Collagen and Collagenase Gene Expression in Three-dimensional Collagen Lattices Are Differentially Regulated by oL 1 1 and Integdns

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts edlM and a2131 integrins are the major receptors responsible for regulating ECM remodeling: a1131 mediates the signals inducing downregulation of collagen gene expression, whereas the a2131 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of ed(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down-regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine-specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other. 1 I~EMODELING of extracellular matrix (ECM) is an im~ ~ portant event during many biological and patho~ physiological processes, as different as organomorphogenesis during embryonal development, wound healing, tumor invasion and metastasis, and fibrosis. All these processes require the disassembly of old and deposition of new ECM components, processes that have to be tightly regulated spatially and temporally. CelI-ECM interactions play a major role in this complex network that regulates the steady state and guarantees connective tissue integrity (for review see Grinnell, 1994). A well-characterized in vitro system representing the in vivo situation during ECM remodeling in connective tissues Address all correspondence to B. Eckes, University of Cologne, Department of Dermatology, Joseph Stelzmann-Strasse 9, D-50924 K61n, Ger many. Ph: 22t 478-4500. Fax: 221 478-4538. 1. Abbreviations used in this paper: ECM, extracellular matrix; MMP-1, matrix metalloproteinase-1; PKC, protein kinase C; VLA, very late antigen. better than conventional monolayer cell culture systems are contracting three-dimensional hydrated collagen matrices, populated by fibroblasts (Bell et al., 1979). When fibroblasts are provided with a three-dimensional matrix consisting mainly of type I collagen, the cells adhere to the collagen fibers and contract the initially loose network to a dense tissuelike structure. This process is accompanied by a fundamental reprogramming of fibroblast morphology and metabolism, resulting in down regulation of type I collagen and induction of matrix-degrading proteases (Nusgens et al., 1984; Unemori and Werb, 1986; Mauch et al., 1988, 1989). In particular, induction of interstitial collagenase (matrix metalloproteinase-1 [MMP-1]) is controlled at the transcriptional level, whereas collagen synthesis is regulated by transcriptional (Mauch et al., 1989) as well as by posttranscriptional mechanisms (Eckes et al., 1993). Integrins are well known as the major cell membrane receptors, which mediate a molecular dialogue between cells and their extracellular matrix (for review see Hynes, i992). They constitute a family of at least 21 distinct molecules, © The Rockefeller University Press, 0021-9525/95/12/1903/13 $2.00 The Journal of Cell Biology, Volume 131, Number 6, Part 2, December 1995 1903-1915 1903 on N ovem er 9, 2017 jcb.rress. org D ow nladed fom which are heterodimeric transmembrane cell surface receptors, assembled by the noncovalent association of one a with one [3 subunit. Both subunits possess a large extracellular domain, a membrane-spanning region and a short cytoplasmic domain. The extracellular domains of one ~x and one [3 chain combine to form the binding region for ECM proteins. The cytoplasmic domains bind to intracellular proteins including talin, vinculin, and c~-actinin, thereby linking the extracellular environment with the cell interior (Horwitz et al., 1986; Burridge et al., 1988; Otey et al., 1990). The exact nature of how integrins transduce information from three-dimensional contact to matrix proteins into cellular events, leading to remodeling of the ECM, however, remains to be elucidated. Phosphorylation of proteins is a common mechanism in signal transduction (Ullrich and Schlessinger, 1990; Glenhey, 1992) and could recently be shown to be part of signal cascades downstream of integrin receptors (reviewed in Juliano and Haskill, 1993; Schaller and Parsons, 1993). A new intracellular tyrosine kinase, focal adhesion kinase, was identified as a downstream target of integrin signaling in platelets (Lipfert et al., 1992; Golden et al., 1990), carcinoma cells (Kornberg et al., 1991), and rodent fibroblasts (Guan et al., 1991; Hanks et al., 1992). The observation that the focal adhesion kinase pp125 FAN is also activated by phosphorylation in response to contact of fibroblasts to collagen in a three-dimensional as well as in a two-dimensional system (R6ckel and Krieg, 1994) suggests that activation is mediated by integrins capable of binding collagen. Binding to native type I collagen by integrins involves mainly txl[31 (very late antigen [VLA]-I) and ~2131 (VLA2) (Wayner and Carter, 1987; Belkin et al., 1990; Kirchhofer et al. 1990; Elices et al., 1989). Cultured fibroblasts from healthy human donors express all of these integrin chains (Schiro et al., 1991). Whether integrin-mediated phosphorylation relates to changes in gene expression which are induced by adhesion or contact to a three-dimensional extracellular matrix environment remains to be determined. Perturbation studies using antibodies blocking [31 integrin showed that this interferes with gel contraction (Gullberg et al., 1990). Klein et al. (1991) proposed ~2131 to be the receptor responsible for the contraction process. Their data were corroborated by transfection experiments introducing full-length a2 cDNA into rhabdomyosarcoma cells (Schiro et al., 1991). Other recent studies demonstrated that among several external factors certain mAb directed to integrin subunits are able to modulate the function of [31 integrins (for review see Hynes, 1992). The proposed mechanism of action of these mAb is to induce a conformational change in the receptors that modulates the affinity and/or avidity for their ligands. For example, Werb and co-workers (1989) could achieve induction of MMP-1 by ligation of the fibronectin receptor with mAb in rabbit synovial fibroblasts in conventional monolayer cultures. The aim of this study was to identify receptors involved in regulating collagen metabolism during extracellular matrix remodeling in contracting collagen lattices. We here present evidence that cd[31 integrin is the receptor responsible for down-regulation of collagen I synthesis in fibroblasts cultured within contracting collagen lattices, whereas tx2131 integrin mediates the induction of MMP-1 in this system. In addition, we show that protein phosphorylation is involved in the further transduction of signals by these integrin heterodimers. Materials and Methods