Biphasic Modulation of Paracellular Claudin-5 Expression in Mouse Brain Endothelial Cells Is Mediated through the Phosphoinositide-3-Kinase/AKT Pathway

Abstract

Blood-brain barrier (BBB) integrity is compromised in many central nervous system disorders. Complex astrocyte and vascular endothelial cell interactions that regulate BBB integrity may be disturbed in these disorders. We previously showed that systemic administration of 3-chloropropanediol [(S)-(1)-3-chloro-1,2-propanediol] induces a transitory glial fibrillary acidic proteinastrocyte loss, reversible loss of tight junction complexes, and BBB integrity disruption. However, the intracellular signaling mechanisms that induce BBB integrity marker loss are unclear. We hypothesize that 3-chloropropanediol-induced modulation of tight junction protein expression is mediated through the phosphoinositide-3-kinase (PI3K)/AKT pathway. To test this hypothesis, we used a mouse brain endothelial cell line (bEnd.3) exposed to 3-chloropropanediol for up to 3 days. Results showed early reversible loss of sharp paracellular claudin-5 expression 90, 105, and 120 minutes after 3-chloropropanediol (500 mM) treatment. Sharp paracellular claudin-5 profiles were later restored, but lost again by 2 and 3 days after 3-chloropropanediol treatment. Western blot and immunofluorescence studies showed increased p85-PI3K expression and transitory increased AKT (Thr308) phosphorylation at 15 and 30 minutes after 3-chloropropanediol administration. PI3K inhibitors LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride; 2.5-25 mM] and PI-828 [2-(4-morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one; 0.1-10 mM] prevented the 3-chloropropanediol-induced AKT (Thr308) phosphorylation and both early and late loss of paracellular claudin-5. However, AKT inhibitors only prevented the early changes in claudin-5 expression. This mechanistic study provides a greater understanding of the intracellular signaling pathways mediating tight junction protein expression and supports a hypothesis that two independent pathways triggered by PI3K mediate early and late loss of paracellular claudin-5 expression.

Picroside II protects the blood-brain barrier by inhibiting the oxidative signaling pathway in cerebral ischemia-reperfusion injury

“…At the same time, MMP-2 can also reduce the expression of claudin-5, which is the constitutive trans-membrane protein of the BBB. All of the above processes harm the structure of the BBB and lead to damage of the BBB[39]. Our experiments confirmed that the expression levels of ROCK, MLCK, and MMP2 were significantly enhanced, while the expression of claudin-5 was significantly decreased after CI/R in rats.…”

Transitory loss of glia and the subsequent modulation in inflammatory cytokines/chemokines regulate paracellular claudin-5 expression in endothelial cells

“…In an earlier study, we have shown that the PI3K/AKT pathway plays a key role in modulating paracellular claudin-5 expression (Camire et al, 2014). To determine whether the PI3K/AKT pathway plays a role in cytokine/chemokine-induced loss of paracellular claudin-5 expression, we inhibited PI3K with the cell permeable inhibitor, LY294002.…”

“…This signaling pathway is activated by a number of receptors, including integrin, cytokine, G protein–coupled receptors, and intracellular signalling molecules (Gonzalez-Mariscal et al, 2008; Hemmings and Restuccia, 2012). Recently we have shown that 3-chloropropanediol (500 μM) alone induced loss of paracellular claudin-5 expression in bEnd.3 cells through the PI3K/AKT pathway (Camire et al, 2014). Pre-treating bEnd.3 cells with the selective cell-permeable PI3K inhibitor, LY294002 (25 μM), prevented TNF-α- and IL-6-induced loss of paracellular claudin-5 (Fig 6), suggesting their actions were mediated through the PI3K pathway (Fig.…”

Junctional proteins of the blood-brain barrier: New insights into function and dysfunction

“…nPKC-u, cPKC-a cPKC-b, PI3Kg, p38MAPK, MLCK and ROCK) is associated with altered claudin-5 localization and expression and ultimately TJ complex disassembly and Tyr kinase activation correlates with claudin-5 disassembly from the TJ complex. [101][102][103]106,107 Among the 23 predicted phosphorylation sites, 6 kinase-specific phosphorylation sites Thr-189, Tyr-200, Thr-207, Thr-209, Tyr-217 and Tyr-218 were observed to be present in the cytoplasmic tail region of human claudin-5. However, only one site, Thr-207, has been experimentally verified so far (RhoK and PKA) and it affects TJ integrity in murine brain endothelial cells and increases permeability.…”