Abstract
Previously, we reported that post-translational modifications (PTMs) of MAGI1, including S741 phosphorylation and K931 de-SUMOylation, both of which are regulated by p90RSK activation, lead to endothelial cell (EC) activation. However, roles for p90RSK and MAGI1-PTMs in regulating EC permeability remain unclear despite MAGI1 being a junctional molecule. Here, we show that thrombin (Thb)-induced EC permeability, detected by the electric cell-substrate impedance sensing (ECIS) based system, was decreased by overexpression of dominant negative p90RSK or a MAGI1-S741A phosphorylation mutant, but was accelerated by overexpression of p90RSK, siRNA-mediated knockdown of magi1, or the MAGI1-K931R SUMOylation mutant. MAGI1 depletion also increased the mRNA and protein expression of the large tumor suppressor kinases 1 and 2 (LATS1/2), which inhibited YAP/TAZ activity and increased EC permeability. Because the endothelial barrier is a critical mediator of tumor hypoxia, we also evaluated the role of p90RSK activation in tumor vessel leakiness by using a relatively low dose of the p90RSK specific inhibitor, FMK-MEA. FMK-MEA significantly inhibited tumor vessel leakiness at a dose that does not affect morphology and growth of tumor vessels in vivo. These results provide novel insights into crucial roles for p90RSK-mediated MAGI1 PTMs and the Hippo pathway in EC permeability, as well as p90RSK activation in tumor vessel leakiness.
Highlights
Endothelial cell-cell junctions are highly dynamic structures that regulate endothelial cell (EC) monolayer integrity and barrier function
We found that p90 ribosomal S6 kinase (p90RSK) overexpression significantly increases Thb-induced EC permeability as seen by the decreased trans-endothelial electrical resistance (TEER) values in comparison to the control (Figures 1A–C)
Since we have shown that the depletion of large tumor suppressor kinases 1 and 2 (LATS1/2) inhibited EC permeability via activating Yes-associated protein (YAP) (Figure 6), this data suggests that the up-regulation of LATS1/2-mediated inhibition of YAP (Figure 6F) may contribute to the part of the depletion of MAGI1-mediated acceleration of EC permeability, further experimentation is necessary to be certain of this relationship
Summary
Endothelial cell-cell junctions are highly dynamic structures that regulate EC monolayer integrity and barrier function. A member of the Ras-like small GTPase family, has been recognized as a key regulator of cell-cell p90RSK-MAGI1 Module and Endothelial Permeability junctional formation at different levels through cadherins [7, 8]. In ECs, the formation of cell-cell contacts induced by Rap activation is hampered by the depletion of Membrane Associated Guanylate Kinase, WW and PDZ domain-containing protein 1 (MAGI1) [13]. These observations suggest that MAGI1 is involved in the regulation of EC permeability
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