Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy

Human Molecular Genetics, 2016, Vol. 25, No. 6 1176–1191 doi: 10.1093/hmg/ddw001 Advance Access Publication Date: 11 January 2016 Original Article ORIGINAL ARTICLE Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy 1 School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK, 2Centre for Dermatology and Genetic Medicine, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Scotland DD1 5EH, UK, 3Microscopy Facility, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK, 4Cathedral Eye Clinic, Academy Street, Belfast BT15 1ED, UK, 5Department of Molecular Biology and Genetics, 6Interdisciplinary Nanoscience Center (iNANO) and Center for Insoluble Protein Structures (inSPIN), Science Park, Aarhus University, Aarhus, Denmark and 7Department of Ophthalmology, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK *To whom correspondence should be addressed. Tel: +44 2870124577; Fax: +44 28703324915; Email: (C.B.T.M.); Tel: +44 1382381047; Fax: +44 1382388535; Email: (W.H.I.M.) Abstract Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro † The authors wish it to be known that, in their opinion, the first two authors should be regarded as co-first authors and the last two authors should be regarded as co-corresponding authors. ‡ Present Address: Northern Ireland Centre for Stratified Medicine, University of Ulster, C-TRIC Building, Altnagelvin Hospital campus, Glenshane Road, Londonderry, BT47 6SB, UK. ¶ Present Address: Department of Dermatology, University of Zürich Hospital, CH-8952 Zürich-Schlieren, Switzerland. Received: October 16, 2015. Revised and Accepted: January 4, 2016 © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1176 Edwin H.A. Allen1,2,†, David G. Courtney1,†, Sarah D. Atkinson1,‡, Johnny E. Moore1,4, Laura Mairs1, Ebbe Toftgaard Poulsen5, Davide Schiroli1, Eleonora Maurizi1, Christian Cole2, Robyn P. Hickerson2, John James3, Helen Murgatroyd7, Frances J.D. Smith2, Carrie MacEwen7, Jan J. Enghild5,6, M. Andrew Nesbit1, Deena M. Leslie Pedrioli2,¶, W.H. Irwin McLean2, * and C.B. Tara Moore1, * Human Molecular Genetics, 2016, Vol. 25, No. 6 | 1177 MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations. Introduction Results Generation of humanized KRT12 mutant knock-in mice A targeting vector was generated that contained the full human KRT12 gene including introns, untranslated regions (UTRs) and a 247 bp sequence downstream of the KRT12 3′-UTR. Exon 1 carried the L132P mutation, and a FLAG-HA coding sequence (21) was placed immediately before the termination codon. A multiple targeting cassette (MTC) was inserted into the KRT12 3′-UTR, consisting of target sequences for four MECD mutations (Supplementary Material, Fig. S1). The targeting strategy is shown in Figure 1. This targeting vector was used to introduce the human Leu132Pro KRT12 mutant allele (KRT12-L132P) into the murine Krt12 locus by homologous recombination in mouse embryonic stem cells, which were used to generate transgenic mice on the C57BL/6 background (Fig. 1). Germline transmission of the targeted allele was confirmed by polymerase chain reaction (PCR) (Supplementary Material, Fig. S4), and recombinant mutant mice were bred with transgenic C57BL/6 mice universally expressing Flp-deleter recombinase to excise the NeoR and PuroR cassettes (Fig. 1) (22). The resultant KRT12-L132P-positive, NeoR/PuroR-negative mouse line was designated Krt12 hL132P. Heterozygous (Krt12 +/hL132P) and homozygous (Krt12 hL132P/hL132P) mutant mice were viable and fertile with no superficial corneal abnormalities (data not shown). Characterization of heterozygous (Krt12 +/hL132P) and homozygous (Krt12 hL132P/hL132P) corneal epithelium Mice of each genotype [wild-type (WT), heterozygous, homozygous; n = 6] were assessed for gross changes in corneal morphology by slit-lamp evaluation at 8, 12, 16 and 30 weeks. No Corneal dystrophies are a group of blinding heritable conditions, with a prevalence of approximately one in 2000 (1). They are noninflammatory conditions restricted to the cornea and usually result in a loss of corneal transparency (2). They have been classified into four separate anatomically defined groups: epithelial and subepithelial dystrophies, epithelial–stromal transforming growth factor beta-induced (TGFBI) dystrophies, stromal dystrophies and endothelial dystrophies (3). Genetic analysis of familial cases has revealed corneal dystrophic mutations arising in TGFBI, KRT3, KRT12, TACSTD2, CHST6, UBIAD1, DCN, PIKFYVE, COL8A2, COL17A1, ZEB1 and SLC4A11 genes (3,4). Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant hereditary disorder of the anterior corneal epithelium. Typically, MECD is characterized by intra-epithelial microcysts in the central cornea, with occasional cases exhibiting grey serpiginous lines. In most cases, MECD remains asymptomatic and is only detected in (...truncated)