Characterization of the Signaling Pathways Implicated in the AMPK-Mediated Assembly of Epithelial Tight Junctions

The formation of tight junctions (TJ) between epithelial cells is a complex and still poorly understood process that involves the cooperation of many proteins and the coordination of different signaling pathways. Disruption of the structure and function of TJ is an important feature in a number of pathologies. It is therefore relevant to understand the processes that promote the integration of epithelial TJ. In the first part of this work, we studied in vitro the signaling pathways that participate downstream of the AMP-activated protein kinase (AMPK) in TJ assembly. Our data suggest that AMPK-mediated TJ formation is a dynamic process and involves the participation of many junction proteins, such as atypical protein kinase C (aPKC), playing a central role in this process. Expression of afadin-phosphodefective mutants (S216A – aPKC consensus phosphorylation site) perturbed ZO-1 localization to the plasma membrane during AMPK-induced TJ assembly. Expression of S216A increased the ZO-1/afadin interaction, while S1083A (AMPK consensus phosphorylation site) reduced this interaction during extracellular calcium-induced TJ assembly. Inhibition of aPKC activity also increased the ZO-1/afadin interaction. Taken together, these data suggest that aPKC-mediated phosphorylation of afadin terminates the ZO-1/afadin interaction, and thus permits the later stages of TJ assembly. In the second part of this work, our in vitro studies aim to determine the respective contribution of AMPK upstream kinases in Ca2+-induced AMPK activation at the time of TJ formation. AMPK activity is modulated by two major upstream kinases: LKB1 and CaMKK. Our observations support a role for CaMKK in the Ca2+-induced AMPK activation, independently of LKB1 activity. Hence, the pharmacological inhibition of CaMKK hampered AMPK phosphorylation and ZO-1 relocation during a Ca2+ switch, whereas the inactivation of LKB1 did not significantly influence these processes. Additionally, we have studied the impact of non-epithelial cells, namely mesenchymal stromal cells (MSC), on the AMPK-mediated TJ assembly. MSC may accelerate the AMPK-mediated formation of epithelial TJ by both direct cell-cell contacts and indirect paracrine ways, as suggested by the exposure to MSC-conditioned medium. In conclusion, the present PhD thesis helps to decipher the down- and up-stream mechanisms involved in the AMPK-mediated cascade of TJ assembly in epithelial cells. Furthermore, we postulate the involvement of non-epithelial cells, i.e. MSC, as positive mediators in TJ modulation.