title: 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)-(+)-3-chloro-1,2-propanediol] induces a transitory glial fibrillary acidic protein-astrocyte 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 μM) 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 μM] and PI-828 [2-(4-morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one; 0.1-10 μM] 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.
authors.affiliation: Westbrook College of Health Professions (R.B.C., E.E.L.), Department of Biomedical Sciences, College of Osteopathic Medicine, and Center for Excellence in the Neurosciences (H.J.B., C.L.W.), and College of Arts and Sciences (S.A.B., A.I.M.), University of New England, Biddeford, Maine.
authors.forename: Ryan B
authors.initials: RB
authors.lastname: Camire
authors.affiliation: Westbrook College of Health Professions (R.B.C., E.E.L.), Department of Biomedical Sciences, College of Osteopathic Medicine, and Center for Excellence in the Neurosciences (H.J.B., C.L.W.), and College of Arts and Sciences (S.A.B., A.I.M.), University of New England, Biddeford, Maine.
authors.forename: Holly J
authors.initials: HJ
authors.lastname: Beaulac
authors.affiliation: Westbrook College of Health Professions (R.B.C., E.E.L.), Department of Biomedical Sciences, College of Osteopathic Medicine, and Center for Excellence in the Neurosciences (H.J.B., C.L.W.), and College of Arts and Sciences (S.A.B., A.I.M.), University of New England, Biddeford, Maine.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Brule
authors.affiliation: Westbrook College of Health Professions (R.B.C., E.E.L.), Department of Biomedical Sciences, College of Osteopathic Medicine, and Center for Excellence in the Neurosciences (H.J.B., C.L.W.), and College of Arts and Sciences (S.A.B., A.I.M.), University of New England, Biddeford, Maine.
authors.forename: Annie I
authors.initials: AI
authors.lastname: McGregor
authors.affiliation: Westbrook College of Health Professions (R.B.C., E.E.L.), Department of Biomedical Sciences, College of Osteopathic Medicine, and Center for Excellence in the Neurosciences (H.J.B., C.L.W.), and College of Arts and Sciences (S.A.B., A.I.M.), University of New England, Biddeford, Maine.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Lauria
authors.affiliation: Westbrook College of Health Professions (R.B.C., E.E.L.), Department of Biomedical Sciences, College of Osteopathic Medicine, and Center for Excellence in the Neurosciences (H.J.B., C.L.W.), and College of Arts and Sciences (S.A.B., A.I.M.), University of New England, Biddeford, Maine clwillis@outlook.com.
authors.forename: Colin L
authors.initials: CL
authors.lastname: Willis
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002461:Cell Line, Transformed; D002867:Chromones / Q000494:pharmacology; D062507:Claudin-5 / Q000096:biosynthesis*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D051379:Mice; D008807:Mice, Inbred BALB C; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
chemical_list: D002867:Chromones; D062507:Claudin-5; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; C491511:Cldn5 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one