Extracellular Matrix Alters PDGF Regulation of Fibroblast Integrins

Extracellular matrix (ECM) and growth factors are potent regulators of cell phenotype. These biological mediators of cellular responses are potentially interactive and as such could drive cells through progressive phenotypes to create new tissue as in morphogenesis and wound repair. In fact, ECM composition changes during tissue formation accompanied by alterations in cell growth and migration. How alterations in the ECM regulate cell activities is poorly defined. To address this question in wound repair, we cultured normal human dermal skin fibroblasts in relaxed collagen gels, fibronectin-rich cultures or stressed fibrin gels, and stressed collagen gels to model normal dermis, early wound provisional matrix, and late granulation tissue, respectively. Integrin subunits, ~2, ~3, and ors, that define receptor specificity for collagen and provisional matrix, respectively, were measured at mRNA steady-state level before and after stimulation with platelet-derived growth factor-BB (PDGF-BB), a potent mitogen and chemoattractant for fibroblasts. Fibronectin-rich cultures and fibrin gels supported PDGF-BB induction of et 3 and et5 mRNA. In contrast, both stressed and relaxed collagen attenuated these responses while promoting maximal a2 m R N A expression. Posttranscriptional regulation was an important mechanism in this differential response. Together PDGF-BB and collagen gels promoted et2, but not et 3 and et 5, mRNA stability. Conversely, when fibroblasts were in fibronectin-rich cultures, PDGF-BB promoted et 3 and o~5, but not a2, mRNA stability. We suggest that ECM alterations during wound healing or any new tissue formation cause cells to respond differently to repeated growth factor stimuli. An ordered progression of cell phenotypes results, ultimately consummating tissue repair or morphogenesis. URING cutaneous wound repair, cells must attach to new matrix molecules and migrate (Welch et al., 1990). Soon after wounding, a provisional matrix of fibronectin, fibrinogen, fibrin, and vitronectin forms in the wound area (Gailit and Clark, 1994a). In the fibrin network, platelets release growth factors such as PDGF that stimulate proliferation and chemoattraction of fibroblasts (Deuel et al., 1991). Activated fibroblasts must move from surrounding collagenous connective tissues into a fibrin/fibronectin-filled wound and subsequently synthesize new collagenous matrix. Clearly, fibroblast responses to these extracellular matrix (ECM) 1 molecules and growth factors are essential for the healing process to progress. To address the mechanisms by which ECM molecules regulate cell activities, three-dimensional ECM-based culture systems are increasingly used. Since type I collagen is a ubiquitous component of dermal ECM much attention has been devoted to cells cultured in stressed and/or relaxed collagen gels. The relaxed collagen gel is considered Please address all correspondence to Dr. A. F. Clark, Department of Dermatology, School of Medicine, SUNY at Stony Brook, Stony Brook, NY 11794-8165. Tel.: (516) 444-3843. Fax: (516) 444-3844. 1. Abbreviat ions used in this paper: ECM, extracellular matrix; RGD, arginine-glycine-aspartic acid; TGF-131, transforming growth factor-131. a reconstituted dermal model whereas the stressed gel is similar to late granulation tissue (Grinnell, 1994). Compared to fibroblast cultures on plastic, cells in a relaxed collagen gel exhibit little proliferative response to PDGF (Lin and Grinnell, 1993) and little collagen production (Eckes et al., 1993). Nevertheless, cells harvested from stressed collagen gels proliferate and synthesize collagen robustly. Collagen matrices can affect fibroblasts function by altering both gene expression and second messenger pathways. Fibroblasts in collagen gels that are stimulated with PDGF decrease Ctl(I ) collagen gene expression (Berthod et al., 1994; Eckes et al., 1993), increase collagen receptor gene expression (Klein et al., 1991), and decrease autophosphorylation of PDGF receptors (Lin and Grinnell, 1993). In contrast, fibroblasts in fibrin gels or in fibronectin-rich tissue culture conditions,, proliferate robustly and produce exuberant ECM (Clark et al., 1995a; Clark, R. A. F., G. A. McCoy, J. M. Folkvord, and J. McPherson, manuscript submitted for publication; Grinnell et al., 1989; Nakagawa et al., 1989). The molecules primarily responsible for cell adherence to ECM are integrins, a family of cell surface receptors. Integrins are heterodimers composed of an o~ chain and a 13 chain. 131 integrin can form a complex with at least ten different et subunits, namely ctl-ot 9 and et v (Hynes, 1992; Haas © The Rockefeller University Press, 0021-9525196101/239/11 $2.00 The Journal of Cell Biology, Volume 132, Numbers 1 & 2, January 1996 239-249 239 on N ovem er 9, 2017 jcb.rress.org D ow nladed fom and Plow, 1994). Among them, 0[2131 is a receptor for various collagen types and laminin, et313t binds to the arginineglycine-aspartic acid (RGD) sequence in fibronectin and laminin. 0t3131 interaction with native collagen is controversial and at best a weak interaction (Pfaff et al., 1993; Yamamoto and Yamamoto, 1994). et5131 is specific for the fibronectin RGD sequence. The expression of integrins is regulated by both growth factors and ECM proteins. For example, transforming growth factor-131 (TGF-131), released from platelets after wounding, upregulates 131 and 133 integrin receptors (Enenstein et al., 1992; Heino et al., 1989; Heino and Massague, 1989). PDGF-BB, also released from platelets, stimulates gene expression of integrin 131 in Swiss 3T3 cells (Bellas et al., 1991), et 5 and 133 in aortic smooth muscle cells (Janat et al., 1992) and ot 2 in human foreskin fibroblasts (Ahlen and Kristofer, 1994). Type I collagen upregulates o[ 2 integrin mRNA level in human fibroblast and melanoma cell lines (Klein et al., 1991). Thus, the level of specific integrins is probably controlled by interactive signals from both ECM molecules and growth factors in the pericellular environment. The global goal of our laboratory is to investigate how variations in ECM composition during wound healing correlates with changes in cell growth and migration. We hypothesized that the alteration in ECM during wound healing differentially regulates cell response to cytokines such as PDGF, resulting in a progression of cell phenotypes necessary for the ordered healing of wounds. Specifically, we hypothesize that fibrin and collagen differentially regulate integrin gene expression, which in turn affects how fibroblasts physically and biochemically relate to ECM. Multiple cycles of these events would lead to a progression of cell-ECM interaction. To test our hypothesis, in vitro systems to model different stages of wound repair were established. Relaxed collagen gels, stressed fibrin gels or fibronectin-rich cultures, and stressed collagen gels were used to model normal dermis, early wound provisional matrix, and late granulation tissue, respectively. Steady-state levels of specific integrin mRNAs, collagen integrin subunit, et 2, and provisional matrix integrin subunits, ot 3 and ets, were analyzed in these different ECM environments. By adding PDGF-BB to these culture systems, we found that fibrin gels and fibronectin-rich cultures supported induction of provisional matrix receptor ~3 and tx5 mRNAs while collagen gels facilitated induction of collagen receptor et 2 mRNAs and attenuated provisional matrix receptor responses. Thus fibroblasts surrounded by fibrin respond to PDGF by expressing receptors necessary for interaction with the wound provisional matrix. In contrast fibroblasts in collagen increase their collagen receptors and fail to support PDGF-induction of provisional matrix receptors. Finally, PDGF-BB was able to prolong half-lives of ~x 3 and ct 5 mRNAs in stressed fibrin gel. Collagen gels failed to support this response. In contrast, ~x 2 mRNA stability was greatly increased by PDGF-BB when the cells were cultured in collagen but not when cultured on plastic. These results suggest that coordinate signals from PDGF-BB and fibrin increase the epigenetic pressure in fibroblasts to express provisional matrix receptors which may be one of the inductive processes of granulation tissue formation. In addition, one new mechanism, modulation of integrin mRNA decay rate, is found to greatly alter integrin mRNA steady state. Materials and Methods