The FRAS/FREM extracellular matrix (ECM) proteins (FRAS1, FREM1 and FREM2) mediate adhesion between the epidermal basement membrane and the underlying dermis during embryonic development

1426 INTRODUCTION The FRAS/FREM extracellular matrix (ECM) proteins (FRAS1, FREM1 and FREM2) mediate adhesion between the epidermal basement membrane and the underlying dermis during embryonic development (reviewed in Short et al., 2007; Petrou et al., 2008). Consequently, their mutation results in the formation of fetal epidermal blisters in a number of human conditions, including Fraser syndrome (FS; FREM2 and FRAS1 mutations), Manitoba oculotrichoanal syndrome (MOTA) and bifid nose/anorectal and renal anomalies syndrome (BNAR) (both caused by mutations in FREM1) (McGregor et al., 2003; Vrontou et al., 2003; Jadeja et al., 2005; Alazami et al., 2009; Vissers et al., 2011). These conditions encompass a wide range of developmental defects, of which cryptophthalmos, syndactyly, renal agenesis, ambiguous genitalia and respiratory tract defects are prominent. The FS-spectrum diseases are phenocopied by the ‘blebs’ family of mouse mutants, whose causative mutations lie in the mouse homologues of these FRAS/FREM genes (McGregor et al., 2003; Vrontou et al., 2003; Smyth et al., 2004; Jadeja et al., 2005). FRAS1 and FREM2 are expressed exclusively by epidermal cells, whereas FREM1 is expressed in both the dermis and epidermis (Petrou et al., 2007; Short et al., 2007). FRAS1, FREM1 and FREM2 are then thought to interact in a complex to stabilize and cross-link epidermal basement membrane attachment to the developing dermis (Kiyozumi et al., 2006). The FRAS/FREM family of proteins share characteristic chondroitin sulphate proteoglycan (CSPG) core repeats similar to those found in the NG2 proteoglycan (Stallcup, 2002). In this protein, they directly bind platelet-derived growth factor A (PDGFA), fibroblast growth factor FGF2, and collagens V and VI (Goretzki et al., 1999). Epidermal blistering defects like those observed in FS syndrome and the blebs mice are also observed in mice lacking either the growth factor PDGFC or its receptor, PDGFRα (Soriano, 1997; Tallquist and Soriano, 2003). PDGFC is expressed in a number of developing epithelia, including the epidermis, with complementary expression of PDGFRα observed in the associated underlying mesenchyme (Ding et al., 2000; Aase et al., 2002). PDGFC signalling acts upstream to drive expression of matrix metalloproteinase-1 (MMP1) and its inhibitor, tissue metalloproteinase inhibitor 1 (TIMP1), in vitro and in transgenic overexpression models (Campbell et al., 2005; Jinnin et al., 2005). Based on the observation that CSPG domains can interact with PDGF proteins and that epidermal blistering caused by Fras/Frem gene mutations in developing blebs mice is ultrastructurally, spatially and temporally similar to that observed in PdgfC mutants, we hypothesised that FREM1 might regulate the capacity of PDGFC to regulate downstream remodelling of the extracellular matrix (ECM). We show that FREM1 binds to PDGFC in vitro and in vivo and demonstrate in mouse embryonic fibroblasts (MEFs) that wild-type (WT) FREM1 maintains the normal duration and amplitude of PI3-kinase (PI3K)/AKT and MAPK activation following PDGFC stimulation. We further demonstrate that this interaction regulates expression of metalloproteinase inhibitor Timp1 and collagen I deposition. We therefore propose that FREM1 potentiates PDGFC signalling, which in turn shapes ECM processing and composition during development. These observations provide a mechanistic basis for basement membrane fragility that leads to epidermal blistering in FS-spectrum diseases and in the blebs mutant mice that model them. Disease Models & Mechanisms 6, 1426-1433 (2013) doi:10.1242/dmm.013748