Role of VASP in reestablishment of epithelial tight junction assembly after Ca switch

Lawrence, Donald W., Katrina M. Comerford, and Sean P. Colgan. Role of VASP in reestablishment of epithelial tight junction assembly after Ca2 switch. Am J Physiol Cell Physiol 282: C1235–C1245, 2002. First published January 9, 2002; 10.1152/ajpcell.00288.2001.—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 phosphoVASP appears at the junction after Ca2 switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actinbinding 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.