EGF-R Regulates MMP Function in Fibroblasts Through MAPK and AP-1 Pathways RISTO KAJANNE, 1,2 PA ¨ IVI MIETTINEN, 3,4 ANNIKA MEHLEM, 1,2 SUVI-KATRI LEIVONEN, 5,6 MICHAEL BIRRER, 7 MARCO FOSCHI, 8 VELI-MATTI KA ¨ HA ¨ RI, 5,6 AND SIRPA LEPPA ¨ 1,2 * 1 Molecular Cancer Biology Research Program, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland 2 Department of Oncology, Helsinki University Central Hospital, Helsinki, Finland 3 Program of Developmental and Reproductive Biology, Biomedicum Helsinki, University of Helsinki, Finland 4 Hospital for Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland 5 Department of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland 6 Department of Dermatology, University of Turku, Turku, Finland 7 Cell and Cancer Biology Department, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 8 Department of Internal Medicine, University of Florence, Florence, Italy EGF-R regulates cell proliferation, migration, and invasion in fibroblasts. However, the connection of EGF-R with downstream signaling pathways mediating these responses has remained elusive. Here we provide genetic and biochemical evidence that EGF-R- and AP-1- mediated signals are required for MMP expression and collagen contraction in fibroblasts. In EGF-R (/) mouse embryonal fibroblasts, basal and inducible expression of several MMPs, including MMP-2, -3, and -14 is impaired in comparison to wild-type counterparts. The loss of MMP expression is associated with a suppression of EGF-induced Erk and Jnk activities, and AP-1 DNA-binding and transactivation capacities. While inhibition of Jnk mainly prevents EGF-induced phosphorylation of c-Jun, inhibition of Erk pathway suppresses both the expression and phosphorylation of c-Jun and c-Fos proteins. Moreover, the expression of MMP-3 and -14, and collagen contraction is partially prevented by Mek/Erk and Jnk inhibitors. However, Jnk inhibitor also suppresses cell growth independently of EGF-R activity. The central role of AP-1 as a mediator of EGF-R signaling in fibroblasts is emphasized by the finding that expression of a dominant negative c-Jun downregulates the expression of MMP-3. Conversely, expression of a constitutively active Mek1 can induce MMP-3 expression independently of upstream signals. The results indicate that ERK pathway and AP-1 are downstream effectors of the EGF-R-mediated MMP-3 expression and collagen contraction in fibroblasts. J. Cell. Physiol. 212: 489–497, 2007. ß 2007 Wiley-Liss, Inc. ErbB family of tyrosine kinase receptors are key regulators of cells by engaging growth factors and triggering downstream signaling events that govern cell proliferation, survival, differentiation, and migration (Prenzel et al., 2001; Yarden and Sliwkowski, 2001). The first identified ErbB family member, epidermal growth factor receptor (EGF-R), is an essential tyrosine kinase receptor for both epithelial cells and fibroblasts. Interestingly, fibroblast mobility and proliferation are regulated to a large extent by epidermal growth factor (EGF), transforming growth factor-a (TGF-a), and heparin-binding EGF-like growth factor (HB-EGF), which are ligands for EGF-R (Chen et al., 1994a,b, 1996; Xie et al., 1998). These factors are known to participate in all stages of wound healing (Wells et al., 1998). Binding of EGF-superfamily growth factors to EGF-R activates four major pathways, that is, the phosphatidylinositol-3 kinase (PI3 kinase), signal transducer and activator of transcription (STAT), phospholipase C-protein kinase C (PLC-PKC), and Ras-mitogen-activated protein kinase (Ras-MAPK) pathways (Prenzel et al., 2001; Yarden and Sliwkowski, 2001). All these pathways have been implicated in growth control and survival. In addition, EGF-R-mediated activation of PLC and MAPKs have been linked to migration and invasion (Chen et al., 1994b; Xie et al., 1998; Cheresh et al., 1999; Glading et al., 2000). In this study, we have investigated the role of MAPKs in EGF-R-mediated signaling in fibroblasts. Major MAPK pathways consist of extracellular signal-regulated kinases (Erk1/2), c-Jun N-terminal kinases (Jnks) and p38s. Erk-type MAPKs are mainly activated by mitogens and growth factors during cell proliferation and differentiation, whereas Jnks and p38 MAPKs, which belong to the stress-activated protein kinases (SAPKs), are activated in response to inflammatory cytokines, ultraviolet irradiation, heat or osmotic shock (Chang and Karin, 2001). More recently, it has been shown, however, that Jnk and p38 MAPKs also play a role in development, proliferation, differentiation and survival (Davis, 2000; Nebreda and Porras, 2000). The biological effects of MAPKs are mediated by downstream phosphorylation of substrates, which in the nucleus are often transcription factors. One of the major downstream effectors for MAPKs is the signal inducible transcription factor activator protein-1 (AP-1) (Whitmarsh and Davis, 1996). Originally, AP-1 was characterized as a ‘‘TPA-inducible’’ factor, which regulates *Correspondence to: Sirpa Leppa ¨, Department of Oncology, Helsinki University Central Hospital, P.O. Box 180, FIN-00029 Helsinki, Finland. E-mail: sirpa.leppa@helsinki.fi Received 27 December 2006; Accepted 4 January 2007 DOI: 10.1002/jcp.21041 ORIGINAL ARTICLE 489 ß 2007 WILEY-LISS, INC.