Non-muscle Mlck is required for β-catenin- and FoxO1-dependent downregulation of Cldn5 in IL-1β-mediated barrier dysfunction in brain endothelial cells.

Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1 beta directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors b-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1 beta has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1 beta-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by b-catenin and FoxO1. We found that treating BMVECs with IL-1 beta induced barrier dysfunction concomitantly with the nuclear translocation of b-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by b-catenin and FoxO1 in IL-1 beta-mediated barrier dysfunction was dependent on nmMlck.