Dominant and recessive compound heterozygous mutations in epidermolysis bullosa simplex demonstrate the role of the stutter region in keratin intermediate filament assembly

Keratin intermediate filaments are important cytoskeletal structural proteins involved in maintaining cell shape and function. Mutations in the epidermal keratin genes, keratin 5 or keratin 14 lead to the disruption of keratin filament assembly, resulting in an autosomal dominant inherited blistering skin disease, epidermolysis bullosa simplex (EBS). We investigated a large EBS kindred who exhibited a markedly heterogeneous clinical presentation and detected two distinct keratin 5 mutations in the proband, the most severely affected. One missense mutation (E170K) in the highly conserved helix initiation peptide sequence of the 1A rod domain was found in all the affected family members. In contrast, the other missense mutation (E418K) was found only in the proband. The E418K mutation was located in the stutter region, an interruption in the heptad repeat regularity, whose function as yet remains unclear. We hypothesized that this mutated stutter allele was clinically silent when combined with the wild type allele but aggravates the clinical severity of EBS caused by the E170K mutation on the other allele. To confirm this in vitro, we transfected mutant keratin 5 cDNA into cultured cells. Although only 12.7% of the cells transfected with the E170K mutation alone showed disrupted keratin filament aggregations, significantly more cells (30.0%) cotransfected with both E170K and E418K mutations demonstrated keratin aggregation (p < 0.05). These transfection assay results corresponded to the heterogeneous clinical findings of the EBS patient in this kindred. We have identified the first case of both compound heterozygous dominant (E170K) and recessive (E418K) mutations in any keratin gene and confirmed the significant involvement of the stutter region in the assembly and organization of the keratin intermediate filament network in vitro.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution- binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

Defining the properties of the nonhelical tail domain in type II keratin 5: insight from a bullous disease-causing mutation

Inherited mutations in the intermediate filament (IF) proteins keratin 5 (K5) or keratin 14 (K14) cause epidermolysis bullosa simplex (EBS), in which basal layer keratinocytes rupture upon trauma to the epidermis. Most mutations are missense alleles affecting amino acids located in the central alpha-helical rod domain of K5 and K14. Here, we study the properties of an unusual EBS-causing mutation in which a nucleotide deletion (1649delG) alters the last 41 amino acids and adds 35 residues to the C terminus of K5. Relative to wild type, filaments coassembled in vitro from purified K5-1649delG and K14 proteins are shorter and exhibit weak viscoelastic properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is purely a function of the K5-1649delG tail domain; in contrast, the cloned 109 residue-long tail domain from wild type K5 is distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5's assembly properties.     

Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.

Previously we demonstrated that transgenic mice expressing mutant basal epidermal keratin genes exhibited a phenotype resembling a group of autosomal dominant human skin disorders known as epidermolysis bullosa simplex (EBS). EBS diseases affect approximately 1: 50,000 and are of unknown etiology, although all subtypes exhibit blistering arising from basal cell cytolysis. We now demonstrate that two patients with spontaneous cases of Dowling-Meara EBS have point mutations in a critical region in one (K14) of two basal keratin genes. To demonstrate function, we engineered one of these point mutations in a cloned human K14 cDNA, and showed that a K14 with an Arg-125----Cys mutation disrupted keratin network formation in transfected keratinocytes and perturbed filament assembly in vitro. Since we had previously shown that keratin network perturbation is an essential component of EBS diseases, these data suggest that the basis for the phenotype in this patient resides in this point mutation.     

Focal activation of a mutant allele defines the role of stem cells in mosaic skin disorders

Stem cells are crucial for the formation and maintenance of tissues and organs. The role of stem cells in the pathogenesis of mosaic skin disorders remains unclear. To study the molecular and cellular basis of mosaicism, we established a mouse model for the autosomal-dominant skin blistering disorder, epidermolytic hyperkeratosis (MIM 113800), which is caused by mutations in either keratin K1 or K10. This genetic model allows activation of a somatic K10 mutation in epidermal stem cells in a spatially and temporally controlled manner using an inducible Cre recombinase. Our results indicate that lack of selective pressure against certain mutations in epidermal stem cells leads to mosaic phenotypes. This finding has important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses.     

Formation of a Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10(-/-) mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10(-/-) mice suggests that there is a considerable redundancy in the keratin gene family.     

KERATINS AND SKIN DISORDERS

The epidermal keratinocytes express two major pairs of keratin polypeptides. One pair (K5/K14) expressed specifically in basal generative compartment and the other (K1/K10) expressed specifically in the differentiating suprabasal compartment. The switch in the expression of the keratins from proliferating to differentiating compartment indicates the changes that occur in the keratin filament organization which in turn influences the functional properties of the epidermis. Proper regulation of keratin gene expression and the filament organization are absolutely necessary for normal functioning of the skin. Keratin gene mutations can influence the filament integrity thereby causing several heritable blistering disorders of the skin such as epidermolysis bullosa, bullous icthyosiform erythroderma, etc. Changes in the keratin gene expression may lead to incomplete differentiation of the epidermal keratinocyte, causing hyperproliferative diseases of the skin such as psoriasis, carcinomas, etc. This review briefly describes the changes in keratin structure or gene expression that are known to result in various disorders of the skin.     

A mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice

Mutations in genes encoding epidermal keratins cause skin disorders, while those in internal epithelial keratins, such as K8 and K18, are risk factors for liver diseases. The effect of dominant mutations in K8 or K18 during embryonic development and tissue homeostasis has not been examined so far. Here we demonstrate that the dominant mutation hK18 R89C, that is highly similar to hK14 R125C, causing EBS in humans, leads to cell type-specific lethality in mice, depending on the ratio of mutant to endogenous keratins. Mice expressing hK18 R89C in the absence of endogenous K19 and K18 died at mid-gestation from defects in trophoblast giant cells, accompanied by haematomas. A single, endogenous K18 allele rescued embryonic lethality but caused aggregation of keratins in all adult internal epithelia, surprisingly without spontaneous cell fragility. Closer analysis revealed that both filaments and aggregates coexisted in the same cell, depending on the ratio of mutant to endogenous keratins. Our results demonstrate that balanced overexpression of a wild-type keratin rescued the lethal consequences of a dominant-negative mutation. This has important implications for therapy approaches of keratinopathies, suggesting that suppressing the mutant allele is not necessary in vivo.     

Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant

The two major intermediate filament proteins in glandular epithelia are keratin polypeptides 8 and 18 (K8/18). To evaluate the function and potential disease association of K18, we examined the effects of mutating a highly conserved arginine (arg89) of K18. Expression of K18 arg89-->his/cys and its normal K8 partner in cultured cells resulted in punctate staining as compared with the typical filaments obtained after expression of wild-type K8/18. Generation of transgenic mice expressing human K18 arg89-->cys resulted in marked disruption of liver and pancreas keratin filament networks. The most prominent histologic abnormalities were liver inflammation and necrosis that appeared at a young age in association with hepatocyte fragility and serum transaminase elevation. These effects were caused by the mutation since transgenic mice expressing wild-type human K18 showed a normal phenotype. A relative increase in the phosphorylation and glycosylation of detergent solubilized K8/18 was also noted in vitro and in transgenic animals that express mutant K18. Our results indicate that the highly conserved arg plays an important role in glandular keratin organization and tissue fragility as already described for epidermal keratins. Phosphorylation and glycosylation alterations in the arg mutant keratins may account for some of the potential changes in the cellular function of these proteins. Mice expressing mutant K18 provide a novel animal model for human chronic hepatitis, and for studying the tissue specific function(s) of K8/18.     

Genomic organization and amplification of the human epidermal type II keratin genes K1 and K5

Keratins are a family of structurally related proteins that form the intermediate filament cytoskeleton in epithelial cells. Mutations in K1 and K5 result in the autosomal dominant disorders epidermolytic hyperkeratosis/bullous congenital ichthyosiform erythroderma and epidermolysis bullosa simplex, respectively. Most disease-associated mutations are within exons encoding protein domains involved in keratin filament assembly. However, some mutations occur outside the mutation hot-spots and may perturb intermolecular interactions between keratins and other proteins, usually with milder clinical consequences. To screen the entire keratin 1 and keratin 5 genes we have characterized their intron-exon organization. The keratin 1 gene comprises 9 exons spanning approximately 5.6 kb on 12q, and the keratin 5 gene comprises 9 exons spanning approximately 6.1 kb on 12q. We have also developed a comprehensive PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.     

Hard α-keratin IF: A structural model lacking a head-to-tail molecular overlap but having hybrid features characteristic of both epidermal keratin and vimentin IF

In intermediate filaments (IF) both epidermal keratin and vimentin molecules have been shown to have an eight residue head to-tail overlap between the rod domains of similarly directed molecules. In the case of the epidermal keratins this region has also been shown to have particular structural/functional significance since it represents a hot-spot for mutations in the four keratinopathies characterized to date. While there is good evidence that this head-to-tail overlap is present in IF containing Type III, IV, and V chains, as well as in the epidermal keratin IF (Ib/IIb), there are no data currently available for the hard α-keratin IF (Ia/IIa). Using a variety of data derived from X-ray diffraction and crosslinking studies, as well as theoretical modeling, it is now possible to demonstrate that the overlap region is not a feature of hard α-keratin IF. Indeed, it is shown that there is a nine residue gap between consecutive parallel molecules in the IF. An explanation for this observation is presented in terms of compensating disulfide bonds that occur both within the IF, and between the IF and the matrix in which the IF are embedded. © 1995 Wiley-Liss, Inc.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.     

Directed Expression of a Chimeric Type II Keratin Partially Rescues Keratin 5-null Mice

The crucial role of structural support fulfilled by keratin intermediate filaments (IFs) in surface epithelia likely requires that they be organized into cross-linked networks. For IFs comprised of keratins 5 and 14 (K5 and K14), which occur in basal keratinocytes of the epidermis, formation of cross-linked bundles is, in part, self-driven through cis-acting determinants. Here, we targeted the expression of a bundling-competent KRT5/KRT8 chimeric cDNA (KRT8bc) or bundling-deficient wild type KRT8 as a control to the epidermal basal layer of Krt5-null mice to assess the functional importance of keratin IF self-organization in vivo. Such targeted expression of K8bc rescued Krt5-null mice with a 47% frequency, whereas K8 completely failed to do so. This outcome correlated with lower than expected levels of K8bc and especially K8 mRNA and protein in the epidermis of E18.5 replacement embryos. Ex vivo culture of embryonic skin keratinocytes confirmed the ability of K8bc to form IFs in the absence of K5. Additionally, electron microscopy analysis of E18.5 embryonic skin revealed that the striking defects observed in keratin IF bundling, cytoarchitecture, and mitochondria are partially restored by K8bc expression. As young adults, viable KRT8bc replacement mice develop alopecia and chronic skin lesions, indicating that the skin epithelia are not completely normal. These findings are consistent with a contribution of self-mediated organization of keratin IFs to structural support and cytoarchitecture in basal layer keratinocytes of the epidermis and underscore the importance of context-dependent regulation for keratin genes and proteins in vivo.     

A 'hot-spot' mutation alters the mechanical properties of keratin filament networks

Keratins 5 and 14 polymerize to form the intermediate filament network in the progenitor basal cells of many stratified epithelia including epidermis, where it provides crucial mechanical support. Inherited mutations in K5 or K14 result in epidermolysis bullosa simplex (EBS), a skin-fragility disorder. The impact that such mutations exert on the intrinsic mechanical properties of K5/K14 filaments is unknown. Here we show, by using differential interference contrast microscopy, that a 'hot-spot' mutation in K14 greatly reduces the ability of reconstituted mutant filaments to bundle under crosslinking conditions. Rheological assays measure similar small-deformation mechanical responses for crosslinked solutions of wild-type and mutant keratins. The mutation, however, markedly reduces the resilience of crosslinked networks against large deformations. Single-particle tracking, which probes the local organization of filament networks, shows that the mutant polymer exhibits highly heterogeneous structures compared to those of wild-type filaments. Our results indicate that the fragility of epithelial cells expressing mutant keratin may result from an impaired ability of keratin polymers to be crosslinked into a functional network.     

Functional Differences between Keratins of Stratified and Simple Epithelia

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.     

Keratin gene mutations in disorders of human skin and its appendages

Keratins, the major structural protein of all epithelia are a diverse group of cytoskeletal scaffolding proteins that form intermediate filament networks, providing structural support to keratinocytes that maintain the integrity of the skin. Expression of keratin genes is usually regulated by differentiation of the epidermal cells within the stratifying squamous epithelium. Amongst the 54 known functional keratin genes in humans, about 22 different genes including, the cornea, hair and hair follicle-specific keratins have been implicated in a wide range of hereditary diseases. The exact phenotype of each disease usually reflects the spatial expression level and the types of mutated keratin genes, the location of the mutations and their consequences at sub-cellular levels as well as other epigenetic and/or environmental factors. The identification of specific pathogenic mutations in keratin disorders formed the basis of our understanding that led to re-classification, improved diagnosis with prognostic implications, prenatal testing and genetic counseling in severe keratin genodermatoses. Molecular defects in cutaneous keratin genes encoding for keratin intermediate filaments (KIFs) causes keratinocytes and tissue-specific fragility, accounting for a large number of genetic disorders in human skin and its appendages. These diseases are characterized by keratinocytes fragility (cytolysis), intra-epidermal blistering, hyperkeratosis, and keratin filament aggregation in severely affected tissues. Examples include epidermolysis bullosa simplex (EBS; K5, K14), keratinopathic ichthyosis (KPI; K1, K2, K10) i.e. epidermolytic ichthyosis (EI; K1, K10) and ichthyosis bullosa of Siemens (IBS; K2), pachyonychia congenita (PC; K6a, K6b, K16, K17), epidermolytic palmo-plantar keratoderma (EPPK; K9, (K1)), monilethrix (K81, K83, K86), ectodermal dysplasia (ED; K85) and steatocystoma multiplex. These keratins also have been identified to have roles in apoptosis, cell proliferation, wound healing, tissue polarity and remodeling. This review summarizes and discusses the clinical, ultrastructural, molecular genetics and biochemical characteristics of a broad spectrum of keratin-related genodermatoses, with special clinical emphasis on EBS, EI and PC. We also highlight current and emerging model tools for prognostic future therapies. Hopefully, disease modeling and in-depth understanding of the molecular pathogenesis of the diseases may lead to the development of novel therapies for several hereditary cutaneous diseases.     

Deficient plakophilin-1 expression due to a mutation in PKP1 causes ectodermal dysplasia-skin fragility syndrome in Chesapeake Bay retriever dogs

In humans, congenital and hereditary skin diseases associated with epidermal cell-cell separation (acantholysis) are very rare, and spontaneous animal models of these diseases are exceptional. Our objectives are to report a novel congenital acantholytic dermatosis that developed in Chesapeake Bay retriever dogs. Nine affected puppies in four different litters were born to eight closely related clinically normal dogs. The disease transmission was consistent with an autosomal recessive mode of inheritance. Clinical signs occurred immediately after birth with superficial epidermal layers sloughing upon pressure. At three month of age, dogs exhibited recurrent superficial skin sloughing and erosions at areas of friction and mucocutaneous junctions; their coat was also finer than normal and there were patches of partial hair loss. At birth, histopathology revealed severe suprabasal acantholysis, which became less severe with ageing. Electron microscopy demonstrated a reduced number of partially formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Sequencing revealed a homozygous splice donor site mutation within the first intron of PKP1 resulting in a premature stop codon, thereby explaining the inability to detect plakophilin-1 in the skin. Altogether, the clinical and pathological findings, along with the PKP1 mutation, were consistent with the diagnosis of ectodermal dysplasia-skin fragility syndrome with plakophilin-1 deficiency. This is the first occurrence of ectodermal dysplasia-skin fragility syndrome in an animal species. Controlled mating of carrier dogs would yield puppies that could, in theory, be tested for gene therapy of this rare but severe skin disease of children.     

Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair

The ability to heal wounds is vital to all organisms. In mammalian tissues, alterations in intermediate filament (IF) gene expression represent an early reaction of cells surviving injury. We investigated the role of keratin IFs during the epithelialization of skin wounds using a keratin 6alpha and 6beta (K6alpha/K6beta)-null mouse model. In skin explant culture, null keratinocytes exhibit an enhanced epithelialization potential due to increased migration. The extent of the phenotype is strain dependent, and is accompanied by alterations in keratin IF and F-actin organization. However, in wounded skin in vivo, null keratinocytes rupture as they attempt to migrate under the blood clot. Fragility of the K6alpha/K6beta-null epidermis is confirmed when applying trauma to chemically treated skin. We propose that the alterations in IF gene expression after tissue injury foster a compromise between the need to display the cellular pliability necessary for timely migration and the requirement for resilience sufficient to withstand the rigors of a wound site.     

Regulated Expression of Human Filaggrin in Keratinocytes Results in Cytoskeletal Disruption, Loss of Cell–Cell Adhesion, and Cell Cycle Arrest

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.