PDIA3/ERp57 promotes a matrix-rich secretome that stimulates fibroblast adhesion through CCN2

Andrew L Hellewell, Kate J Heesom, Mark A Jepson, Josephine C Adams

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Abstract

The matricellular glycoprotein thrombospondin1 (TSP1) has complex roles in the extracellular matrix and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulphide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalise in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3-/- mouse embryo fibroblasts (Pdia3-/-MEF), led to alterations in the composition of cell-derived ECM, involving changed abundance of fibronectin and TSP1, and was correlated with reduced cell spreading, altered organisation of F-actin and reduced focal adhesions. These cellular phenotypes of Pdia3-/-MEF were normalised by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEF. Rescue depended on PDIA3 activity in WT-MEF, and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEF and Pdia3-/- MEF identified multiple ECM and growth factor proteins to be down-regulated in the CM of Pdia3-/- MEF. Of these, CCN2 was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3-/- MEF and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, pro-adhesive microenvironment, with implications for PDIA3 as a translational target.

Original languageEnglish
Pages (from-to)C624–C644
Number of pages21
JournalAmerican journal of physiology. Cell physiology
Volume322
Issue number4
Early online date28 Mar 2022
DOIs
Publication statusPublished - 1 Apr 2022

Bibliographical note

Funding Information:
This work was supported by MRC K018043. BBSRC/EPSRC L01386X supported the HyVolution imaging.

Funding Information:
We thank Prof. Marek Michalak, University of Alberta, for the gift of wild-type and Pdia3−/− mouse embryonic fibroblasts; Keshvi Haria and David Marshall, undergraduates in the Bristol Biochemistry Honours B.Sc. programme, for their assistance with cell adhesion analyses; the MRC and Wolfson Bioimaging Facility for support of confocal and HyVolution imaging; and BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (grant number L01386X), for support of the HyVolution imaging.

Publisher Copyright:
© 2022 The Authors.

Keywords

  • cell adhesion
  • extracellular matrix
  • interactome
  • secretome
  • hrombospondin-1

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