Phosphatase 2A is involved in endothelial cell microtubule remodeling and barrier regulation

We have recently shown that rnicrotubule (MT) inhibitor, nocodazole (2-5 muM) significantly increases endothelial cells (EC) actomyosin contraction and permeability indicating the importance of MT in maintaining the EC barrier (Verin et al. [2001]: Cell Mol Physiol 281:L565-L574). Okadaic acid (OA, 2-5 nM), a powerful inhibitor of protein phosphatase 2A (PP2A), significantly potentiates the effect of submaximal concentrations of nocodazole (50-200 nM) on transendothelial electrical resistance (TER) suggesting the involvement of PP2A activity in the MT-mediated EC barrier regulation. Immunofluorescent staining of EC revealed that in control cells PP2A distributes in a pattern similar to MT. Consistent with these results, we demonstrated that significant amounts of PP2A were present in MT-enriched EC fractions indicating tight association of PP2A with MT in endothelium. Treatment of EC with OA leads to disappearance of MT-like PP2A staining suggesting dissociation of PP2A from the MT network. Next, we examined the effect of PP2A inhibition on phosphorylation status of MT-associated protein tau, which in its unphosphorylated form promotes MT assembly. OA caused significant increases in tau phosphorylation confirming that tau is a substrate for PP2A in endothelium. Immunofluorescent experiments demonstrated that the OA-induced increases in tau phosphorylation strongly correlated with translocation of phospho-tau to cell periphery and disassembly of peripheral MT. These results suggest the involvement of PP2A-mediated tau dephosphorylation in alteration of EC MT structure and highlight the potential importance of PP2A in the regulation of EC the MT cytoskeleton and barrier function.(C) 2004 Wiley-Liss, Inc.