Molecular Biology of the Cell Vol. 5, 439-453, April 1994 The Extracellular Matrix Ligands Fibronectin and Tenascin Collaborate in Regulating Collagenase Gene Expression in Fibroblasts Patrice Tremble,* Ruth Chiquet-Ehrismann,t and Zena Werb* *Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, California 94143-0750; and tFriedrich Meischer Institute, CH-4002 Basel, Switzerland Submitted June 14, 1993; Accepted February 14, 1994 Monitoring Editor: Richard Hynes Tenascin (TN) is a large oligomeric glycoprotein that is present transiently in the extracellular matrix (ECM) of cells and is involved in morphogenetic movements, tissue patterning, and tissue repair. It has multiple domains, both adhesive and anti-adhesive, that interact with cells and with fibronectin (FN) and other ECM macromolecules. We have studied the consequences of the interaction of TN with a FN matrix on gene expression in rabbit synovial fibroblasts. Fibroblasts plated on a mixed substrate of FN and TN, but not on FN alone, upregulated synthesis of four genes: collagenase, stromelysin, the 92-kDa gelatinase, and c-fos. Although the fibroblasts spread well on both FN and FN/TN substrates, nuclear c-Fos increased within 1 h only in cells that were plated on FN/TN. TN did not induce the expression of collagenase in cells plated on substrates of type I collagen or vitronectin (VN). Moreover, soluble TN added to cells adhering to a FN substrate or to serum proteins had no effect, suggesting that TN has an effect only in the context of mixed substrates of FN and TN. Collagenase increased within 4 h of plating on a FN/TN substrate and exhibited kinetics similar to those for induction of collagenase gene expression by signaling through the integrin FN receptor. Arg-Gly-Asp peptide ligands that recognize either the FN receptor or the VN receptor and function-perturbing anti-integrin monoclonal antibodies diminished the interaction of fibroblasts with a mixed substrate of FN, TN, and VN, but had no effect on the adhesion of fibroblasts to a substrate of FN and VN, suggesting that both receptors recognize the complex. Anti-TN68, an antibody that recognizes an epitope in the carboxyl- terminal type III repeats involved in the interaction of TN with both FN and cells, blocked the inductive effect of the FN/TN substrate, whereas anti-TNM1, an antibody that rec- ognizes an epitope in the amino-terminal anti-adhesive region of epidermal growth factor- like repeats, had no effect. These data suggest that transient alteration of the composition of ECM by addition of proteins like TN may regulate the expression of genes involved in cell migration, tissue remodeling, and tissue invasion, in regions of tissue undergoing phe- notypic changes. INTRODUCTION in the morphogenetic movement of cells and the pat- of tissues The structure Tenascin (TN)' is a large oligomeric glycoprotein in the terning during development. of extracellular matrix (ECM) that is thought to play a role hydrolysate; mAb, monoclonal antibody; MMP, matrix metallopro- teinase; PBS, phosphate-buffered saline; PCR, polymerase chain re- 1 Abbreviations used: BSA, bovine serum albumin; CM, conditioned action; RGD, Arg-Gly-Asp; RSF, rabbit synovial fibroblasts; RT, reverse medium; DME, Dulbecco's modified Eagle's medium; ECM, extra- transcription; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel cellular matrix; EGF, epidermal growth factor; FN, fibronectin; FNR, electrophoresis; TN, tenascin; TPA, 12-0-tetradecanoylphorbol-13- fibronectin receptor; GAPDH, glyceraldehyde-3-phosphate dehy- acetate; VN, vitronectin; VNR, vitronectin receptor; 12OFN, 120-kDa drogenase; LDLR, low-density-lipoprotein receptor; LH, lactalbumin fragment of fibronectin. © 1994 by The American Society for Cell Biology 439 P. Tremble et al. TN (also called GMEM, cytotactin, hexabrachion, or Jl) (Chiquet-Ehrismann et al., 1986). In adults, the expres- (reviewed by Erickson and Bourdon, 1989) is deter- sion of TN is more limited; it is present in regions of mined by a linear array of four motifs: a cysteine-rich, continuous tissue renewal, such as in the intestinal amino-terminal domain that plays a role in TN arm as- crypts (Weller et al., 1991), and is strikingly induced in sociation; a number of epidermal growth factor (EGF)- the dermis beneath a wound epithelium after tissue in- like repeats; a series of fibronectin (FN) type III repeats, jury (Mackie et al., 1988; Whitby et al., 1991). The tran- the number of which is determined by alternative splic- sient expression of TN in discrete developmental pat- ing; and a carboxyl-terminal fibrinogen-like globular terns and its upregulation in regions of repairing tissues domain. The cDNA sequence of chicken, mouse, and suggest an important role for TN in remodeling tissue. human TN has shown that TN isoforms are a result of However, the contributions of a TN-supplemented ma- alternative splicing of FN type III repeats (Spring et al., trix to a remodeling cascade may be functionally du- 1989; Nies et al., 1991; Nishi et al., 1991; Siri et al., plicated by other interactions, because TN-null mice 1991; Weller et al., 1991). Although the number of al- develop normally, with no obvious abnormalities (Saga ternatively spliced type III repeats varies with species, et al., 1992). the type III repeats in similar positions share consid- The function of TN in remodeling tissue has not yet erable homology (Gulcher et al., 1991). been clearly defined. However, a common response of In vitro, TN binds to immobilized FN (Chiquet-Eh- fibroblasts, epithelial cells, neurons, and glia cultured rismann et al., 1991) and to cell surface proteoglycans in the presence of TN is a diminution of cell adhesion (Salmivirta et al., 1991). There is some tissue specificity (for review see Chiquet-Ehrismann, 1991; Sage and in the interaction of heparan sulfate proteoglycans and Bornstein, 1991). This destabilization of cell adhesion TN, because the integral membrane heparan sulfate to the ECM could enhance cell motility as suggested by proteoglycan syndecan, isolated from toothbud extracts, Tucker and McKay (1991). Alternatively, the addition interacts with immobilized TN in cell-free binding as- of TN to an ECM could change the structure of the says, whereas preparations of syndecan isolated from mesenchymal matrix as suggested by Whitby et al. mammary gland do not (Salmivirta et al., 1991). The (1991) and Ekblom and Aufderheide (1989). Finally, carbohydrate side chains of heparan sulfate and chon- the presence of TN in ECM could directly or indirectly droitin sulfate proteoglycans play a role in the inter- signal cells to alter their expression of genes involved action with TN (Murphy-Ullrich et al., 1991; Salmivirta in ECM remodeling. This possibility is supported by et al., 1991); however, the core protein of cytotactin- similar patterns of distribution of TN and ECM-de- binding proteoglycan, which contains chondroitin sul- grading matrix metalloproteinases (MMPs) in repairing fate proteoglycan, mediates the interaction with TN and remodeling tissue (reviewed by Alexander and (Hoffman et al., 1988). Werb, 1991). The interaction of FN and TN molecules TN expression is seen in two types of tissues. The is clearly documented (Chiquet-Ehrismann et al., 1991), smallest isoform of TN is expressed in regions of dense and other studies demonstrate that TN inhibits or per- connective tissue such as gizzard or myotendinous turbs adhesion of cells to intact FN and FN fragments junctions, where tensile strength is important (Chiquet- that contain the classical Arg-Gly-Asp (RGD) sequence Ehrismann et al., 1991). In both adult and embryonic (Chiquet-Ehrismann et al., 1988; Lotz et al., 1989). In tissues the expression of TN is also seen in regions of fibroblasts, MMP expression is upregulated in response tissue in which ECM remodeling, cell division, and cell to stimuli that subtly perturb cell adhesion or alter the migrations take place. The temporal and spatial distri- actin cytoskeleton, as in cells treated with anti-fibro- bution of TN expression in the early embryo (Chiquet- nectin receptor (FNR) antibodies or plated on the 120- Ehrismann et al., 1986; Prieto et al., 1990), coupled with kDa chymotryptic fragment of FN (12OFN) (Aggeler et the observations that TN diminishes cell adhesion and al., 1984; Unemori and Werb, 1986; Werb et al., 1989). migration (Chiquet-Ehrismann et al., 1986; Friedlander We therefore investigated whether perturbations in cell et al., 1988; Halfter et al., 1989; Lotz et al., 1989; Riou behavior generated by addition of TN to a substrate of et al., 1990), suggest that TN is an important molecule FN regulate cellular gene expression. As our model sys- used in directed migrations early in development. The tem, we chose rabbit fibroblasts, which respond to FN transient expression of specific TN isoforms in the de- fragments or anti-FNR antibodies by induction of MMP veloping nervous system suggests that TN has important expression, and we investigated the regulation of col- functions here as well (Steindler et al., 1989; Prieto et lagenase as a model gene. al., 1990). TN may also play a role in condensing tissue, because the expression of TN is upregulated during ep- ithelial mesenchymal induction (Ekblom and Aufder- MATERIALS AND METHODS heide, 1989; Salmivirta et al., 1991); the distribution of Cells and Cell Culture TN in condensing tissues may also change, because TN Rabbit synovial fibroblasts (RSF), isolated as described previously localization shifts from a diffuse presence to a peri- (Aggeler et al., 1984), were cultured in Dulbecco's modified Eagle's chondral localization during cartilage condensation medium (DME) (Cell Culture Facility, University of California, San 440 Molecular Biology of the Cell Fibronectin-Tenascin Regulates Collagenase Francisco) supplemented with 10% fetal bovine serum (FBS) (Hyclone, of 50 ,g/ml pepsin-treated bovine skin collagen (Vitrogen, Collagen, Denver, CO). RSF were used between passages 2 and 10. Early-passage Palo Alto, CA) in distilled water then washed with PBS, and uncoated fibroblasts in culture often synthesize and secrete TN into the culture sites were blocked with 0.2% BSA in PBS.