Assembly of Exogenous Fibronectin by Fibronectin-null Cells Is Dependent on the Adhesive Substrate

Eunnyung Bae,Takao Sakai,Deane F Mosher

doi:10.1074/jbc.m406283200

Eunnyung Bae, Takao Sakai + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m406283200

Copy DOI

Abstract

The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III(7-10)) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and beta(1) integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (beta(1)-expressing) cells immediately adjacent to (FN-secreting) beta(1)-null cells. In co-cultures on vitronectin of FN-null cells and beta(1)-null cells expressing a chimera with the extracellular domain of beta(1) and the cytoplasmic domain of beta(3), preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III(7-10), supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a beta(1) integrin, presumably alpha(5)beta(1), substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN.

Highlights

Deposition of fibronectin (FN)1 into extracellular matrix is a dynamic process that is tightly regulated and controlled despite the presence of high concentrations of FN in plasma (200 – 600 ␮g/ml, 440 –1320 nM) and other body fluids [1, 2]
These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN and suggest that efficient interaction of freshly secreted cellular FN with a ␤1 integrin, presumably ␣5␤1, substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or plasma FN (pFN) to support assembly of FN
VN had a suppressive effect that was overcome by surface-adsorbed pFN or laminin-1 (LN), but not by exogenously added soluble pFN or recombinant EDaϩ or EDaϪ FN, whereas III7–10 was unable to support FN assembly by FN-null cells. cellular FN (cFN) was preferentially assembled by cFN-expressing cells regardless of the adhesive ligand

Summary

EXPERIMENTAL PROCEDURES

Cells—The derivation of FNϪ/Ϫ mouse fibroblastic cells (FN-null cells) and FNϩ/Ϫ cells (cFN-expressing cells) from FNϪ/Ϫ or FNϩ/Ϫ mouse embryonic stem cells was described previously [27]. ␤1-null GD25 cells and GD10 cells deficient in the integrin ␤1 subunit had been derived by a similar technique and transfected with the ␤1A splice variant to give ␤1-expressing ␤1A GD25 cells [19] or ␤1A GD10 cells. ␤1A␤3 GD10 cells were generated by transfection of GD10 cells by a ␤1A/␤3 chimeric construct in which the cytoplasmic domain of ␤1A was replaced with the cytoplasmic domain of ␤3 [28]. Trypsin was quenched by washing with 10% fetal bovine serum in DMEM (Cellgro Mediatech, VA), followed by washing with PBS, and cells were cultured at 60ϳ70% confluence for 4 or 18 h in DMEM supplemented with 0.2% BSA (Sigma) on glass coverslips coated with pFN (3 ␮g/ml), LN extracted from Engelbreth-Holm-Swarm mouse tumor (10 ␮g/ml) (BD Biosciences, Bedford, MA), VN (3 ␮g/ml) [33], or III7–10 (3 ␮g/ml) [34, 35] unless indicated. FN-null cells in DMEM containing 0.2% BSA and 9 ␮g/ml Rx-pFN were incubated for 4 h on coverslips precoated as described above (VN/vFN, vVN/FN, VN/vLN, or vVN/LN). Cells were incubated for 30 min at 37 °C in a suspension of DMEM containing 0.2% BSA with or without 40 ␮g/ml of GoH3 anti-mouse ␣6 monoclonal antibody or 15–30 ␮g/ml cRGDfV. Non-adherent cells were removed by washing, and adherent cells were quantified by colorimetric detection at 595 nm using a microplate reader (Model EL340, BIO-TEK Instruments, Inc.), and data were obtained with DELTA Soft IITM (BioMetallics, Inc.)

RESULTS

To corroborate the finding that endogenous cFN expression

DISCUSSION