Receptor Protein Tyrosine Phosphatase μ Regulates the Paracellular Pathway in Human Lung Microvascular Endothelia

Abstract

The pulmonary vascular endothelial paracellular pathway and zonula adherens (ZA) integrity are regulated, in part, through protein tyrosine phosphorylation. ZA-associated protein tyrosine phosphatase (PTP)s are thought to counterregulate tyrosine phosphorylation events within the ZA multiprotein complex. One such receptor PTP, PTPmu, is highly expressed in lung tissue and is almost exclusively restricted to the endothelium. We therefore studied whether PTPmu, in pulmonary vascular endothelia, associates with and/or regulates both the tyrosine phosphorylation state of vascular endothelial (VE)-cadherin and the paracellular pathway. PTPmu was expressed in postconfluent human pulmonary artery and lung microvascular endothelial cells (ECs) where it was almost exclusively restricted to EC-EC boundaries. In human lung microvascular ECs, knockdown of PTPmu through RNA interference dramatically impaired barrier function. In immortalized human microvascular ECs, overexpression of wild-type PTPmu enhanced barrier function. PTPmu-VE-cadherin interactions were demonstrated through reciprocal co-immunoprecipitation assays and co-localization with double-label fluorescence microscopy. When glutathione S-transferase-PTPmu was incubated with purified recombinant VE-cadherin, and when glutathione S-transferase-VE-cadherin was incubated with purified recombinant PTPmu, PTPmu directly bound to VE-cadherin. Overexpression of wild-type PTPmu decreased tyrosine phosphorylation of VE-cadherin. Therefore, PTPmu is expressed in human pulmonary vascular endothelia where it directly binds to VE-cadherin and regulates both the tyrosine phosphorylation state of VE-cadherin and barrier integrity.

Synergistic effects of methylnaltrexone with 5-fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor–induced angiogenesis

“…RPTPA is highly expressed in the vasculature where it is localized to EC junctions (17 -19). Consistent with our results, researchers have shown that silencing RPTPA expression inhibits EC barrier function (19), a prerequisite for angiogenesis. We have extended these findings to show that RPTPA can regulate VEGF-induced Src activation.…”

“…One possible mechanism of attenuating Src tyrosine phosphorylation is through regulation of tyrosine phosphatase activity. An important tyrosine phosphatase implicated in regulating human pulmonary EC contacts is the RPTPA (18,19). MNTX, but not naloxone, treatment of HPMVEC enhanced RPTPA tyrosine phosphatase activity (Fig.…”

“…17). Structurally, RPTPA is composed of extracellular Meprin A5 protein M-type RPTP (RPTPA), immunoglobulin-like, and fibronectin type 3 -like domains and intracellular PTP catalytic domains (18,19). RPTPA is localized at EC junctions and regulates vascular integrity (18,19).…”

TLR4 Signaling Is Coupled to SRC Family Kinase Activation, Tyrosine Phosphorylation of Zonula Adherens Proteins, and Opening of the Paracellular Pathway in Human Lung Microvascular Endothelia

“…Identification of Phosphotyrosine-containing Proteins-An immunoprecipitation strategy was employed to identify ZA substrates for LPS-induced tyrosine phosphorylation as described (43). Lysates of ECs treated with LPS or medium alone were precleared by incubation with either anti-murine or anti-rabbit IgG cross-linked to agarose (Sigma) for 1 h at 4°C and then incubated overnight at 4°C with specific murine monoclonal antibodies raised against ␤-, ␥-, or p120-catenin (BD Biosciences) or a rabbit polyclonal antibody raised against VE-cadherin (Cayman, Ann Arbor, MI).…”

Angiogenesis: The VE-Cadherin Switch

“…Among them, protein tyrosine phosphatases VE-PTP and PTP-mu directly interact with VE-cadherin, and there is convincing evidence showing that the activities of either enzymes alter VE-cadherin phosphorylation state ( , ). In addition, Csk binding to phospho-VE-cadherin may inactivate Src by phosphorylation of the Src Nawroth et al 2002Sui et al 2005 inhibitory site (Tyr527). This mechanism may represent a negative feedback regulation of VE-cadherin activation.…”