Thrombospondin-1-induced Focal Adhesion Disassembly in Fibroblasts Requires Thy-1 Surface Expression, Lipid Raft Integrity, and Src Activation

Thomas H Barker,Manuel A Pallero,Mark W Macewen,Samuel G Tilden,Anne Woods,Joanne E Murphy-Ullrich,James S Hagood

doi:10.1074/jbc.m402169200

Thomas H Barker, Manuel A Pallero + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m402169200

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Abstract

The hep I peptide of thrombospondin-1 is known to induce the disassembly of focal adhesions, a critical step in regulating cellular adhesive changes needed for cell motility. Fibroblasts that are heterogeneous with respect to the surface expression of Thy-1 differ markedly in morphology, cytoskeletal organization, and migration, suggesting differential regulation of focal adhesion dynamics. Here we demonstrate that disassembly of focal adhesions mediated by both full-length thrombospondin-1 and the hep I peptide in fibroblasts requires the expression of Thy-1, although it does not appear to function as a stable member of the hep I receptor complex. Consistent with a known function of Thy-1 in regulating lipid raft-associated signaling, intact lipid rafts are necessary for hep I-mediated focal adhesion disassembly. Furthermore, we establish Src family kinase (SFK) activation as a novel component required for hep I-induced signaling leading to focal adhesion disassembly. hep I induces transient phosphorylation of SFKs in Thy-1-expressing fibroblasts only. Therefore, we conclude that Thy-1 surface expression is required for thrombospondin-1-induced focal adhesion disassembly in fibroblasts through an SFK-dependent mechanism. This represents a novel role for Thy-1 in the regulation of fibroblast-matrix interactions critical to tissue homeostasis and remodeling.

Highlights

Induce the disassembly of focal adhesions, a critical step TSP-1 binding to the co-receptor complex of calreticulin and in regulating cellular adhesive changes needed for cell low density lipoprotein receptor-related protein (LRP) [2,3,4]
We describe clearly discordant responses of Thy-1 fibroblast subpopulations to TSP-1-mediated focal adhesion disassembly, identify that both lipid raft integrity and Src family kinase (SFK) activation are critical to the TSP-1-mediated focal adhesion disassembly signaling cascade, and demonstrate that Thy-1 surface expression is required for SFK activation by TSP-1 and subsequent focal adhesion disassembly in fibroblasts
We demonstrate that Thy-1-dependent TSP-1-mediated focal adhesion disassembly requires Src family kinase signaling

Summary

Introduction

Induce the disassembly of focal adhesions, a critical step TSP-1 binding to the co-receptor complex of calreticulin and in regulating cellular adhesive changes needed for cell low density lipoprotein receptor-related protein (LRP) [2,3,4]. We demonstrate that disassembly of focal adhesions mediated by both full-length thrombospondin-1 and the hep I peptide in fibroblasts requires the expression of Thy-1, it does not appear to function as a stable member of the hep I receptor complex. We identified a requirement for Src family kinase downstream signaling in Thy-1-dependent hep I-mediated fibroblast focal adhesion disassembly by the addition of the Src-specific inhibitor PP2 (10 nM, Fig 4A).

Results

Conclusion