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

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.

Protein disulfide isomerase A3 activity promotes extracellular accumulation of proteins relevant to basal breast cancer outcomes in human MDA-MB-A231 breast cancer cells

“…To further investigate the functional properties of CM, we investigated the role of heparin-binding proteins in the cell-spreading activity of CM from control MDA-MB-231 cells. Many secreted proteins that have roles in cell attachment and cell spreading contain heparin-binding domains ( 47 ), and we have shown that heparin-binding proteins are essential for the stimulation of spreading of Pdia3 −/− MEFs by CM from WT-MEFs ( 25 ). Serum-free CM was produced from control MDA-MB-231 cells as above and then mixed with either control agarose beads or heparin-agarose beads, the beads removed by centrifugation and filtration, and the residual supernatants tested in MDA-MB-231 cell spreading for 4 h. The CM depleted for heparin-binding proteins was markedly less effective at supporting MDA-MB-231 cell spreading assays and F-actin organization than the control CM or fresh FGM, as apparent from cell morphology ( Fig.…”

“…3 B , blue arrow). In addition, IGFBP7 is less abundant in the CM of Pdia3 -null MEFs compared with CM from WT-MEFs ( 25 ). IGFBP7 was detected principally in CM, with minor amounts in cell lysate, as a band of the expected molecular mass of 30 kDa.…”

“…3 B , magenta arrow). LOXL2 has roles in collagen cross linking by oxidative deamidation of lysine residues ( 48 ) and has previously been identified as a PDIA3 substrate ( 49 ), and its abundance is quantitatively reduced in the CM of Pdia3 -null MEFs compared with CM from WT-MEFs ( 25 ). All blots for LOXL2 (87 kDa) showed a band below 100 kDa and a second band ∼75 kDa in the CM, and very little LOXL2 was detected in cell lysates ( Fig.…”

“…We have also established by comparative studies of mouse embryo fibroblasts (MEFs) that gene knockout of Pdia3 correlated with reduced cell spreading relative to wild-type MEFs (WT-MEFs) and strongly affects the secretome of MEFs, resulting in reduced extracellular abundance of a number of heparin-binding proteins in the serum-free conditioned medium (CM) of Pdia3 −/− MEFs compared with the CM of WT-MEFs. The affected proteins included several ECM proteins and growth factors ( 25 ). In addition, whereas exposure to CM of WT-MEFs restored cell spreading of Pdia3 −/− MEFs, the CM of Pdia3 −/− MEFs poorly supported adhesion, F-actin organization, and focal adhesion formation by WT-MEFs ( 25 ).…”

“…The affected proteins included several ECM proteins and growth factors ( 25 ). In addition, whereas exposure to CM of WT-MEFs restored cell spreading of Pdia3 −/− MEFs, the CM of Pdia3 −/− MEFs poorly supported adhesion, F-actin organization, and focal adhesion formation by WT-MEFs ( 25 ).…”