ORIGINAL ARTICLE BPAG1-e Restricts Keratinocyte Migration through Control of Adhesion Stability Magdalene Michael1,2,3,RumenaBegum1,2,KennethFong2, Celine Pourreyrone4,AndrewP.South4, John A. McGrath2 and Maddy Parsons1 Bullous pemphigoid antigen 1 (BPAG1-e, also known as BP230) is a member of the plakin family of hemidesmosome cytoskeletal linker proteins that is encoded by an isoform of the dystonin (DST) gene. Recently, we reported two unrelated families with homozygous nonsense mutations in this DST isoform that led to ultrastructural loss of hemidesmosomal inner plaques and clinical features of trauma-induced skin fragility. We now demonstrate that keratinocytes isolated from these individuals have significant defects in adhesion, as well as increased cell spreading and migration. These mutant keratinocytes also display reduced levels of b4 integrins at the cell surface but increased total protein levels of keratin-14 and b1 integrins. These alterations in cell behavior and protein expression were not seen in control keratinocytes in which BPAG1-e expression had been silenced by stable expression of short hairpin RNA to target DST. The failure of knockdown approaches to recapitulate the changes in morphology, adhesion, and migration seen in patient cells therefore suggests such approaches are not appropriate to study loss of this protein in vivo. The contrasting findings in keratinocytes harboring naturally occurring mutations, however, demonstrate a previously unappreciated key role for BPAG1-e in regulating keratinocyte adhesion and migration and suggest a requirement for this protein in controlling functional switching between integrin types in epithelial cells. Journal of Investigative Dermatology (2014) 134, 773–782; doi:10.1038/jid.2013.382; published online 17 October 2013 INTRODUCTION known as BP230), a key component of hemidesmosomes Epidermolysis bullosa (EB) represents a diverse collection of and a member of the plakin family with cytoskeletal linker autosomal dominant and autosomal recessive conditions with properties (Borradori and Sonnenberg, 1999; Leung et al., varying degrees of trauma-induced skin and mucous membrane 2001a; Litjens et al., 2006; Sonnenberg and Liem, 2007). fragility and, in some cases, extracutaneous abnormalities (Fine BPAG1-e is encoded by the dystonin (DST) gene, the et al., 2008; Fine and Mellerio, 2009a, b). The disease spectrum alternative splicing of which may give rise to multiple tissue of EB (or EB-like disorders) encompasses inherited disorders of isoforms with variable expression in the skin, neurons, hemidesmosome attachment complexes, keratin intermediate muscles, and the central nervous system (Leung et al., filaments, focal adhesions, desmosome cell junctions, keratino- 2001b; Jefferson et al., 2006; Young and Kothary, 2007, cyte vesicle transport, and enzymes involved in cornification, 2008). The main isoform expressed in the central nervous and currently involves pathogenic mutations in at least 18 system is BPAG1-a, although a further neural variant, BPAG1- different genes (Bruckner-Tuderman et al., 2013). n, may also exist; whether BPAG1-n is expressed in vivo, One protein that was recently implicated in the pathogen- however, is uncertain (Leung et al., 2001b). In muscle, the esis of EB, is bullous pemphigoid antigen 1 (BPAG1-e, also main isoform is BPAG1-b. In the skin, there is a predominance of BPAG1-e, and some BPAG1-a and perhaps BPAG1-n may 1Randall Division of Cell and Molecular Biophysics, King’s College London, also be present (Sonnenberg and Liem, 2007). Guy’s Campus, London, UK; 2St Johns Institute of Dermatology, King’s College We recently reported two unrelated cases with a form of London, Guys Campus, London, UK; 3Division of Molecular Cell Biology, autosomal recessive EB simplex because of homozygous Institute for Molecular Bioscience, The University of Queensland, St Lucia, nonsense mutations in BPAG1-e (Groves et al., 2010; Liu Brisbane, Queensland, Australia and 4Jacqui Wood Cancer Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK et al., 2012). The mutations detected were c.3478C4Tand Correspondence: Maddy Parsons, Randall Division of Cell and Molecular c.3853A4T (GenBank NM_001723.4), which result in the Biophysics, King’s College London, Guy’s Campus, London SE11UL, UK. amino-acid changes p.Gln1124X and p.Arg1249X, E-mail: [email protected] respectively. Clinically, affected individuals had trauma- Abbreviations: BPAG1, bullous pemphigoid antigen 1; DST, dystonin; EB, induced blistering (generalized but mainly on the feet), and epidermolysis bullosa; KD, knockdown; K14, keratin 14; PBS, phosphate- skin biopsy revealed a lack of hemidesmosomal inner plaques buffered saline; shRNA, short hairpin RNA; WT, wild type on transmission electron microscopy and a complete absence Received 31 May 2013; revised 12 August 2013; accepted 27 August 2013; accepted article preview online 11 September 2013; published online of immunostaining for the BPAG1-e protein using two 17 October 2013 isoform-specific monoclonal antibodies. & 2014 The Society for Investigative Dermatology www.jidonline.org 773 MMichaelet al. BPAG1-e Regulation of Keratinocyte Migration In contrast to the clinicopathologic consequences of homo- antibody used to detect BPAG1-e in these experiments zygous nonsense mutations in BPAG1-e, a recent study recognized an epitope within the C-terminal region of the demonstrated that depleting BPAG1-e levels in keratinocytes protein, beyond the mutation site identified in both DST resulted in rather subtle defects in cell polarity and no patients. Owing to the lack of reliable antibodies specifically apparent changes in migration speed or adhesion (Hamill recognizing BPAG1-e N-terminal epitopes, we were unable to et al., 2009). Thus, the role of BPAG1-e in controlling various determine whether a truncated version of the BPAG1-e protein aspects of keratinocyte behavior is unclear. Here, we show remained in DST1 and DST2 patient cells. Further analysis of that cells derived from two individuals with loss-of-function these cells was conducted using in vitro organotypic assays in mutations in DST show significant defects in cell morphology, which keratinocytes are grown on top of collagen/Matrigel adhesion, and migration. Contrary to previous knockdown three-dimensional scaffolds containing human fibroblasts and (KD) studies, however, these cells show increased migration cultured at an air–liquid interface for 2 weeks to mimic a that results from a loss of surface b4 integrin and increased tissue-like environment (Nystrom et al., 2005). Analysis of active b1 integrin. Our findings confirm a key role for organotypic cultures post-fixation revealed a flattened BPAG1-e in controlling keratinocyte adhesion and motility morphology of basal keratinocytes in contact with the under- and further suggest this protein has an important role lying basement membrane in both DST patients compared in coordinating maturation of hemidesmosomes and with controls (Supplementary Figure S1i online). However, suppression of focal adhesion dynamics. there were no gross changes in the localization of differentia- tion markers observed (Supplementary Figure S1j online), in RESULTS agreement with previous findings in the patient skin. These DST mutant keratinocytes display altered adhesion and data demonstrate that loss of BPAG1-e expression in kerati- migration nocytes leads to alterations in basal keratinocyte morphology As the DST gene is known to give rise to multiple isoforms, that may be a consequence of altered stability or adhesion to gene expression analysis of known tissue-specific isoforms basement membrane. were analyzed by quantitative PCR in wild-type (WT) kerati- In order to analyze these potential attachment defects in nocytes. In comparison with total DST gene expression (total more detail, we quantified adhesion of WT or DST mutant BPAG1), nearly undetectable levels of the muscle-specific and keratinocytes to the basement membrane matrix Matrigel. neuronal isoforms, BPAG1-b and BPAG1-a, respectively were Both DST mutant keratinocyte cell lines showed a significant detected; however, high expression of BPAG1-e was observed delay in adhesion at 30 and 60 minutes post-plating compared confirming it as the predominant BPAG1 isoform expressed in with control cells (Figure 1a and b). Similarly, analysis of cell keratinocytes (Supplementary Figure S1a online). Comparison detachment demonstrated a significant increase in DST mutant of WT keratinocytes with those isolated from the first DST cell detachment rates compared with controls, suggesting that patient (DST1) revealed a reduction in total BPAG1 isoform adhesions formed in these cells are less stably connected to expression as well as a significant loss of BPAG1-e isoform the substratum (Figure 1c). Adhesion strengthening is known expression in DST1 keratinocytes (Supplementary Figure S1b to be required for normal keratinocyte migration (Margadant and c online) confirming the impact of the homozygous et al., 2010). We therefore analyzed random migration nonsense mutation on BPAG1-e isoform expression. To con- behavior of control and DST mutant cells plated at low firm the loss of BPAG1-e in both individuals with loss-of- density by time-lapse microscopy. Tracking single cells over function mutations in DST and assess potential changes in time revealed an unexpected increase in migration speed and adhesion protein localization, immunostaining for BPAG1-e, directional migration persistence in both DST