title: Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition.
abstract: Epidermal barrier acquisition during late murine gestation is accompanied by an increase in Akt kinase activity and cJun dephosphorlyation. The latter is directed by the Ppp2r2a regulatory subunit of the Pp2a phosphatase. This was accompanied by a change of Claudin-1 localisation to the cell surface and interaction between Occludin and Claudin-1 which are thought to be required for tight junction formation. The aim of this study was to determine the nature of the barrier defect caused by the loss of AKT/Ppp2r2a function. There was a paracellular barrier defect in rat epidermal keratinocytes expressing a Ppp2r2a siRNA. In Ppp2r2a knockdown cells, Claudin-1 was located to the cytoplasm and its expression was increased. Inhibiting cJun phosphorylation restored barrier function and plasma membrane localisation of Claudin-1. Expression of the Rab3 GTPase activating protein, Rab3Gap1, was restored in Ppp2r2a siRNA cells when cJun phosphorylation was inhibited. During normal mouse epidermal development, Claudin-1 plasma membrane localisation and Rab3Gap1 cell surface expression were co-incident with Akt activation in mouse epidermis, strongly suggesting a role of Rab3Gap1 in epidermal barrier acquisition. Supporting this hypothesis, siRNA knockdown of Rab3Gap1 prevented plasma membrane Claudin-1 expression and the formation of a barrier competent epithelium. Replacing Rab3Gap1 in Ppp2r2a knockdown cells was sufficient to rescue Claudin-1 transport to the cell surface. Therefore these data suggest Rab3Gap1 mediated exocytosis of Claudin-1 is an important component of epidermal barrier acquisition during epidermal development.
authors.affiliation: Livingstone Skin Research Centre for Children, UCL Institute of Child Health, London WC1N 1EH, UK.
authors.forename: G
authors.initials: G
authors.lastname: Youssef
authors.forename: L
authors.initials: L
authors.lastname: Gerner
authors.forename: A S
authors.initials: AS
authors.lastname: Naeem
authors.forename: O
authors.initials: O
authors.lastname: Ralph
authors.forename: M
authors.initials: M
authors.lastname: Ono
authors.forename: C A
authors.initials: CA
authors.lastname: O'Neill
authors.forename: R F L
authors.initials: RF
authors.lastname: O'Shaughnessy
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002478:Cells, Cultured; D062445:Claudin-1 / Q000032:analysis / Q000378:metabolism*; D004817:Epidermis / Q000378:metabolism*; D005089:Exocytosis; D051379:Mice; D062793:Occludin / Q000032:analysis; D054648:Protein Phosphatase 2 / Q000502:physiology; D051381:Rats; D019108:Tight Junctions / Q000502:physiology*; D020692:rab3 GTP-Binding Proteins / Q000502:physiology*
chemical_list: D000873:Anthracenes; D062445:Claudin-1; C568114:Cldn1 protein, rat; D062793:Occludin; C568987:Ocln protein, rat; C432165:pyrazolanthrone; D054648:Protein Phosphatase 2; D020692:rab3 GTP-Binding Proteins
keywords: Epidermal barrier; Epidermis; Keratinocyte; Rab3Gap1; Signalling