title: Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability.
abstract: We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses that ERM proteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylation-deficient ERM mutants, exhibit less attenuation of 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubule-disrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.
authors.affiliation: Vascular Biology Center, Georgia Health Sciences University, Augusta, 30912, USA. nbogatcheva@georgiahealth.edu
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Bogatcheva
authors.forename: Marina A
authors.initials: MA
authors.lastname: Zemskova
authors.forename: Boris A
authors.initials: BA
authors.lastname: Gorshkov
authors.forename: Kyung Mi
authors.initials: KM
authors.lastname: Kim
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Daglis
authors.forename: Christophe
authors.initials: C
authors.lastname: Poirier
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
mesh_terms: D000077584:2-Methoxyestradiol; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology; D050257:Tubulin Modulators / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism; D000099149:Ezrin
chemical_list: D000077584:2-Methoxyestradiol; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D004958:Estradiol; D007211:Indoles; D008301:Maleimides; D008565:Membrane Proteins; D008840:Microfilament Proteins; D017239:Paclitaxel; D011493:Protein Kinase C; D011758:Pyrroles; D050257:Tubulin Modulators; D048051:p38 Mitogen-Activated Protein Kinases; D054460:rho-Associated Kinases; D000099149:Ezrin; C071058:moesin; C071776:radixin; C085989:Ro 32-0432; C064755:Ro 31-7549