Enhancement of Cell Adhesion and Spreading by a Cartilage-specific Noncollagenous Protein, Cartilage Matrix Protein (CMP/Matrilin-1), via Integrin α1β1

Takeshi Kawamoto,Seicho Makihira,Taizo Hamada,Katsumi Fujimoto,Weiqun Yan,Eri Yoshida,Shigeru Ohno,Yukio Kato,Mitsuhide Noshiro,Akinobu Okimura

doi:10.1074/jbc.274.16.11417

Abstract

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.

Highlights

Cartilage matrix protein (CMP1/matrilin-1) was originally isolated as a protein that binds to aggrecan and thereafter was shown to bind to type II collagen [1,2,3]
Unlabeled CMP competed for the binding to integrin ␣1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, in the presence of type II collagen, and that integrin ␣1␤1 is involved in CMP-mediated cell adhesion and spreading
We examined the effect of coating culture dishes with CMP on cell adhesion and investigated whether CMP interacts with integrins using 125I-CMP and various antiintegrin antibodies

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Mouse neutralizing monoclonal antibodies (mAbs) to human integrins ␣1 (FBI2), ␣2 (P1E6) [17], ␣3 (ASC-6) [18], ␣5 (P1D6) [19], ␤2 (P4H9-A11) [20], ␣5␤1 (JBS5), and ␣V␤5 (P1F6) [21]; rabbit antisera to human integrins ␣1, ␣2, ␣V, ␤1, and ␤5; and rabbit antiserum to collagen type I were purchased from Chemicon International, Inc. (Temecula, CA). Mouse neutralizing mAbs to human integrin ␣1 (5E8D9) [22] and ␤1 (DE9) [19] were purchased from Upstate Biotechnology, Inc. Mouse neutralizing mAbs to human integrin ␣2 (P1E6) [23] and ␣3 (P1B5) [23] were purchased from Becton Dickinson (Lincoln Park, NJ). SDSPAGE analysis showed that purified CMP migrated as a single band at a position corresponding to 215 and 60 kDa under nonreducing and reducing conditions, respectively (Fig. 1), as expected from previous studies [2].

Effects of Cartilage Matrix Protein on Cell Adhesion

RESULTS

DISCUSSION