signal through cyclic nucleotides, might be involved in modulation of inter-TEC GJIC. When we evaluated VIP and adenosine, we did not observe any change in the degree of cell coupling in the mouse TEC preparation in any concentration of each molecule used. Similar results were seen despite the various concentrations applied for each molecule.
We should mention however, that our finding on VIP is at variance with the data reported by Head and co-workers [44
]. These authors reported an inhibition of GJIC in a rat TEC. Although by now we cannot explain such a difference, it may be related to cell line variations or presumably due to receptor signaling dynamics that cannot be merely mimicked by a single agonist [78
], or may be related to differences in methodological approaches given that the authors treated the cells before GJIC formation.
In any case, adenosine, which may also signal through cyclic nucleotides depending on its concentration and the activated P1 receptor [67
], did not modify the basal level of inter-TEC GJIC as well. These results demonstrate that VIP and adenosine possibly are not physiological modulators of GJIC among TECs. A recent review indicates that adrenoreceptor agonists mediate thymus homeostasis and local T cell development [79
]. In addition, reports have suggested a role for catecholamines on thymic epithelial cells controlling proliferation and cytokine secretion [68
]. Our data demonstrate that epinephrine, and activation of cAMP, lead to an increasing dye coupling in TEC cells suggesting that this mediator could endogenously control communication between thymic epithelial cells and contribute to thymus physiology. We are currently investigating other endogenous molecules which also signal through cAMP, in order to evaluate its potential to modulate inter-TEC GJIC.
In a second set of experiments, we demonstrated that PKC activation (induced herein by the phorbol ester, PMA) significantly inhibits the dye coupling in both mouse and human TEC in vitro models.
The fact that the same inhibitory effect was seen in a mouse TEC line as well as in primary cultures of human TEC deserve further discussion. The study by Chanson et al. [56
] demonstrated that GJIC inhibition was induced by phorbol esters in a liver-derived cell line but not in differentiated primarily cultures of pancreatic exocrine cells. Similarly, the inhibitory effect of phorbol esters on GJIC was observed in primary cultured developing lens cells, which express Cx43 and Cx49, but not in lentoid cells (differentiated lens cells), which express Cx46 and Cx49, demonstrating that the effects of these compounds might be dependent on the cellular differentiation stage and its pattern of Cx expression [81
By contrast, the presence of thymocytes apparently is not involved in the control of inter-TEC GJIC, at least in the co-culture experimental conditions that we used. Nevertheless, further studies are still necessary in order to completely discard a role for thymocytes in the control of inter-TEC communication mediated by gap junctions.