Blood-brain barrier (BBB) integrity is compromised in many CNS disorders. Complex astrocyte and vascular endothelial cell interactions that regulate BBB integrity may be disturbed in these disorders. We have previously shown that systemic administration of 3-chloropropanediol induces a transitory glial fibrillary acidic protein (GFAP)-astrocyte loss, reversible loss of tight junction complexes, and BBB integrity disruption. However, the intracellular signaling mechanisms that induce BBB integrity marker loss are unclear. We hypothesize that 3-chloropropanediol-induced modulation of tight junction protein expression is mediated through the phosphoinositide-3-kinase (PI3K)/AKT pathway. To test this hypothesis we have used a mouse brain endothelial cell line (bEnd.3) exposed to 3-chloropropanediol for up to 3 days. Results showed early reversible loss of sharp paracellular claudin-5 expression 90, 105, and 120 min following 3-chloropropanediol (500 μM) treatment. Sharp paracellular claudin-5 profiles were later restored, but lost again by 2 and 3 days post 3-chloropropanediol treatment. Western blot and immunofluorescence studies showed increased p85-PI3K expression and transitory increased AKT (Thr308) phosphorylation at 15 and 30 min after 3-chloropropanediol administration. PI3K inhibitors LY294002 (2.5-25 μM) and PI-828 (0.1-10 μM) prevented the 3-chloropropanediol-induced AKT (Thr308) phosphorylation and both early and late loss of paracellular claudin-5. However, AKT inhibitors only prevented the early changes in claudin-5 expression. This mechanistic study provides a greater understanding of the intracellular signaling pathways mediating tight junction protein expression and supports a hypothesis that two independent pathways triggered by PI3K mediate early and late loss of paracellular claudin-5 expression.
- The American Society for Pharmacology and Experimental Therapeutics