Related Experiment Videos
Protein tyrosine phosphatase activity regulates endothelial cell-cell interactions, the paracellular pathway, and
Bradford A Young1, Xiufen Sui, Timothy D Kiser
1Division of Infectious Diseases, Department of Veterans Affairs Medical Center, Baltimore 21201, USA.
Abstract:
Protein tyrosine phosphorylation is tightly regulated through the actions of both protein tyrosine kinases and protein tyrosine phosphatases. In this study, we demonstrate that protein tyrosine phosphatase inhibition promotes tyrosine phosphorylation of endothelial cell-cell adherens junction proteins, opens an endothelial paracellular pathway, and increases both transendothelial albumin flux and neutrophil migration. Tyrosine phosphatase inhibition with sodium orthovanadate or phenylarsine oxide induced dose- and time-dependent increases in [14C]bovine serum albumin flux across postconfluent bovine pulmonary artery endothelial cell monolayers. These increases in albumin flux were coincident with actin reorganization and intercellular gap formation in both postconfluent monolayers and preformed endothelial cell capillary tubes. Vanadate (25 microM) increased tyrosine phosphorylation of endothelial cell proteins 12-fold within 1 h. Tyrosine phosphorylated proteins were immunolocalized to the intercellular boundaries, and several were identified as the endothelial cell-cell adherens junction proteins, vascular-endothelial cadherin, and beta-, gamma-, and p120-catenin as well as platelet endothelial cell adhesion molecule-1. Of note, these tyrosine phosphorylation events were not associated with disassembly of the adherens junction complex or its uncoupling from the actin cytoskeleton. The dose and time requirements for vanadate-induced increases in phosphorylation were comparable with those defined for increments in transendothelial [14C]albumin flux and neutrophil migration, and pretreatment with the tyrosine kinase inhibitor herbimycin A protected against these effects. These data suggest that protein tyrosine phosphatases and their substrates, which localize to the endothelial cell-cell boundaries, regulate adherens junctional integrity, the movement of macromolecules and cells through the endothelial paracellular pathway, and capillary tube stability.
Insights
Inhibiting protein tyrosine phosphatases increases endothelial cell tyrosine phosphorylation, opening cell junctions and enhancing albumin and neutrophil passage. This highlights phosphatases
Area of Science:
- Cell Biology
- Biochemistry
- Physiology
Background:
- Protein tyrosine phosphorylation is crucial for cellular regulation.
- Protein tyrosine kinases and phosphatases are key regulators of this process.
- Endothelial cell junctions control paracellular transport and cell migration.
Purpose of the Study:
- To investigate the role of protein tyrosine phosphatases in regulating endothelial cell-adherens junction integrity.
- To determine the effect of phosphatase inhibition on endothelial barrier function and cell migration.
- To identify specific proteins affected by tyrosine phosphatase inhibition at endothelial cell boundaries.
Main Methods:
- Inhibition of protein tyrosine phosphatases using sodium orthovanadate and phenylarsine oxide.
- Measurement of transendothelial albumin flux across endothelial cell monolayers.
- Immunolocalization and identification of tyrosine phosphorylated proteins at intercellular boundaries.
- Assessment of actin reorganization and neutrophil migration.
Main Results:
- Phosphatase inhibition significantly increased tyrosine phosphorylation of endothelial cell proteins, including adherens junction components like VE-cadherin and catenins.
- This inhibition led to increased albumin flux and intercellular gap formation.
- Tyrosine phosphorylation events did not disrupt adherens junction complex integrity or actin cytoskeleton coupling.
- Pretreatment with a tyrosine kinase inhibitor blocked the effects of phosphatase inhibition.
Conclusions:
- Protein tyrosine phosphatases regulate endothelial adherens junction integrity and barrier function.
- Phosphatase activity influences the paracellular passage of macromolecules and cells.
- Targeting protein tyrosine phosphatases may modulate endothelial permeability and stability.