Integrin Beta 4E Promotes Endothelial Phenotypic Changes and Attenuates Lung Endothelial Cell Inflammatory Responses.

Weiguo Chen,Yulia Epshtein,Joe G N Garcia,Anne E Cress,Jeffrey R Jacobson,Jamie M C Gard,Sara M Camp

doi:10.3389/fphys.2022.769325

Weiguo Chen, Yulia Epshtein + Show 5 more

Open Access

https://doi.org/10.3389/fphys.2022.769325

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Abstract

We previously reported integrin beta 4 (ITGB4) is an important mediator of lung vascular protection by simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitor. In this study, we report increased endothelial cell (EC) expression specifically of ITGB4E, an ITGB4 mRNA splice variant, by simvastatin with effects on EC protein expression and inflammatory responses. In initial experiments, human pulmonary artery ECs were treated using simvastatin (5 μM, 24 h) prior to immunoprecipitation of integrin alpha 6 (ITGA6), which associates with ITGB4, and Western blotting for full-length ITGB4 and ITGB4E, uniquely characterized by a truncated 114 amino acid cytoplasmic domain. These experiments confirmed a significant increase in both full-length ITGB4 and ITGB4E. To investigate the effects of increased ITGB4E expression alone, ECs were transfected with ITGB4E or control vector, and cells were seeded in wells containing Matrigel to assess effects on angiogenesis or used for scratch assay to assess migration. Decreased angiogenesis and migration were observed in ITGB4E transfected ECs compared with controls. In separate experiments, PCR and Western blots from transfected cells demonstrated significant changes in EC protein expression associated with increased ITGB4E, including marked decreases in platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin) as well as increased expression of E-cadherin and N-cadherin along with increased expression of the Slug and Snail transcription factors that promote endothelial-to-mesenchymal transition (EndMT). We, then, investigated the functional effects of ITGB4E overexpression on EC inflammatory responses and observed a significant attenuation of lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, including decreased phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as reduced inflammatory cytokines (IL-6 and IL-8), expressed in the media of EC after either LPS or excessive cyclic stretch (CS). Finally, EC expression-increased ITGB4E demonstrated decreased barrier disruption induced by thrombin as measured by transendothelial electrical resistance. Our data support distinct EC phenotypic changes induced by ITGB4E that are also associated with an attenuation of cellular inflammatory responses. These findings implicate ITGB4E upregulation as an important mediator of lung EC protection by statins and may lead to novel therapeutic strategies for patients with or at risk for acute lung injury (ALI).

Highlights

The statin drugs are a class of 3-hydroxy-3-methylglutarylcoenzyme A-reductase inhibitors (HMG CoA-reductase inhibitors) most commonly used clinically to lower serum cholesterol levels and for their protective effects with respect to cardiovascular disease
integrin beta 4 (ITGB4) has been identified as a mediator of mitogen-activated protein kinase (MAPK) signaling in a variety of cell types (Nikolopoulos et al, 2005; Meng et al, 2020), and we previously reported that ITGB4 regulates lung endothelial cell (EC) inflammatory responses via effects on MAPK signaling mediated by SHP-2, a protein tyrosine phosphatase (Chen et al, 2010)
Immunoprecipitation of integrin alpha 6 (ITGA6) followed by Western blotting for ITGB4 confirmed increased heterodimer formation of both ITGB4 and ITGB4E with ITGA6 after simvastatin treatment

Summary

Introduction

The statin drugs are a class of 3-hydroxy-3-methylglutarylcoenzyme A-reductase inhibitors (HMG CoA-reductase inhibitors) most commonly used clinically to lower serum cholesterol levels and for their protective effects with respect to cardiovascular disease. We previously reported lung vascularprotective effects of simvastatin in acute inflammatory lung injury models with protection mediated by simvastatin-mediated upregulation of integrin beta 4 (ITGB4), a key transmembrane protein, in lung endothelial cells (ECs) (Jacobson et al, 2005; Chen et al, 2012). There are 8 separate integrin beta subunits with the laminin-5 receptor ITGB4, which forms a heterodimer only with integrin α6, uniquely characterized by an extended cytoplasmic tail (>1,000 amino acids). This tail is comprised of a proximal Calx Na-Ca exchanger domain followed by two pairs of fibronectin type III repeats separated by a tyrosine activation motif (TAM) (Hogervorst et al, 1990). Subsequent studies identified variable effects on EC inflammatory responses associated with the overexpression of ITGB4 mutants characterized by specific mutations or deletions within the regions of the cytoplasmic tail (Chen et al, 2015)

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