Abstract 17429: Phosphorylation of Myosin Light Chain Mediates Dissociation of Adherens Junctions of Endothelial Cells Following Monocyte Attachment
The molecular mechanisms underlying cytoskeletal alterations in endothelial cells (ECs) during transendothelial migration (TEM) of monocytes are not determined. Tyrosine (Tyr) phosphorylation of VE-cadherin (VE-cad) has been implicated in the dissociation of adherens junctions and increased paracellular permeability of ECs. We hypothesized that phosphorylation of EC myosin light chain (MLC) is crucial for Tyr phosphorylation of VE-cad and TEM of monocytes. Our studies on ECs indicated that inhibition or induction of MLC phosphorylation inhibits or enhances TEM of monocytes, respectively. Attachment of monocytes to human umbilical vein or aortic ECs leads to activation of HRasRafMekERK, increase in phosphorylation of MLC, Tyr phosphorylation of VE-cad, and formation of gaps in EC monolayers. Through a combination of techniques involved expression of dominant negative and active mutants and siRNA we determined signal transduction pathways in ECs regulating Tyr phosphorylation VE-cad after attachment of monocyte. We found that monocyte-induced VE-cad Tyr phosphorylation in ECs is due to activation of Src and proline-rich tyrosine kinase 2 (Pyk2). Activation of HRasRafMekERK pathway or MLC phosphorylation also leads to VE-cad, Src and Pyk2 Tyr phosphorylation. Phosphorylation of MLC after attachment of monocyte is mediated by ERK phosphorylation via activation of MLC kinase and inhibition of MLC phosphatase. Interaction of integrin α4β1 with VCAM-1 triggers signal transduction that leads to VE-cad Tyr phosphorylation, as function blocking antibodies to either molecule prevented monocyte-induced VE-cad Tyr phosphorylation. In fact, artificially cross-linking VCAM-1 on ECs lead to Tyr phosphorylation of VE-cad, Src, Pyk2, and phosphorylation of ERK and MLC. Immuno-precipitation studies indicated that phosphorylation of MLC increases VE-cad Tyr phosphorylation by recruiting Src to VE-cad. We found that atorvastatin inhibits VE-cad Tyr phosphorylation by inhibiting phosphorylation of MLC and ERK in ECs, suggesting a novel pleiotropic effect for statins. Our studies indicate that after attachment of monocytes to ECs, phosphorylation of ERK and MLC leads to an increase in VE-cad Tyr phosphorylation that enhances TEM of monocytes.
- © 2010 by American Heart Association, Inc.