Role of Protein Phosphatase 2A inhibition in modulation of VE-cadherin and Claudin-5 in human brain microvascular endothelial cells and the potential reversal by small molecule activators of PP2A (SMAPs)

Abstract

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase which plays a key role in modulating signalling, metabolism and cell growth, and is implicated in various pathologies. There is conflicting evidence regarding the role of PP2A in regulating blood brain barrier function. This study investigates if okadaic acid and inflammation modulate VEcadherin and claudin-5 through PP2A, and whether this can be reversed by novel small molecule activators of PP2A as a possible therapeutic intervention. Human brain microvascular endothelial cells (hCMEC/d3) were exposed to IFNg/TNFa (10ng/mL each) or okadaic acid (OA, 10 nM; PP2A inhibitor) for 24 h. mRNA and protein abundance were determined using qPCR and western blotting, respectively. Data were analysed using one-way ANOVA (P < 0.05) with post hoc analysis. IFNg/TNFa decreased claudin-5 and VEcadherin mRNA expression by 68 ± 6 % and 54 ± 8 % respectively, and VEcadherin protein by 83 ± 6 %. Interestingly, OA decreased VE-cadherin mRNA and protein expression by 80 ± 5 % and 80 ± 3 %, respectively, but had no effect on claudin-5. PP2A activators FTY-720 (1 µM) and DBK-1154 (1 µM) did not alter the responses to OA or IFNg/TNFa. Additionally, IFNg/TNFa decreased demethylated PP2Ac and potentially increased phosphorylated PP2Ac. Furthermore, transfection experiments were carried out in order to overexpress PP2Ac, SET or CIP2A in hCMEC/d3 cells, however the protocol needs to be further optimized. In conclusion, this study has shown IFNg/TNFa and OA to differentially modulate claudin-5 mRNA expression but to have similar effects on VEcadherin. This indicates a role for PP2A in transcriptional regulation of VEcadherin but not claudin-5. Importantly, the VE-cadherin response was insensitive to DBK-1154. The role of PP2A in modulating junctional proteins requires further investigation.