Endothelial junction proteins that regulate movement across the vascular system, are modulated through phosphorylation.1 However, the role of protein phosphatase 2A (PP2A) mediated dephosphorylation in modulating permeability of the blood-brain barrier remains unclear. This study investigates the role of PP2A inhibition on VE-cadherin and PECAM-1 abundance, and whether it affects brain microvascular permeability.
Human brain microvascular endothelial cells (hBMECs) were exposed to okadaic acid (OA, 10 nM), or dimethylsulphoxide (DMSO; 0.01% v/v) for 24 hour. Protein expression was determined using immunoblotting. PP2Ac activity was measured by an immunoprecipitation assay (Millipore). Proteasomal degradation was investigated using MG-132 (2 µM). hCMEC/D3 cells were transfected with CIP2A and SET plasmids (pcDNA3.1) using polyfect. Transendothelial permeability was determined using FITC-dextran. Data are presented as mean ±S.E.M. (n=5) and analysed by one-way ANOVA with post hoc (p<0.05).
OA (PP2A inhibitor) reduced abundance of VE-cadherin to undetectable levels and decreased PECAM-1 abundance by 50% (p<0.05). OA decrease PP2A activity (58.9%±5.5%, p<0.05) without effecting protein abundance. OA increased demethylation of PP2Ac and reduced abundance of leucine carboxyl methyltransferase-1 (LCMT-1) (p<0.05); protein phosphatase methylesterase-1 (PME-1) abundance was not altered. Overexpression of the PP2A inhibitors CIP2A and SET decreased (p<0.05) VE-cadherin and PECAM-1 abundance compared to the pcDNA3.1 control. OA and overexpression of CIP2A and SET increased (p<0.05) transendothelial permeability.
In conclusion, inhibiting PP2A decreases VE-cadherin and PECAM-1 abundance due to proteasomal degradation. This loss is associated with increased microvascular permeability consistent with loosening of tight junctions. The inhibition of PP2A is not due to the loss of abundance but instead an increase in PP2A methylation by LCMT-1, preventing the assembly of the holoenzyme. As such, PP2A-mediated regulation of the blood brain barrier, might be a target of therapeutic value.
. Bertocchi C, et al. J. Signal Transduct2012;12:ID125295.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.