SPARC Regulates Extracellular Matrix Organization through Its Modulation of Integrin-linked Kinase Activity

doi:10.1074/jbc.M504663200

SPARC Regulates Extracellular Matrix Organization through Its Modulation of Integrin-linked Kinase Activity*

^‡Hope Heart Program, Benaroya Research Institute at Virginia Mason, and the ^§Department of Bioengineering, University of ^∥ Washington, Seattle, Washington 98101, the ^¶University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, the Barbara Jane Levy Laboratory, Hermelin Brain Tumor Center, Henry Ford Hospital, Detroit, Michigan 48202, the ^**BC Cancer Agency, Jack Bell Research Centre, Vancouver, British Columbia V5Z 1L3, Canada, and the ^‡‡Swiss Federal Institute of Technology, ETH, CH-8093 Zurich, Switzerland

1 To whom correspondence should be addressed: Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1201 9th Ave. Seattle, WA 98101. Tel.: 206-341-1314; Fax: 206-341-1375; E-mail: hsage{at}benaroyaresearch.org.

Abstract

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

Footnotes

↵2 The abbreviations used are: ECM, extracellular matrix; Ad, adenovirus; Ad.SP, adenovirus containing mouse SPARC cDNA; Ad.RLuc, adenovirus containing Renilla luciferase cDNA; AF, AlexaFluor©; Fn, fibronectin; Fn-D/A, doubly labeled fibronectin; GdnHCl, guanidine hydrochloride; HA, hemagglutinin; I_A, intensity of acceptor fluore; I_D, intensity of donor fluore; ILK, integrin-linked kinase; MBP, myelin basic protein; MLC, myosin light chain; MLCP, myosin light chain phosphatase; S-/-, SPARC-null; WT, wild-type; TRITC, tetramethylrhodamine isothiocyanate; FRET, fluorescence resonance energy transfer; FBS, fetal bovine serum; PBS, phosphate-buffered saline; rhuSPARC, recombinant human SPARC; rhuILK, recombinant human ILK; BSA, bovine serum albumin.
↵3 H. Xenias, R. Faessler, and M. Sheetz, personal communication.
↵5 M. Antia, G. Baneyx, G. Young, and V. Vogel, submitted for publication.
↵* This work was supported by National Institutes of Health Grants GM40711 and EY13180 (to E. H. S.) and CA 086997 (to S. A. R.) and National Science Foundation Grant EEC-9529161 (to E. H. S. and V. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received April 28, 2005.
- Revision received August 19, 2005.
The American Society for Biochemistry and Molecular Biology, Inc.