Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) loss causes neurodegeneration by altering protein turnover in the first postnatal weeks.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

Tripartite Separation of Glomerular Cell Types and Proteomes from Reporter-Free Mice.

BACKGROUND: The glomerulus comprises podocytes, mesangial cells, and endothelial cells, which jointly determine glomerular filtration. Understanding this intricate functional unit beyond the transcriptome requires bulk isolation of these cell types for biochemical investigations. We developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). METHODS: We separated glomerular cell types from wild-type or mT/mG mice via a novel FACS approach, and validated their purity. Cell type proteomes were compared between strains, ages, and sex. We applied timMEP to the podocyte-targeting, immunologic, THSD7A-associated, model of membranous nephropathy. RESULTS: timMEP enabled protein-biochemical analyses of podocytes, mesangial cells, and endothelial cells derived from reporter-free mice, and allowed for the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. We identified marker proteins for mesangial and endothelial proteins, and outlined protein-based, potential communication networks and phosphorylation patterns. The analysis detected cell type-specific proteome differences between mouse strains and alterations depending on sex, age, and transgene. After exposure to anti-THSD7A antibodies, timMEP resolved a fine-tuned initial stress response, chiefly in podocytes, that could not be detected by bulk glomerular analyses. The combination of proteomics with super-resolution imaging revealed a specific loss of slit diaphragm, but not of other foot process proteins, unraveling a protein-based mechanism of podocyte injury in this animal model. CONCLUSION: timMEP enables glomerular cell type-resolved investigations at the transcriptional and protein-biochemical level in health and disease, while avoiding reporter-based artifacts, paving the way toward the comprehensive and systematic characterization of glomerular cell biology.

ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage.

BACKGROUND: Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury. METHODS: Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, in vitro and in vivo, in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice. RESULTS: ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner. CONCLUSIONS: ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.

Evaluating a Targeted Cancer Therapy Approach Mediated by RNA trans-Splicing In Vitro and in a Xenograft Model for Epidermolysis Bullosa-Associated Skin Cancer.

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.

Deubiquitinating Enzyme UCH-L1 Promotes Dendritic Cell Antigen Cross-Presentation by Favoring Recycling of MHC Class I Molecules.

The deubiquitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) is required for the maintenance of axonal integrity in neurons and is thought to regulate the intracellular pool of ubiquitin in the brain. In this study, we show that UCH-L1 has an immunological function in dendritic cell (DC) Ag cross-presentation. UCH-L1 is expressed in mouse kidney, spleen, and bone marrow-derived DCs, and its expression and activity are regulated by the immune stimuli LPS and IFN-γ. UCH-L1-deficient mice have significantly reduced ability to cross-prime CD8 T cells in vivo and in vitro because of a reduced ability of DCs to generate MHC class I (MHC I) peptide complexes for cross-presented Ags. Mechanistically, Ag uptake by phagocytosis and receptor-mediated endocytosis as well as phagosome maturation are unaffected by loss of UCH-L1 in DCs. Rather, MHC I recycling is reduced by loss of UCH-L1, which affects the colocalization of intracellular MHC I with late endosomal/lysosomal compartments necessary for cross-presentation of Ag. These results demonstrate a hitherto unrecognized role of the deubiquitinating enzyme UCH-L1 in DC Ag processing.

A cancer stem cell-like phenotype is associated with miR-10b expression in aggressive squamous cell carcinomas.

BACKGROUND: Cutaneous squamous cell carcinomas (cSCC) are the primary cause of premature deaths in patients suffering from the rare skin-fragility disorder recessive dystrophic epidermolysis bullosa (RDEB), which is in marked contrast to the rarely metastasizing nature of these carcinomas in the general population. This remarkable difference is attributed to the frequent development of chronic wounds caused by impaired skin integrity. However, the specific molecular and cellular changes to malignancy, and whether there are common players in different types of aggressive cSCCs, remain relatively undefined. METHODS: MiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimized in situ hybridization protocol. Functional impact of overexpression or knock-out of a dysregulated miRNA was assessed in migration and 3D-spheroid assays. Sample-matched transcriptome data was generated to support the identification of disease relevant miRNA targets. RESULTS: Several miRNAs were identified as dysregulated in cSCCs compared to control skin. These included the metastasis-linked miR-10b, which was significantly upregulated in primary cell cultures and in archival biopsies. At the functional level, overexpression of miR-10b conferred the stem cell-characteristic of 3D-spheroid formation capacity to keratinocytes. Analysis of miR-10b downstream effects identified a novel putative target of miR-10b, the actin- and tubulin cytoskeleton-associated protein DIAPH2. CONCLUSION: The discovery that miR-10b mediates an aspect of cancer stemness - that of enhanced tumor cell adhesion, known to facilitate metastatic colonization - provides an important avenue for future development of novel therapies targeting this metastasis-linked miRNA.

Arginine- but not alanine-rich carboxy-termini trigger nuclear translocation of mutant keratin 10 in ichthyosis with confetti.

Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant-negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)-terminus, from poly-glycine to either a poly-arginine or -alanine tail. Previous studies on the type of C-terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C-termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C-terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine-rich C-terminus. However, this was not observed with K10 featuring an alanine-rich C-terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild-type and truncated forms of K10. This study demonstrates that, of the various 3' frameshift variants of KRT10, exclusively arginine-rich C-termini lead to nuclear localization of K10.

Epidermolysis bullosa: Advances in research and treatment.

Epidermolysis bullosa (EB) is the umbrella term for a group of rare inherited skin fragility disorders caused by mutations in at least 20 different genes. There is no cure for any of the subtypes of EB resulting from different mutations, and current therapy only focuses on the management of wounds and pain. Novel effective therapeutic approaches are therefore urgently required. Strategies include gene-, protein- and cell-based therapies. This review discusses molecular procedures currently under investigation at the EB House Austria, a designated Centre of Expertise implemented in the European Reference Network for Rare and Undiagnosed Skin Diseases. Current clinical research activities at the EB House Austria include newly developed candidate substances that have emerged out of our translational research initiatives as well as already commercially available medications that are applied in off-licensed indications. Squamous cell carcinoma is the major cause of death in severe forms of EB. We are evaluating immunotherapy using an anti-PD1 monoclonal antibody as a palliative treatment option for locally advanced or metastatic squamous cell carcinoma of the skin unresponsive to previous systemic therapy. In addition, we are evaluating topical calcipotriol and topical diacerein as potential agents to improve the healing of skin wounds in EBS patients. Finally, the review will highlight the recent advancements of gene therapy development for EB.

An RNA-targeted therapy for dystrophic epidermolysis bullosa.

Functional impairment or complete loss of type VII collagen, caused by mutations within COL7A1, lead to the severe recessive form of the skin blistering disease dystrophic epidermolysis bullosa (RDEB). Here, we successfully demonstrate RNA trans-splicing as an auspicious repair option for mutations located in a wide range of exons by fully converting an RDEB phenotype in an ex vivo pre-clinical mouse model based on xenotransplantation. Via a self-inactivating (SIN) lentiviral vector a 3' RNA trans-splicing molecule, capable of replacing COL7A1 exons 65-118, was delivered into type VII collagen deficient patient keratinocytes, carrying a homozygous mutation in exon 80 (c.6527insC). Following vector integration, protein analysis of an isolated corrected single cell clone showed secretion of the corrected type VII collagen at similar levels compared to normal keratinocytes. To confirm full phenotypic and long-term correction in vivo, patches of skin equivalents expanded from the corrected cell clone were grafted onto immunodeficient mice. Immunolabelling of 12 weeks old skin specimens showed strong expression of human type VII collagen restricted to the basement membrane zone. We demonstrate that the RNA trans-splicing technology combined with a SIN lentiviral vector is suitable for an ex vivo molecular therapy approach and thus adaptable for clinical application.

Ubiquitin C-Terminal Hydrolase L1 is required for regulated protein degradation through the ubiquitin proteasome system in kidney.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.

Gene editing for skin diseases: designer nucleases as tools for gene therapy of skin fragility disorders.

NEW FINDINGS: What is the topic of this review? This review concerns current gene editing strategies for blistering skin diseases with respect to individual genetic constellations and distinct conditions. What advances does it highlight? Specificity and safety dominate the discussion of gene editing applications for gene therapy, where a number of tools are implemented. Recent developments in this rapidly progressing field pose further questions regarding which tool is best suited for each particular use. The current treatment of inherited blistering skin diseases, such as epidermolysis bullosa (EB), is largely restricted to wound care and pain management. More effective therapeutic strategies are urgently required, and targeting the genetic basis of these severe diseases is now within reach. Here, we describe current gene editing tools and their potential to correct gene function in monogenetic blistering skin diseases. We present the features of the most frequently used gene editing techniques, transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), determining their preferential application for specific genetic conditions, including the type of mutational inheritance, the targeting site within the gene or the possibility to target the mutation specifically. Both tools have traits beneficial in specific situations. Promising developments in the field engender gene editing as a potentially powerful therapeutic option for future clinical applications.

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases.

With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.

RNA Trans-Splicing Modulation via Antisense Molecule Interference.

In recent years, RNA trans-splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher trans-splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA trans-splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the trans-splicing efficiency. Thus, we assume that besides the RNA trans-splicing molecule, short antisense molecules can act as splicing modulators, thereby increasing the trans-splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases.

The formation of endoderm-derived taste sensory organs requires a Pax9-dependent expansion of embryonic taste bud progenitor cells.

In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair.

RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3' and 5' RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders.

Deletion of K1/K10 does not impair epidermal stratification but affects desmosomal structure and nuclear integrity.

Keratins K1 and K10 are the most abundant proteins in the upper epidermis where they polymerize to form intermediate filaments (IFs). In addition to their well-established function in providing epidermal stability, K1/K10 (i.e. the dimer between K1 and K10) IFs are supposed to be important for terminal epidermal differentiation and barrier formation. It was previously shown that the imbalanced deletion of one of the partner keratins, K10, disturbed epidermal homoeostasis, although stability was provided by compensatory upregulation of K5/K14, which formed IFs together with the remaining K1. Here, we show that deletion of both partner keratins, K1 and K10, results in lethal postnatal skin fragility in mice. Krt1(-/-);Krt10(-/-) mice revealed that K1/K10 IFs are unexpectedly dispensable for epidermal stratification. Although the stratum corneum was less compact and cornified envelope differentiation was impaired, a dye exclusion assay showed that the development of a functional water barrier was surprisingly independent from the presence of K1/K10 IFs. The deletion of K1/K10 was not compensated by any other keratin pair such as the basal epidermal keratins K5/K14, and electron microscopy revealed total absence of IFs in the suprabasal epidermis. Although plakoglobin was unchanged, the expression of the desmosomal proteins desmoplakin, desmocollin 1 and desmoglein 1 were altered and suprabasal desmosomes were smaller in Krt1(-/-);Krt10(-/-) than in wild-type epidermis suggesting an involvement of K1/K10 IFs in desmosome dynamics. Furthermore, Krt1(-/-);Krt10(-/-) mice showed premature loss of nuclei during epidermal differentiation and lower levels of emerin, lamin A/C and Sun1, revealing a previously unknown function for IFs in maintaining nuclear integrity in the upper epidermis.

Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.

Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. KRT1 mutations cause epidermolytic ichthyosis in humans, characterized by loss of barrier integrity and recurrent erythema. In search of the largely unknown pathomechanisms and the role of keratins in barrier formation and inflammation control, we show here that Krt1 is crucial for maintenance of skin integrity and participates in an inflammatory network in murine keratinocytes. Absence of Krt1 caused a prenatal increase in interleukin-18 (IL-18) and the S100A8 and S100A9 proteins, accompanied by a barrier defect and perinatal lethality. Depletion of IL-18 partially rescued Krt1(-/-) mice. IL-18 release was keratinocyte-autonomous, KRT1 and caspase-1 dependent, supporting an upstream role of KRT1 in the pathology. Finally, transcriptome profiling revealed a Krt1-mediated gene expression signature similar to atopic eczema and psoriasis, but different from Krt5 deficiency and epidermolysis bullosa simplex. Our data suggest a functional link between KRT1 and human inflammatory skin diseases.