Regulation of PDGFC Signalling and Extracellular Matrix Composition by FREM1 in Mice

RESEARCH ARTICLE Disease Models & Mechanisms 6, 1426-1433 (2013) doi:10.1242/dmm.013748 Regulation of PDGFC signalling and extracellular matrix composition by FREM1 in mice Fenny Wiradjaja1, Denny L. Cottle1, Lynelle Jones1 and Ian Smyth1,2,* SUMMARY Fras1-related extracellular matrix protein 1 (FREM1) is required for epidermal adhesion during embryogenesis, and mice lacking the gene develop fetal skin blisters and a range of other developmental defects. Mutations in members of the FRAS/FREM gene family cause diseases of the Fraser syndrome spectrum. Embryonic epidermal blistering is also observed in mice lacking PdgfC and its receptor, PDGFRα. In this article, we show that FREM1 binds to PDGFC and that this interaction regulates signalling downstream of PDGFRα. Fibroblasts from Frem1-mutant mice respond to PDGFC stimulation, but with a shorter duration and amplitude than do wild-type cells. Significantly, PDGFC-stimulated expression of the metalloproteinase inhibitor Timp1 is reduced in cells with Frem1 mutations, leading to reduced basement membrane collagen I deposition. These results show that the physical interaction of FREM1 with PDGFC can regulate remodelling of the extracellular matrix downstream of PDGFRα. We propose that loss of FREM1 function promotes epidermal blistering in Fraser syndrome as a consequence of reduced PDGFC activity, in addition to its stabilising role in the basement membrane. DMM INTRODUCTION The FRAS/FREM family of proteins share characteristic The FRAS/FREM extracellular matrix (ECM) proteins (FRAS1, chondroitin sulphate proteoglycan (CSPG) core repeats similar FREM1 and FREM2) mediate adhesion between the epidermal to those found in the NG2 proteoglycan (Stallcup, 2002). In this basement membrane and the underlying dermis during embryonic protein, they directly bind platelet-derived growth factor A development (reviewed in Short et al., 2007; Petrou et al., 2008). (PDGFA), fibroblast growth factor FGF2, and collagens V and Consequently, their mutation results in the formation of fetal VI (Goretzki et al., 1999). Epidermal blistering defects like those epidermal blisters in a number of human conditions, including observed in FS syndrome and the blebs mice are also observed Fraser syndrome (FS; FREM2 and FRAS1 mutations), Manitoba in mice lacking either the growth factor PDGFC or its receptor, oculotrichoanal syndrome (MOTA) and bifid nose/anorectal and PDGFRα (Soriano, 1997; Tallquist and Soriano, 2003). PDGFC renal anomalies syndrome (BNAR) (both caused by mutations in is expressed in a number of developing epithelia, including the FREM1) (McGregor et al., 2003; Vrontou et al., 2003; Jadeja et al., epidermis, with complementary expression of PDGFRα observed 2005; Alazami et al., 2009; Vissers et al., 2011). These conditions in the associated underlying mesenchyme (Ding et al., 2000; encompass a wide range of developmental defects, of which Aase et al., 2002). PDGFC signalling acts upstream to drive cryptophthalmos, syndactyly, renal agenesis, ambiguous genitalia expression of matrix metalloproteinase-1 (MMP1) and its and respiratory tract defects are prominent. The FS-spectrum inhibitor, tissue metalloproteinase inhibitor 1 (TIMP1), in vitro diseases are phenocopied by the ‘blebs’ family of mouse mutants, and in transgenic overexpression models (Campbell et al., 2005; Disease Models & Mechanisms whose causative mutations lie in the mouse homologues of these Jinnin et al., 2005). FRAS/FREM genes (McGregor et al., 2003; Vrontou et al., 2003; Based on the observation that CSPG domains can interact with Smyth et al., 2004; Jadeja et al., 2005). FRAS1 and FREM2 are PDGF proteins and that epidermal blistering caused by Fras/Frem expressed exclusively by epidermal cells, whereas FREM1 is gene mutations in developing blebs mice is ultrastructurally, expressed in both the dermis and epidermis (Petrou et al., 2007; spatially and temporally similar to that observed in PdgfC Short et al., 2007). FRAS1, FREM1 and FREM2 are then thought mutants, we hypothesised that FREM1 might regulate the to interact in a complex to stabilize and cross-link epidermal capacity of PDGFC to regulate downstream remodelling of the basement membrane attachment to the developing dermis extracellular matrix (ECM). We show that FREM1 binds to (Kiyozumi et al., 2006). 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 1Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia demonstrate that this interaction regulates expression of 2Department of Anatomy and Developmental Biology, Monash University, metalloproteinase inhibitor Timp1 and collagen I deposition. We Wellington Road, Clayton, Victoria 3800, Australia therefore propose that FREM1 potentiates PDGFC signalling, *Author for correspondence ([email protected]) which in turn shapes ECM processing and composition during Received 25 July 2013; Accepted 14 August 2013 development. These observations provide a mechanistic basis for © 2013. Published by The Company of Biologists Ltd basement membrane fragility that leads to epidermal blistering This is an Open Access article distributed under the terms of the Creative Commons Attribution in FS-spectrum diseases and in the blebs mutant mice that model License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. them. 1426 dmm.biologists.org FREM1 regulates ECM via PDGFC signalling RESEARCH ARTICLE antibody was performed as a control and confirmed the specificity TRANSLATIONAL IMPACT of the rat anti-FREM1 signal (data not shown). Consistent with reported literature, we also observed PDGFC Clinical issue Fraser syndrome (FS), an autosomal recessive developmental disorder, is caused enriched in the epidermis and weakly in the dermis; however, most by mutations in members of the FRAS and FREM family of extracellular matrix significantly, we observed PDGFC on the basement membrane (ECM) proteins. The FRAS and FREM proteins are thought to function (Fig. 1B). Thus, there is substantial overlap in the localisation of collectively and cooperatively to structurally cross-link components of the FREM1 and PDGFC within keratinocytes, on the basement basement membrane in developing epithelia. FRAS and FREM mutations found membrane and within local ECM surrounding dermal fibroblasts. in individuals with FS (or in the blebs family of mouse mutants that phenocopy We did not see a change in the distribution of PDGFC in bat mice the disorder) lead to defects in epidermal adhesion and the formation of large (Fig. 1B). skin blisters in utero. These blisters are thought to contribute to the formation To further explore potential interactions between the proteins, of a number of craniofacial and soft tissue malformations, including the signature feature of FS, cryptophthalmos, in which skin covers the globe of the NIH3T3 cells were transfected with plasmid constructs encoding eye. Based on earlier studies conducted in mice, it was noted that mutation of full-length FREM1-FLAG (Fig. 1A) and PDGFC-V5. Detection of FREM1, one of the key proteins implicated in FS, leads to phenotypes that extracellular deposits of either factor was rare; however, FREM1- paralleled those caused by loss of platelet-derived growth factor receptor alpha FLAG localised with secretory vesicles marked by the ECM (PDGFR) signalling, and might therefore control ECM remodelling. However, component laminin γ1. PDGFC-V5 showed a broad intracellular this hypothesis has not been directly tested. distribution but was also enriched in laminin-γ1-positive vesicles, Results where it colocalised with FREM1-FLAG (Fig. 1C, see white boxes). The authors focussed on PDGFC, a key component of the PDGFR pathway. This indicates that FREM1 and PDGFC can colocalise Using immunoprecipitation, cell culture and transfection experiments in a intracellularly, as well as in ECM components in the absence of a mutant mouse line, they show that FREM1 binds to PDGFC in vitro and in vivo. basement membrane in vitro. They show that FREM1 colocalises with PDGFC in the basement membrane, in To test for a physical interaction between FREM1 and PDGFC, close proximity to PDGFR in dermal fibroblasts. Furthermore, they DMM HEK293 cells were transfected with constructs encoding full-length demonstrate that the interaction between PDGFC and FREM1 potentiates signalling via PDGFRThis signalling cascade controls expression of the matrix FREM1-FLAG and PDGFC-V5, or empty vector controls. metalloproteinase inhibitor TIMP1, which in turns plays an active Immunoprecipitation using a mouse V5 antibody revealed the developmental role in regulating ECM deposition, including that of collagen I, presence of FREM1-FLAG in the PDGFC-V5 immunoprecipitate, part of the foundation of the basement membrane. but not in the V5 control pull-down (Fig. 1D). FREM1-FLAG and Implications and future directions PDGFC-V5 ran as a 260-kDa and 55-kDa species, respectively, as This study provides some of the first evidence that FREM proteins bind to demonstrated in earlier studies (Li et al., 2000; Kiyozumi et al., additional ECM factors in the extracellular milieu. By establishing a link 2006). To determine which domain mediates binding to PDGFC, between FREM1, PDGFC growth factor

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