Mutant Keratin Research Articles

597 Interim results of a phase I/II, closed label, randomized pilot study for the safety and efficacy of TolaSure gel, 5% w/w targeting aggregated mutant keratin in severe epidermolysis bullosa simplex (TAMES)

597 Interim results of a phase I/II, closed label, randomized pilot study for the safety and efficacy of TolaSure gel, 5% w/w targeting aggregated mutant keratin in severe epidermolysis bullosa simplex (TAMES)

580 Vers la compréhension du lien entre le profil des cytokines et les phénotypes pathologiques associés à l’épidermolyse bulleuse simple à l’aide de cellules souches pluripotentes induites

Epidermolysis bullosa simplex (EBS) is a skin disorder caused by dominant missense mutations in KERATIN5 (KRT5) or KERATIN14 (KRT14) gene, which encode intermediate filament (IF) proteins mainly expressed in epidermal basal keratinocytes. Incorporation of mutant keratins in network of IF makes the network sensitive to stress inducing inflammation and skin blistering formation. Currently, no therapeutic approach is known. Significant cytokine imbalance in blister fluid samples from different patients was demonstrated, suggesting that EB could be considered as inflammatory disorder rather than skin disorder.

Interleukin-18 as a severity marker and novel potential therapeutic target for epidermolytic ichthyosis.

Epidermolytic ichthyosis (EI) is a major form of nonsyndromic inherited ichthyosis, characterized by erythroderma, marked hyperkeratosis and scale, bulla and erosion at birth, associated with KRT1/KRT10 mutations. The cytokine and chemokine profiles in EI are poorly understood, and specific treatment options have not been established. To explore novel biomarkers and therapeutic targets in patients with EI. We analysed cytokine levels in serum and skin samples from 10 patients with inherited ichthyosis, including seven patients with EI. Wild-type and mutant KRT1 constructs were established and transfected into HaCaT cells, an immortalized keratinocyte cell line, for in vitro immunoblotting and immunocytochemistry analyses. Multiplex cytokine/chemokine analysis revealed that 10 cytokines/chemokines [interleukin (IL)-1β, IL-4, IL-17A, IL-16, IL-18, IL-1 receptor-α, macrophage colony-stimulating factor, interferon-α2, basic fibroblast growth factor and monocyte chemotactic protein-3] were significantly increased in patients with EI. Furthermore, IL-18 levels were significantly higher in patients with EI [n = 7; 2714.1 (1438.0) pg mL-1] than in healthy controls [n = 11; 218.4 (28.4) pg mL-1, P < 0.01]. Immunohistochemical analyses showed that IL-18 expression was elevated in skin samples from patients with EI. Serum IL-18 levels correlated with the severity of ichthyosis, as measured by the Ichthyosis Scoring System. Immunoblotting analysis revealed that mature IL-18 levels were increased in the supernatant of mutant KRT1 expressing HaCaT cells. Additionally, these cells showed NLRP3 aggregation in the cytoplasm and ASC clustered around mutant keratin aggregations. These findings suggest that mutant keratin might promote the activation of the NLRP3 inflammasome and its downstream caspase-1-mediated IL-18 release in keratinocytes from patients with EI. Our results suggest that serum IL-18 is a severity marker released from the skin of patients with EI. Blockade of IL-18 may be a useful novel therapeutic option for patients with EI.

586 New class of mTOR inhibitor stabilizes intermediate filament networks in severe epidermolysis bullosa simplex keratinocytes

Epidermolysis Bullosa Simplex (EBS) is a rare genodermatosis caused by mutations in either KRT5 or KRT14. Severe EBS (i.e., EBS Dowling-Meara) is characterized by extremely fragile skin with frequent and severe herpetiform blistering. Genetic mutations cause poor keratin 5/14 dimerization resulting in mechanically weakened intermediate filament (IF) networks that collapse into intracellular aggregates upon stress (e.g., scratching, UV). Enhanced cellular removal and degradation of mutant keratins through stimulation of autophagy and the UPS system has the potential to strengthen the IF networks, reducing blistering and erosions. Previously, UPS cochaperones (e.g., 4-phenylbuterate) were shown to decreased keratin aggregation but also decreased keratinocyte adhesion and migration. The purpose of this research was to evaluate if BM-3103, (4-Hydroxy 4'-methyoxytolan), a known wound healing promoter, anti-inflammatory, and autophagy inducer, could remove mutant keratins/aggregates thereby strengthening the IF network without adversely affecting cell survival, adhesion, or migration. Assays tested on patient derived EBS keratinocytes included: (1) Assessment of cytotoxicity, adhesion, and migration effects of BM-3103 using MTT, LDH, and scratch assays. (2) Autophagy induction (LC3-II conversion) and keratin aggregation (heat stress assay). There were no signs of cytotoxicity, loss of cell adhesion or slowed migration. Cells were exposed to escalating concentrations of BM-3103 for 24 hrs then heat shocked (43°C for 30min) generating an abundance of aggregates visualized by immunofluorescent microscopy. Untreated keratinocytes displayed keratin aggregate formation in 72% of KRT5 and 87% of KRT14 mutant cells. Treatment with BM-3103 reduced the incidence of keratin aggregates to 16% (p<0.01) of KRT5 and 24% (p<0.01) KRT14 mutant cells. BM-3103 is the first compound identified that is capable of stabilizing IF networks in keratinocytes from severe EBS patients. A clinical trial is ongoing to assess if BM-3103, can heal blistering areas and strengthen EBS patient skin.

A mathematical model for the dependence of keratin aggregate formation on the quantity of mutant keratin expressed in EGFP-K14 R125P keratinocytes.

We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.

Growth, lifetime, directional movement and myosin-dependent motility of mutant keratin granules in cultured cells

Intermediate filament polypeptides (IFPs) are prominent components of cytoplasmic aggregates, which are pathognomonic for multiple diseases. Recent observations in cultured cells suggest that they are dynamic and subject to regulated turnover. The emerging concept is that multiple factors contribute to motility and turnover of IFP-containing aggregates. To understand their relative contribution, quantitative tools are needed. The current study addresses this need using epithelial cells producing mutant keratin IFPs that have been identified as the cause of the hereditary blister-forming skin disease epidermolysis bullosa simplex. Digital image analysis of individual granules allowed mapping of their complete life cycle, with information on multiple characteristics at any given time-point. The deduced signet features revealed rapid granule fusion and directed transport from the periphery towards the cell centre, and a limited, ~ 30 min lifetime with a slow, continuous growth phase followed by fast disassembly. As paradigmatic proof-of-principle, we demonstrate that inhibition of myosin II selectively reduces granule movement, linking keratin granule motility to retrograde cortical acto-myosin flow. The newly developed methods and established parameters will help in the characterization of known and the identification of novel regulators of IFP-containing aggregates.

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.

The role of keratins in the digestive system: lessons from transgenic mouse models.

Keratins are the largest subfamily of intermediate filament proteins. They are either type I acidic or type II basic keratins. Keratins form obligate heteropolymer in epithelial cells and their expression patterns are tissue-specific. Studies have shown that keratin mutations are the cause of many diseases in humans or predispose humans to acquiring them. Using mouse models to study keratin-associated human diseases is critical, because they allow researchers to get a better understanding of these diseases and their progressions, and so many such studies have been conducted. Acknowledging the importance, researches with genetically modified mice expressing human disease-associated keratin mutants have been widely done. Numerous studies using keratin knockout mice, keratin-overexpressed mice, or transgenic mice expressing keratin mutants have been conducted. This review summarizes the mouse models that have been used to study type I and type II keratin expression in the digestive organs, namely, the liver, pancreas, and colon.

CRISPR/Cas9-Mediated Treatment Ameliorates the Phenotype of the Epidermolytic Palmoplantar Keratoderma-like Mouse

CRISPR/Cas9 has been confirmed as a distinctly efficient, simple-to-configure, highly specific genome-editing tool that has been used to treat monogenetic disorders. Epidermolytic palmoplantar keratoderma (EPPK) is a common autosomal dominant keratin disease resulting from dominant-negative mutation of the KRT9 gene, and it has no effective therapy. We performed CRISPR/Cas9-mediated treatment on a knockin (KI) transgenic mouse model that carried a small indel heterozygous mutation of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), which caused a humanized EPPK-like phenotype. The mutation within exon 1 of Krt9 generated a novel protospacer adjacent motif site, TGG, for Cas9 recognition and cutting. By delivering lentivirus vectors (LVs) encoding single-guide RNAs (sgRNAs) and Cas9 that targeted Krt9 sequence into HeLa cells engineered to constitutively express wild-type and mutant keratin 9 (K9), we found the sgRNA was highly effective in reducing expression of the mutant K9 protein in vitro. We injected the LV into the fore-paws of adult KI-Krt9 mice three times every 8 days and found that the expression of K9 decreased ∼14.6%. The phenotypic mitigation was revealed by restoration of the abnormal differentiation and aberrant proliferation of the epidermis. Our data are the first to show that CRISPR/Cas9 is a potentially powerful therapeutic option for EPPK and other PPK subtypes.

Skin Stem Cells in Silence, Action, and Cancer.

In studying how stem cells make and maintain tissues, nearly every chapter of a cell biology textbook takes on special interest. The field even allows us to venture where no chapters have yet been written. In studying this basic problem, we are continually bombarded by nature's surprises and challenges. Stem cell biology has captured my interest for nearly my entire scientific career. Below, I focus on my laboratory's contributions to this fascinating field, to which so many friends and colleagues have made seminal discoveries equally deserving of this award.

178 Cooperation of keratin mutations and posttranslational modifications

The skin fragility disorder Epidermolysis bullosa simplex (EBS) mostly results from KRT5 and KRT14 mutations which cause aggregation of the keratin cytoskeleton and render the skin sensitive to mild trauma, followed by recurrent blisters. The prevalence of more serious disease during warmer summer months in most EBS patients suggests that temperature-sensing mechanisms cooperating with keratin mutations contribute to this phenomenon. In order to dissect the underlying mechanism and to exploit this for a therapy approach, we analyzed several KRT5 and KRT14 mutations in a cell culture based heat stress assay. Hence, we identified a subset of KRT5 mutations forming extensive keratin networks with KRT14 at normal culture conditions but reversible aggregation of the keratin cytoskeleton upon short exposure to elevated temperature. This heat-induced aggregation of the keratin cytoskeleton subsequently led to impaired desmosomes and diminished intercellular adhesion. A phosphoproteomic approach revealed elevated Ser phosphorylation of mutant KRT5, but not KRT14, upon brief heat exposure, indicating the involvement of specific kinases in the heat-induced keratin aggregation. Currently, we are investigating kinase inhibitors to prevent the heat-induced filament collapse. Moreover, parthenolide, a plant sesquiterpene lactone that exerts anti-cancer and anti-inflammatory properties and modulates sirtuin function, was found to affect keratin organization, likely by reducing elevated Lys acetylation of mutant keratins. Most importantly, pre-treatment of a temperature-sensitive KRT5 mutant with parthenolide prevented heat-induced keratin aggregation and strengthened desmosome adhesion. Collectively, we show that keratin mutations promote select posttranslational modifications, partially in combination with elevated temperature. Our assay system and results enable the development of a rational therapy for the improvement of EBS.

Traceless Targeting and Isolation of Gene-Edited Immortalized Keratinocytes from Epidermolysis Bullosa Simplex Patients

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events and devise a strategy that allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here we report on TALENs that efficiently disrupt the KRT5 locus in immortalized patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.

Keratin gene mutations influence the keratinocyte response to DNA damage and cytokine induced apoptosis.

The keratin filament cytoskeleton is vital to the normal function of epithelial cells. It provides structural support and regulates different aspects of cell metabolism. Mutations in keratins 5 and 14 cause a skin fragility disorder, epidermolysis bullosa simplex (EBS). Patients with severe EBS have an increased cumulative risk for basal cell carcinoma. In this study, we tested how keratin 5 and 14 mutant EBS patient-derived keratinocytes behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-α and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. When case of DNA damage, the ATM/Chk2-pathway is one of the two main tracks that can prevent the progression of mitosis and so allow repair. This was altered in all investigated keratin mutants with a particular down-regulation of the activated form of checkpoint kinase 2 (pChk2). Keratin mutants also appear less sensitive than normal cells to treatment with TNF-α or TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. Such changes are likely to have a profound effect on mutant keratinocytes ability to survive and withstand stress, and in theory this may be also a contributing factor to cell transformation.

LB795 TALEN-mediated elimination of mutant keratin 14 as a gene therapy for epidermolysis bullosa simplex

LB795 TALEN-mediated elimination of mutant keratin 14 as a gene therapy for epidermolysis bullosa simplex

Assays to Study Consequences of Cytoplasmic Intermediate Filament Mutations: The Case of Epidermal Keratins.

The discovery of the causative link between keratin mutations and a growing number of human diseases opened the way for a better understanding of the function of the whole intermediate filament families of cytoskeleton proteins. This chapter describes analytical approaches to identification and interpretation of the consequences of keratin mutations, from the clinical and diagnostic level to cells in tissue culture. Intermediate filament pathologies can be accurately diagnosed from skin biopsies and DNA samples. The Human Intermediate Filament Database collates reported mutations in intermediate filament genes and their diseases, and can help clinicians to establish accurate diagnoses, leading to disease stratification for genetic counseling, optimal care delivery, and future mutation-aligned new therapies. Looking at the best-studied keratinopathy, epidermolysis bullosa simplex, the generation of cell lines mimicking keratinopathies is described, in which tagged mutant keratins facilitate live-cell imaging to make use of today's powerful enhanced light microscopy modalities. Cell stress assays such as cell spreading and cell migration in scratch wound assays can interrogate the consequences of the compromised cytoskeletal network. Application of extrinsic stresses, such as heat, osmotic, or mechanical stress, can enhance the differentiation of mutant keratin cells from wild-type cells. To bring the experiments to the next level, 3D organotypic human cultures can be generated, and even grafted onto the backs of immunodeficient mice for greater in vivo relevance. While development of these assays has focused on mutant K5/K14 cells, the approaches are often applicable to mutations in other intermediate filaments, reinforcing fundamental commonalities in spite of diverse clinical pathologies.

Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75

Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele specific siRNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific shRNA persistently suppressed this phenotype. The phenotypic correction was associated with significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of the mutant keratin genes.

A Drosophila Model of Epidermolysis Bullosa Simplex

The blistering skin disorder Epidermolysis bullosa simplex (EBS) results from dominant mutations in K5 or K14 genes, encoding the intermediate filament network of basal epidermal keratinocytes. The mechanisms governing keratin network formation and collapse due to EBS mutations remain incompletely understood. Drosophila lacks cytoplasmic intermediate filaments, providing a ‚null’ environment to examine the formation of keratin networks and determine mechanisms by which mutant keratins cause pathology. Here, we report that ubiquitous co-expression of transgenes encoding wild-type human K14 and K5 resulted in the formation of extensive keratin networks in Drosophila epithelial and non-epithelial tissues, causing no overt phenotype. Similar to mammalian cells, treatment of transgenic fly tissues with phosphatase inhibitors caused keratin network collapse, validating Drosophila as a genetic model system to investigate keratin dynamics. Co-expression of K5 and a K14R125C mutant that causes the most severe form of EBS resulted in widespread formation of EBS-like cytoplasmic keratin aggregates in epithelial and non-epithelial fly tissues. Expression of K14R125C/K5 caused semi-lethality; adult survivors developed wing blisters and were flightless due to lack of intercellular adhesion during wing heart development. This Drosophila model of EBS is valuable for the identification of pathways altered by mutant keratins and for development of EBS therapies.

Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin.

Small interfering RNAs (siRNAs) specifically and potently inhibit target gene expression. Pachyonychia congenita (PC) is a skin disorder caused by mutations in genes encoding keratin (K) 6a/b, K16, and K17, resulting in faulty intermediate filaments. A siRNA targeting a single nucleotide, PC-relevant mutation inhibits K6a expression and has been evaluated in the clinic with encouraging results. To better understand the pathophysiology of PC, and develop a model system to study siRNA delivery and visualize efficacy in skin, wild type (WT) and mutant K6a complementary DNAs (cDNAs) were fused to either enhanced green fluorescent protein or tandem tomato fluorescent protein cDNA to allow covisualization of mutant and WT K6a expression in mouse footpad skin using a dual fluorescence in vivo confocal imaging system equipped with 488 and 532 nm lasers. Expression of mutant K6a/reporter resulted in visualization of keratin aggregates, while expression of WT K6a/reporter led to incorporation into filaments. Addition of mutant K6a-specific siRNA resulted in inhibition of mutant, but not WT, K6a/reporter expression. Intravital imaging offers subcellular resolution for tracking functional activity of siRNA in real time and enables detailed analyses of therapeutic effects in individual mice to facilitate development of nucleic acid-based therapeutics for skin disorders.

Keratins and skin disease.

Mutations in keratin genes cause a diverse spectrum of skin, hair and mucosal disorders. Cutaneous disorders include epidermolysis bullosa simplex, palmoplantar keratoderma, epidermolytic ichthyosis and pachyonychia congenita. Both clinical and laboratory observations confirm a major role for keratins in maintaining epidermal cell-cell adhesion. When normal tissue homeostasis is disturbed, for example, during wound healing and cancer, keratins play an important non-mechanical role. Post-translational modifications including glycosylation and phosphorylation of keratins play an important role in protection of epithelial cells from injury. Keratins also play a role in modulation of the immune response. A current focus in the area of keratins and disease is the development of new treatments including small inhibitory RNA (siRNA) to mutant keratins and small molecules to modulate keratin expression.

Collapse of the keratin filament network through the expression of mutant keratin 6c observed in a case of focal plantar keratoderma

Focal palmoplantar keratoderma (PPK) with severe pain is a hallmark of pachyonychia congenita, a rare autosomal dominant disorder involving PPK and hypertrophic nail dystrophy. Some families present focal PPK with either minimal or no nail changes. Dominant-negative mutations in any of the four identified keratin genes, KRT6A, KRT6B, KRT16 or KRT17, lead to pachyonychia congenita. However, the majority of families with focal PPK showing minimal or no nail changes do not harbor mutations in these genes. Recently, mutations of KRT6C were identified in families with focal PPK alone. Here, we report a 26-year-old Japanese man with focal plantar hyperkeratosis that developed at approximately 10years of age with no palmar involvement and no nail alterations. We identified a missense KRT6C mutation c.1414G>A resulting in an p.Glu472Lys substitution, as reported in other Japanese patients. When the mutant keratin 6c protein is exogenously expressed in human HaCaT cells, a collapse of the keratin filament network is observed in a dose-dependent manner, suggesting the mutation has a dominant-negative effect on keratin filament network formation. The mutated residue is located at the helix termination motif of keratin 6c. The peptide sequence around this residue is highly conserved among type II, III and IV intermediate filament proteins. Glu to Lys mutations of the equivalent residue have been reported in a variety of inherited diseases, including neurodegenerative diseases, corneal dystrophy and skin disorders, suggesting that this residue is vital to keratin function.