title: Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch.
abstract: Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.
authors: Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors: Donald W
authors: DW
authors: Lawrence
authors: Katrina M
authors: KM
authors: Comerford
authors: Sean P
authors: SP
authors: Colgan
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019108:Tight Junctions / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D062826:Zonula Occludens-1 Protein; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D015815:Cell Adhesion Molecules; D002614:Chelating Agents; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D000098662:Vasodilator-Stimulated Phosphoprotein; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium