[Frontiers In Bioscience, Landmark, 23, 1612-1627, March 1, 2018]

Contribution of activated beta3 integrin in the PDI release from endothelial cells

Halszka Ponamarczuk1, Marcin Popielarski1, Marta Stasiak1, Radosław Bednarek1, Maciej Studzian2, Lukasz Pulaski2,3, Anna Babinska4, Maria Swiatkowska1

1Department of Cytobiology and Proteomics, Medical University of Lodz, Lodz, Poland, 2Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland, 3Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS, Lodz, Poland, 4Department of Medicine, SUNY Downstate Medical Center, Brooklyn, New York, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and methods
3.1. Reagents and antibodies
3.2. EA.hy926 cell culture
3.3. Secretion of PDI from endothelial cells
3.4. Cell adhesion assay
3.5. Wound healing migration assay
3.6. Coimmunoprecipitation and immunoblotting
3.7. Coimmunoprecipitation beta1/PDI
3.8. Confocal microscopy
3.9. Sulfhydryl group labeling
3.10. In vitro angiogenesis assay - endothelial cell spheroids
3.11. Statistical analysis
4. Results
4.1. Secretion of PDI from endothelial cells
4.2. Association of PDI with alphaVbeta3 integrin on endothelial cells during adhesion
4.3. Influence of PDI inhibitors and thiol group blockers on the functions of endothelial cells
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

Protein disulfide isomerase (PDI) is an abundant reticulum endoplasmic protein but also acts as an important functional regulator of some extracellular surface proteins. Recent studies suggest that PDI plays a role in integrin activation and thrombus formation. The aim of this study was to examine whether activation of integrin is the first stage leading to release of PDI from the subcellular compartments of endothelial cells to extracellular space. Our results show that endothelial cells which adhere to fibronectin or fibrinogen release significantly more PDI than those which adhere to poly-L-lysine. Cells treated with RGD peptide, Src and FAK kinase inhibitors and anti alphaVbeta3 antibody display lower PDI secretion. The destruction of the actin cytoskeleton of endothelial cells by cytochalasin D inhibits PDI release. When the endothelial cells adhere to fibrinogen or fibronectin, PDI and alphaVbeta3 gain free thiol groups. Our data suggest that upon activation of integrins, PDI is released from endothelial cells and forms a disulfide bond complex with alphaVbeta3 integrin.

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Abbreviations: BSA, bovine serum albumin; DENV, dengue virus; DMEM, Dulbecco’s modified Eagle medium; DTNB, 5,5’-dithio-bis(2-nitrobenzoic acid); FAK, focal adhesion kinase; FBS, fetal bovine serum; GRO-alpha, growth-related oncogene-alpha; GSH, reduced glutathione; HAT, hypoxanthine, aminopterin, thymidine; HUVEC, human umbilical vein endothelial cells; MPB, N-(3-Maleimidopropionyl)biocytin; PCMBS, p-chloromercuribenzenesulfonic acid; PDI, protein disulfide isomerase; RGD, Arg-Gly-Asp amino acid sequence; RDE, Arg-Gly- Glu amino acid sequence; Src, sarcoma-family kinases

Key words: PDI, Thiol Groups, Integrins, Adhesion, Endothelial Cells

Send correspondence to: Maria Swiatkowska, Department of Cytobiology and Proteomics, Medical University of Lodz, 6/8 Mazowiecka St. Lodz, Poland, Tel: 48-42-272-5721, Fax:48-42-272-5730, E-mail: maria.swiatkowska@umed.lodz.pl