Developmental Biology 237, 116–129 (2001) doi:10.1006/dbio.2001.0363, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE ␣11␤1 Integrin Is a Receptor for Interstitial provided by Elsevier - Publisher Connector Collagens Involved in Cell Migration and Collagen Reorganization on Mesenchymal Nonmuscle Cells Carl-Fredrik Tiger,* Francoise Fougerousse,† Gunilla Grundstro¨m,‡ Teet Velling,‡ and Donald Gullberg‡,1 *Department of Cell and Molecular Biology, Biomedical Center, Box 596, Uppsala University, S-75124 Uppsala, Sweden; †Laboratoire de Histoembryologie et de Cytoge´ne´tique, Faculte´ Cochin Port Royal, Paris 75014, France; and ‡Department of Medical Biochemistry and Microbiology, Biomedical Center, Box 582, Uppsala University, S-75123 Uppsala, Sweden ␣11␤1 integrin constitutes a recent addition to the integrin family. Here, we present the first in vivo analysis of ␣11 protein and mRNA distribution during human embryonic development. ␣11 protein and mRNA were present in various mesenchymal cells around the cartilage anlage in the developing skeleton in a pattern similar to that described for the transcription factor scleraxis. ␣11 was also expressed by mesenchymal cells in intervertebral discs and in keratocytes in cornea, two sites with highly organized collagen networks. Neither ␣11 mRNA nor ␣11 protein could be detected in myogenic cells in human embryos. The described expression pattern is compatible with ␣11␤1 functioning as a receptor for interstitial collagens in vivo. To test this hypothesis in vitro, full-length human ␣11 cDNA was stably transfected into the mouse satellite cell line C2C12, lacking endogenous collagen receptors. ␣11␤1 mediated cell adhesion to collagens I and IV (with a preference for collagen I) and formed focal contacts on collagens. In addition, ␣11␤1 mediated contraction of fibrillar collagen gels in a manner similar to ␣2␤1, and supported migration on collagen I in response to chemotactic stimuli. Our data support a role for ␣11␤1 as a receptor for interstitial collagens on mesenchymally derived cells and suggest a multifunctional role of ␣11␤1 in the recognition and organization of interstitial collagen matrices during development. © 2001 Academic Press Key Words: ␣11␤1 integrin; immunohistochemistry; in situ hybridization; human embryogenesis; collagen binding; collagen gel contraction; cell migration. INTRODUCTION subunits, which can assemble into 24 different het- erodimers, are known (Velling et al., 1999). Collagens are major constituents of various types of Cellular interactions with the extracellular matrix (ECM) specialized ECM. Until recently, only two members of the are important for fundamental biological processes such as integrin family, ␣1␤1 and ␣2␤1, were known to act as cell proliferation, cell differentiation, cell migration, apo- collagen receptors. More recently, ␣10␤1 (Camper et al., ptosis, morphogenesis, and organogenesis. Integrins are one 1998) and ␣11␤1 (Velling et al., 1999) have been added to family of proteins mediating cell–ECM interactions, and this subfamily of collagen-binding integrins. The ␣3␤1 many members have proven essential for embryonic devel- integrin, initially described as a collagen receptor (Wayner opment (Brakebusch et al., 1997; Hynes, 1996; Sheppard, and Carter, 1987), has later been shown to serve as an 2000). Integrins are heterodimeric transmembrane recep- important laminin-5 receptor (Carter et al., 1991). Genetic tors consisting of noncovalently assembled ␣ and ␤ sub- data, however, have indicated that ␣3␤1 can affect the units (Hynes, 1992). To date, 8 different ␤ and 18 different ␣ activity of the collagen receptor ␣2␤1 through receptor cross-talk (Hodivala-Dilke et al., 1998). 1 To whom correspondence should be addressed. Fax: ϩ46-18- The distribution of the ␣1␤1 and ␣2␤1 in vivo has been 471-4862. E-mail: [email protected]. studied in detail. During embryonic development, ␣1␤1 and 0012-1606/01 $35.00 Copyright © 2001 by Academic Press 116 All rights of reproduction in any form reserved. ␣11␤1 Integrin Is an Interstitial Collagen Receptor 117 ␣2␤1 are widely expressed (Duband et al., 1992; Gardner et heart and skeletal muscle (Velling et al., 1999). However, al., 1996; Wu and Santoro, 1994). Postnatally, the ␣1 chain neither the cellular origin of the ␣11 mRNA in these tissues is strongly expressed on microcapillary endothelium and nor the expression pattern of ␣11 during development is pericytes as well as in smooth muscle cells (Voigt et al., known. 1995). The ␣2 chain is present on platelets, haematopoetic In the current study, we have determined ␣11 protein and cells, and a variety of epithelia (Gardner et al., 1996; Keely mRNA expression in human embryos and have performed et al., 1995; Wu and Santoro, 1994). Both ␣1 and ␣2 are in vitro studies of cell adhesion, cell migration, and colla- present on fibroblasts (Voigt et al., 1995; Wu and Santoro, gen gel contraction. The distribution pattern shows that 1994). ␣11 during embryonic development displays a restricted The ligand specificities of ␣1␤1 and ␣2␤1 differ for expression on mesenchymal nonmuscle cells in areas of collagens I, IV, and XIII (Dickeson and Santoro, 1998; highly organized interstitial collagen networks. The re- Kapyla et al., 2000; Kern et al., 1994; Nykvist et al., 2000; stricted distribution suggests that ␣11 is under strict tran- Tuckwell et al., 1996). Whereas the binding of the intersti- scriptional control and might be one of the sought-after cell tial collagen I by ␣1␤1 and ␣2␤1 involves helical GFOGER adhesion genes acting downstream of scleraxis, involved in and related sequences (Knight et al., 1998, 2000; Xu et al., prefiguring the axial and appendicular skeleton. The in 2000), the ␣1␤1 binding of the basement membrane colla- vitro data show that ␣11␤1 can take part in collagen- gen IV involves specific R and D residues present on mediated events such as cell migration, collagen deposi- different collagen ␣-chains (Eble et al., 1993; Golbik et al., tion, and collagen reorganization, suggesting that ␣11␤1 in 2000). The residues in collagen XIII recognized by ␣1␤1 vivo could fulfill these important functions and play an (Nykvist et al., 2000) have not been identified. Neither the important collagen organizing role during cartilage, inter- exact collagen specificity of ␣10␤1 and ␣11␤1 nor the vertebral disc, and cornea organogenesis. regions within collagens recognized by ␣10␤1 and ␣11␤1 are known. An important in vivo function of cell–collagen interac- tions is to reorganize the collagen matrix within developing EXPERIMENTAL PROCEDURES tendons, ligaments, periosteum, capsules of organs, and in healing wounds (Harris et al., 1981; Stopak and Harris, Cells 1982). The ability of cells to contract three-dimensional collagen matrices in vitro reflects their potential to modu- Normal diploid human fibroblasts (AG1518; Genetic Mutant late a collagen-rich matrix. ␣2␤1 serves as a major ␤1 Cell Repository, Camden, NJ) were obtained from Dr. Kristofer integrin receptor involved in contraction of collagen gels Rubin (Uppsala University). Skin fibroblasts and keratocytes were (Klein et al., 1991), whereas the ability of ␣1␤1 to contract obtained from Natalie Isnard (Laboratoire de Recherche en collagen gels seems to depend on the cell type (Carver et al., Opthamologie, Paris). Human satellite cells (XXVI) isolated from a 1995; Gotwals et al., 1996; Jenkins et al., 1999; Kagami et 2.5-year-old child were provided by Dr. Helen Blau (Stanford ␣ al., 1999; Racine-Samson et al., 1997). For some cell types, University). Other human cell lines tested for 11 expression an ␣v␤3-dependent contraction of collagen gels has been included Caco-2 (colon adenocarcinoma, ATCC No. HTB-37), JAR (human choriocarcinoma cells, ATCC No. HTB-144), RD (rhabdo- observed (Agrez et al., 1991; Cooke et al., 2000; Jones et al., myosarcoma, ATCC No. CCL-136), HT-29 cells (human colon 1997; Nunohiro et al., 1999). Induction of collagenases adenocarcinoma, ATCC No. HTB-38), and HT1080 (kindly pro- leading to denaturation of collagen or deposition of RGD- vided by S. Johansson). Murine C2C12 myoblast cells from the containing ligands has been suggested to underlie the ␣v␤3- American Type Culture Collection were provided by A. Starzinski- dependency of this contraction. Powitz. Cells were cultured at 37°C in Dulbecco’s modified Eagle’s The biological significance of cell–collagen interactions medium, supplemented with 10% fetal bovine serum (FBS) and during development and in adult tissues is still far from antibiotics (Statens veterina¨rmedicinska anstalt, Uppsala). The clear. Mice deficient in ␣1 integrin chain are viable, but cells were grown to subconfluency and passaged every 2–3 days. display a proliferation defect and a disturbed regulation of collagen synthesis in skin fibroblasts (Gardner et al., 1996, 1999; Pozzi et al., 1998). Tumor angiogenesis is also defec- Antibodies tive in mice lacking ␣1 integrin chain (Pozzi et al., 2000). Data from inactivation of ␣2, ␣10, and ␣11 integrin genes in The rabbit antibodies to the cytoplasmic tail of ␣11 integrin have mice are not yet available. been described previously (Velling et al., 1999). To immunoprecipi- ␤ ␤ ␣11␤1 was first identified as a major integrin in cultured tate 1 integrins, a polyclonal antibody to rat integrin 1 chain was used (Gullberg et al., 1989). Monoclonal antibodies (MoAb) to skeletal muscle cells (Gullberg et al., 1995b; Velling et al., ␣ ␣ ␣ integrin 1 chain (FB12, MAB 1973) and integrin 2 chain (BHA2.1, 1999). During myogenic differentiation in vitro, the 11 MAB 1998) were obtained from Chemicon International Inc. As a chain is up-regulated at both the protein and the mRNA marker for forming cartilage, two collagen II MoAbs were used: levels. Analysis of mRNA from a panel of adult human mouse MoAb CIIC1 (Klareskog et al., 1986) and rat MoAb 126.7 (P. tissues reveals expression of ␣11 in a wide variety of tissues Wernhoff, unpublished observations), both obtained from Dr.