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.     

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.     

The mechanical behavior of mutant K14-R125P keratin bundles and networks in NEB-1 keratinocytes

Epidermolysis bullosa simplex (EBS) is an inherited skin-blistering disease that is caused by dominant mutations in the genes for keratin K5 or K14 proteins. While the link between keratin mutations and keratinocyte fragility in EBS patients is clear, the exact biophysical mechanisms underlying cell fragility are not known. In this study, we tested the hypotheses that mutant K14-R125P filaments and/or networks in human keratinocytes are mechanically defective in their response to large-scale deformations. We found that mutant filaments and networks exhibit no obvious defects when subjected to large uniaxial strains and have no negative effects on the ability of human keratinocytes to survive large strains. We also found that the expression of mutant K14-R125P protein has no effect on the morphology of the F-actin or microtubule networks or their responses to large strains. Disassembly of the F-actin network with Latrunculin A unexpectedly led to a marked decrease in stretch-induced necrosis in both WT and mutant cells. Overall, our results contradict the hypotheses that EBS mutant keratin filaments and/or networks are mechanically defective. We suggest that future studies should test the alternative hypothesis that keratinocytes in EBS cells are fragile because they possess a sparser keratin network.     

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.     

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.     

Severe keratin 5 and 14 mutations induce down-regulation of junction proteins in keratinocytes

The intermediate filament cytoskeleton is essential for the development and maintenance of normal tissue function. A number of diverse recent observations implicate these filament systems in sensing stress and protecting cells against its worst consequences. Cells expressing severely disruptive keratin mutations, characteristic of Dowling-Meara EBS, were previously reported to show elevated responses to physiological stress, and partial disassembly of cell junctions was reported upon direct mechanical stress to the cells. Gene expression microarray analysis has therefore been used here to examine the broad spectrum of effects of mutant keratins. Many genes associated with keratins and other components of the cytoskeleton showed altered expression levels; in particular, many cell junction components are down-regulated in EBS cells. That this is due to the expression of the mutant keratins, and not to other genetic variables, is supported by observation of the same effects in isogenic cells generated from wild type keratinocytes transfected with the same keratin mutations in the helix boundary motifs of K14 or K5. Whilst the mechanism underlying this is unclear, these findings may help to explain other aspects of EBS-associated pathology, such as faster scratch wound migration, or acantholysis (cell-cell separation) in patients' skin. Constitutive stress combined with constitutively weakened cell junctions may also contribute to a recently reported increased risk of non-melanoma skin cancer in EBS patients.     

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.     

The molecular basis of human keratin disorders

Keratins are cytoskeletal proteins that provide structural support to epithelial cells and tissues. Perturbation causes cell and tissue fragility and accounts for a large number of genetic disorders in humans. In humans, 54 functional keratin genes exist and 21 different keratin genes including hair keratins and hair follicle-specific epithelial keratins have been associated with hereditary disorders. Moreover, keratins have been implicated in more complex traits such as liver disease and inflammatory bowel disease. Understanding the molecular basis of keratin disorders has been the basis for improved diagnosis with prognostic implications, genetic counseling and prenatal testing for severe disorders. Besides their mechanical role, keratins have newly identified functions in apoptosis, cell growth, tissue polarity, wound healing and tissue remodeling. Improved understanding of the regulatory functions of keratins may offer novel approaches to overcome current treatment limitations.     

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.     

Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex]

The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti-keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human keratins: biology and pathology

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins--including numerous keratins characterized only recently--are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family.     

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis

The claw of lizards is largely composed of beta‐keratins, also referred to as keratin‐associated beta‐proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta‐keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha‐keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha‐keratins resembled that of beta‐keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

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.     

Lessons from disorders of epidermal differentiation-associated keratins

A number of diseases have been associated with mutations in genes encoding keratin intermediate filaments. Several of these disorders have skin manifestations, in which histological changes highlight the role of various different keratins in epidermal differentiation. For example, mutations in either K1 or K10 (the major keratin pair expressed in differentiated keratinocytes) usually lead to clumped keratin filaments and cytolysis. Furthermore, the precise nature of the mutation has direct implications for disease phenotype. Specifically, mutations in the H1 and alpha-helical rod domains of K1/K10 result in bullous congenital ichthyosiform erythroderma, underscoring the critical role for this keratin filament domain in maintaining cellular integrity. However, a lysine to isoleucine substitution in the V1 domain of K1 underlies a form of palmoplantar keratoderma, which has different cell biological implications. Keratins are cross-linked into the cornified cell envelopes through this particular lysine residue and the consequences of the mutation lead to changes in keratin-desmosome association and cornified cell morphology, suggesting a role for this keratin subdomain in cornified cell envelope formation. Recently, to extend genotype-phenotype correlation, a frameshift mutation in the V2 region of the K1 tail domain was identified in ichthyosis hystrix (Curth-Macklin type), in which keratin filaments show a characteristic shell-like structure and fail to form proper bundles. In this case, the association of desmosomes with loricrin was also altered, implicating this keratin domain in organizing the intracellular distribution of loricrin during cornification. Collectively, these mutations in K1/K10 provide a fascinating insight into both normal and abnormal processes of epidermal differentiation.     

Diseases of epidermal keratins and their linker proteins

Epidermal keratins, a diverse group of structural proteins, form intermediate filament networks responsible for the structural integrity of keratinocytes. The networks extend from the nucleus of the epidermal cells to the plasma membrane where the keratins attach to linker proteins which are part of desmosomal and hemidesmosomal attachment complexes. The expression of specific keratin genes is regulated by differentiation of the epidermal cells within the stratifying squamous epithelium. Progress in molecular characterization of the epidermal keratins and their linker proteins has formed the basis to identify mutations which are associated with distinct cutaneous manifestations in patients with genodermatoses. The precise phenotype of each disease apparently reflects the spatial level of expression of the mutated genes, as well as the types and positions of the mutations and their consequences at mRNA and protein levels. Identification of specific mutations in keratinization disorders has provided the basis for improved diagnosis and subclassification with prognostic implications and has formed the platform for prenatal testing and preimplantation genetic diagnosis. Finally, precise knowledge of the mutations is a prerequisite for development of gene therapy approaches to counteract, and potentially cure, these often devastating and currently intractable diseases.     

A function for keratins and a common thread among different types of epidermolysis bullosa simplex diseases.

Previously we demonstrated that transgenic mice expressing a mutant keratin in the basal layer of their stratified squamous epithelia exhibited a phenotype bearing resemblance to a subclass (Dowling Meara) of a heterogeneous group of human skin disorders known as epidermolysis bullosa simplex (EBS) (Vassar, R., P. A. Coulombe, L. Degenstein, K. Albers, E. Fuchs. 1991. Cell. 64:365-380.). The extent to which subtypes of EBS diseases might be genetically related is unknown, although they all exhibit skin blistering as a consequence of basal cell cytolysis. We have now examined transgenic mice expressing a range of keratin mutants which perturb keratin filament assembly to varying degrees. We have generated phenotypes which include most subtypes of EBS, demonstrating for the first time that at least in mice, these diseases can be generated by different mutations within a single gene. A strong correlation existed between the severity of the disease and the extent to which the keratin filament network was disrupted, implicating perturbations in keratin networks as an essential component of these diseases. Some keratin mutants elicited subtle perturbations, with no signs of the tonofilament clumping typical of Dowling-Meara EBS and our previous transgenic mice. Importantly, basal cell cytolysis still occurred, thereby uncoupling cytolysis from the generation of large, insoluble cytoplasmic protein aggregates. Moreover, cell rupture occurred in a narrowly defined subnuclear zone, and seemed to involve three factors: (a) filament perturbation, (b) the columnar shape of the basal cell, and (c) physical trauma. This work provides the best evidence to date for a structural function of a cytoplasmic intermediate filament network, namely to impart mechanical integrity to the cell in the context of its tissue.     

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.     

Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes

The polypeptide composition of epidermal keratin varies in disease. To better understand the biological meaning of these variations, we have analyzed keratins from a number of human epidermal diseases by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The results reveal a continuous spectrum of keratin expression ranging from one closely resembling the normal in vivo pattern to one almost identical to cultured epidermal keratinocytes. Specifically, a 50-kilodalton (kd) (AE1-positive) and a 58-kd (AE3-positive) keratin are present in all diseases, supporting the concept that they represent "permanent" markers for keratinocytes. A 56.5-kd (AE1) and a 65-67-kd (AE3) keratin, previously shown to be markers for keratinization, are expressed only by lesions retaining a keratinized morphology. A 48-kd (AE1) and a 56-kd (AE3) keratin are present in all hyperproliferative (para- or nonkeratinized) disorders, but not in normal abdominal epidermis or in ichthyosis vulgaris which is a nonhyperproliferative disease. These two keratins have previously been found in various nonepidermal keratinocytes undergoing hyperproliferation, suggesting that these keratins are not epidermis-specific and may represent markers for hyperproliferative keratinocytes in general. In various epidermal diseases, there is a reciprocal expression of the (keratin) markers for hyperproliferation and keratinization, supporting the mutual exclusiveness of the two cellular events. Moreover, our results indicate that, as far as keratin expression is concerned, cultured human epidermal cells resemble and thus may be regarded as a model for epidermal hyperplasia. Finally, the apparent lack of any major, disease-specific keratin changes in the epidermal disorders studied so far implies that keratin abnormalities probably represent the consequence, rather than the cause, of these diseases.