Phosphorylation of Claudin-5 and Occludin by Rho Kinase in Brain Endothelial Cells

Abstract

Critical to the proper maintenance of blood-brainbarrier (BBB) integrity are the endothelial tight junctions (TJs). Posttranslational modifications of essential endothelial TJ proteins, occludin and claudin-5, contribute and possibly disrupt BBB integrity. Our previous work has shown that Rho kinase (RhoK) activation mediates occludin and claudin-5 phosphorylation resulting in diminished barrier tightness and enhanced monocyte migration across BBB in the setting of human immunodeficiency virus-1 encephalitis (HIVE). To determine whether RhoK can directly phosphorylate TJ proteins, we examined phosphorylation of cytoplasmic domains of recombinant claudin-5 and occludin by RhoK. We found that RhoK predominately phosphorylated two sites on occludin (T382 and S507) and one site on claudin-5 (T207). The blood-brain-barrier (BBB) is composed of specialized nonfenestrated brain microvascular endothelial cells (BMVECs) connected by tight junctions (TJs) in an impermeable monolayer devoid of transcellular pores.1 TJs are composed of claudins and occludin (OCC, integral membrane proteins) and intracellular proteins, zonula occludens (ZO-1 to ZO-3).2 OCC (65-kDa protein) is highly expressed in BMVECs, and it is consistently found along the cell borders of brain endothelium.3,4 OCC is composed of four transmembranous domains with the carboxyl and amino terminals oriented to the cytoplasm and two extracellular loops (44 amino acids and 45 amino acids) spanning the intercellular cleft.5 OCC content is much lower in endothelial cells outside of the central nervous system 6,7 suggesting its active role in BBB function. The phosphorylation state of OCC regulates its association with the cell membrane and barrier permeability, and multiple phosphorylation sites have been identified on OCC serine and threonine residues.8 -10 The cytoplasmic C-terminal domain provides the connection of OCC with the cytoskeleton via accessory proteins, ZO-1 and ZO-2. 11Up to 24 claudins (20-to 24-kDa proteins) sharing the high sequence homology in the first and fourth transmembranous domains and extracellular loops have been identified in mammals.12 Contiguous staining for claudins is found along endothelial cell borders in and outside the central nervous system. BMVECs express predominantly claudin-3 and -5 (CLD5).3,13 The homophilic and heterophilic interactions between the extracellular loops of clauSupported in part by the National Institutes of Health (research grants P01 NS043985, R01 AA015913, and R01 MH65151 to Y.P.; R01 MH072539 to T.I.; and National Center for Research Resources P20RR15635 to T.I. and R.L.C.).M.Y. and S.H.R. contributed equally in this study.

Dynamic interactions ofPlasmodiumspp. with vascular endothelium

“…In general, endothelial barrier function is maintained by preserving transmembrane AJ and TJ proteins VE-cadherin, claudin-5 and occludin in an unphosphorylated state. 2,3 AJ serve as the primary regulators of endothelial barrier function in most organs, with the notable exception of the blood brain barrier (BBB) where the thin microvascular endothelial cells are bound together to a large extent by TJ, creating a high resistance paracellular barrier. Permeability results when junctional proteins are relocated or their expression downregulated, as occurs in many inflammatory conditions.…”

Molecular and Genetic Basis of Cerebral Cavernous Malformations

“…Bu yolağın aktivasyonu, beyindeki damar endotelinde yer alan okludin ve klaudin-5'i fosforilleyerek yapılarında konformasyonel bir değişime sebep olmakta ve görevlerini yapamaz hale getirmektedir (14). Okludin ve klaudin-5'in hücreler arası sıkı bağlantı komplekslerinden olduğu dikkate alınınca endotelyal hücreler arasındaki bu bağlantıların bozulması lezyona kan akımının girişini kolaylaştırmaktadır (15). KRIT1 geninin kaybı, SKM lezyonlarının ortaya çıkışında oldukça önemli olup; hücresel morfolojinin belirlenmesinde dinamik bir yapı sergilemektedir (16).…”

Poly(ADP-ribose) Polymerase-1 Inhibition in Brain Endothelium Protects the Blood—Brain Barrier under Physiologic and Neuroinflammatory Conditions

“…[24][25][26] The microvascular endothelium regulates selective permeability of the BBB to fluids and solutes. Since small GTPases, RhoA and Rac1, control cytoskeleton, TJ and adhesion molecule expression in BMVEC and endothelial cells of other organs, 25,27,28 we examined the ability of PARP inhibitors to decrease their activation. Using a G-LISA assay that measures active, GTP-bound RhoA and Rac1, we demonstrated significant increases in both RhoA and Rac1 activation, 1.8-fold in RhoA-GTP ( Figure 5A) and 1.6-fold in Rac1-GTP ( Figure 5B) in BMVEC stimulated with TNFα; activity of both GTPases was diminished 25-35% by PARP suppression.…”