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.     

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.     

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.     

Chromosomal localization of acidic and basic keratin genes of the domestic dog

Our laboratories are interested in characterizing genes involved in the myriad of heritable diseases affecting the domestic dog, Canis lupus familiaris, and in development of detailed genetic and physical maps of the canine genome. Included in these efforts is examination of conservation of the genetic organization, structure, and function of gene families involved in diseases of the canine skin, skeleton, and eye. To that end, study of the highly conserved keratin gene family was undertaken. Keratins belong to the superfamily of intermediate filaments and are the major structural proteins of the epidermis, hair, and nail. The keratins are highly conserved throughout vertebrate evolution both at the DNA and amino acid sequence levels. Mutations in genes encoding epithelial keratins are known to cause various diseases in humans, and similar histopathological presentations have been reported in the dog. The keratins are divided into two groups, type I (acidic) and type II (basic). In the human, the genes encoding the acidic and basic keratins are clustered on Chrs 17 and 12, respectively. The same genetic arrangement is seen in the mouse with the acidic and basic keratin gene clusters found on Chrs 11 and 15, respectively. Reported here are the chromosomal localization of acidic and basic canine keratin genes as well as supportive sequence data. Fluorescence in situ hybridization (FISH) experiments with clones isolated from a canine genomic library suggest that the acidic keratin gene cluster resides on CFA9 and the basic keratin gene cluster is located on CFA27. Received: 25 September 1998 / Accepted: 1 December 1998     

'Hard' and 'soft' principles defining the structure, function and regulation of keratin intermediate filaments.

Keratins make up the largest subgroup of intermediate filament proteins and represent the most abundant proteins in epithelial cells. They exist as highly dynamic networks of cytoplasmic 10-12 nm filaments that are obligate heteropolymers involving type I and type II keratins. The primary function of keratins is to protect epithelial cells from mechanical and nonmechanical stresses that result in cell death. Other emerging functions include roles in cell signaling, the stress response and apoptosis, as well as unique roles that are keratin specific and tissue specific. The role of keratins in a number of human skin, hair, ocular, oral and liver diseases is now established and meshes well with the evidence gathered from transgenic mouse models. The phenotypes associated with defects in keratin proteins are subject to significant modulation by functional redundancy within the family and modifier genes as well. Keratin filaments undergo complex regulation involving post-translational modifications and interactions with self and with various classes of associated proteins.     

The organization of the keratin I and II gene clusters in placental mammals and marsupials show a striking similarity

The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials are sister groups a corresponding organization is also expected for the archetype mammal. Since hair is a mammalian acquisition the following features of the cluster refer to its origin. In both clusters hair keratin genes arose at an interior position. While we do not know from which epithelial keratin genes the first hair keratins type-I and -II genes evolved, subsequent gene duplications gave rise to a subdomain of the clusters with many neighboring hair keratin genes. A second subdomain accounts in both clusters for 4 neighboring genes encoding the keratins of the inner root sheath (irs) keratins. Finally the hair keratin gene subdomain in the type-I gene cluster is interrupted after the second gene by a region encoding numerous genes for the high/ultrahigh sulfur hair keratin-associated proteins (KAPs). We also propose a tentative synteny relation of opossum and human genes based on maximal sequence conservation of the encoded keratins. The keratin gene clusters of the opossum seem to lack pseudogenes and display a slightly increased number of genes. Opossum keratin genes are usually longer than their human counterparts and also show longer intergenic distances.     

Genomic organization and amplification of the human keratin 15 and keratin 19 genes

Keratin intermediate filaments are the major components of the cytoskeleton in epithelial cells. Mutations in keratin genes have been documented in many disorders of the skin, nails, hair, and mucous membranes. Although no mutations have been described in either keratin 15 or keratin 19, they are good candidates for other as yet uncharacterized genetic disorders of keratinization, particularly as the skin, nails, hair, and conjunctiva are sites of keratin 15 and 19 expression. To facilitate future mutation detection analyses, we have therefore characterized the intron-exon organization of the human keratin 15 and keratin 19 genes. The keratin 15 gene comprises 8 exons spanning approximately 5.1 kb on 17q21, and the keratin 19 gene consists of 6 exons covering approximately 4.7 kb on 17q21. We have also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.     

Human keratin hydrogels support fibroblast attachment and proliferation in vitro

Human hair keratins have a strong potential for development as clinically relevant biomaterials because they are abundant and bioactive and are a realistic source of autologous proteins. Specifically, keratins have the propensity to polymerize in an aqueous environment to form hydrogels. In order to evaluate the suitability of keratin hydrogels as substrates for cell culture, we have fabricated hydrogels using keratins extracted from human hair by inducing polymerization with Ca²⁺; these hydrogels exhibit highly branched and porous micro-architectures. L929 murine fibroblasts have been used in a preliminary cell culture study to compare the in vitro biocompatibility of the keratin hydrogels with collagen type 1 hydrogels of similar viscoelastic properties. Our results reveal that keratin hydrogels are comparable with collagen hydrogels in terms of the promotion of cell adhesion, proliferation and the preservation of cell viability. Interestingly, cells remain clustered in proliferative colonies within the keratin hydrogels but are homogeneously distributed as single cells in collagen hydrogels. Collectively, our results demonstrate that keratin hydrogels can be used as substrates for cell culture. Such gels might find applications as templates for soft tissue regeneration.     

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.     

Phenotypes,genotypes and their contribution to understanding keratin function

A large number of mutations in keratin genes underlie inherited tissue fragility disorders of epithelia. The genotype-phenotype correlations emerging from these studies provide a rich source of information about the function of keratins that would have taken decades to achieve by a purely transgenic approach. Human disease studies are being supplemented by engineered mouse mutant studies, which give access to the effects of genetic alterations unlikely to occur naturally. Evidence is emerging that the great diversity of keratins might be required to enable cells to adapt their structure in response to different signalling pathways.     

Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.     

Intermediate filaments: a role in epithelial polarity

Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and Caenorhabditis elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratin-null or overexpression phenotypes are still missing, it is hoped that investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments.     

Human type I hair keratin pseudogene ϕ hHaA has functional orthologs in the chimpanzee and gorilla: evidence for recent inactivation of the human gene after the Pan-Homo divergence

In addition to nine functional genes, the human type I hair keratin gene cluster contains a pseudogene, phihHaA (KRTHAP1), which is thought to have been inactivated by a single base-pair substitution that introduced a premature TGA termination codon into exon 4. Large-scale genotyping of human, chimpanzee, and gorilla DNAs revealed the homozygous presence of the phihHaA nonsense mutation in humans of different ethnic backgrounds, but its absence in the functional orthologous chimpanzee (cHaA) and gorilla (gHaA) genes. Expression analyses of the encoded cHaA and gHaA hair keratins served to highlight dramatic differences between the hair keratin phenotypes of contemporary humans and the great apes. The relative numbers of synonymous and non-synonymous substitutions in the phihHaA and cHaA genes, as inferred by using the gHaA gene as an outgroup, suggest that the human hHaA gene was inactivated only recently, viz., less than 240,000 years ago. This implies that the hair keratin phenotype of hominids prior to this date, and after the Pan-Homo divergence some 5.5 million years ago, could have been identical to that of the great apes. In addition, the homozygous presence of the phihHaA exon 4 nonsense mutation in some of the earliest branching lineages among extant human populations lends strong support to the "single African origin" hypothesis of modern humans.     

Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation

Keratins K14 and K5 have long been considered to be biochemical markers of the stratified squamous epithelia, including epidermis (Moll, R., W. Franke, D. Schiller, B. Geiger, and R. Krepler. 1982. Cell. 31:11-24; Nelson, W., and T.-T. Sun. 1983. J. Cell Biol. 97:244-251). When cells of most stratified squamous epithelia differentiate, they downregulate expression of mRNAs encoding these two keratins and induce expression of new sets of keratins specific for individual programs of epithelial differentiation. Frequently, as in the case of epidermis, the expression of differentiation-specific keratins also leads to a reorganization of the keratin filament network, including denser bundling of the keratin fibers. We report here the use of monospecific antisera and cRNA probes to examine the differential expression of keratin K14 in the complex tissue of human skin. Using in situ hybridizations and immunoelectron microscopy, we find that the patterns of K14 expression and filament organization in the hair follicle are strikingly different from epidermis. Some of the mitotically active outer root sheath (ORS) cells, which give rise to ORS under normal circumstances and to epidermis during wound healing, produce only low levels of K14. These cells have fewer keratin filaments than basal epidermal cells, and the filaments are organized into looser, more delicate bundles than is typical for epidermis. As these cells differentiate, they elevate their expression of K14 and produce denser bundles of keratin filaments more typical of epidermis. In contrast to basal cells of epidermis and ORS, matrix cells, which are relatively undifferentiated and which can give rise to inner root sheath, cuticle and hair shaft, show no evidence of K14, K14 mRNA expression, or keratin filament formation. As matrix cells differentiate, they produce hair-specific keratins and dense bundles of keratin filaments but they do not induce K14 expression. Collectively, the patterns of K14 and K14 mRNA expression and filament organization in mitotically active epithelial cells of the skin correlate with their relative degree of pluripotency, and this suggests a possible basis for the deviation of hair follicle programs of differentiation from those of other stratified squamous epithelia.     

Embryonic simple epithelial keratins 8 and 18: chromosomal location emphasizes difference from other keratin pairs.

The keratins 8 and 18 of simple epithelia differ from stratified epithelial keratins in tissue expression and regulation. To examine the specific properties of human keratin 8, we cloned and sequenced the cDNA from a placental mRNA expression library and defined the optimum state of such clones for expression in bacterial plasmid vectors. Using the polymerase chain reaction we identified and sequenced three introns and located the single active gene for keratin 8, out of a background of 9 to 24 pseudogenes, on chromosome 12. This chromosome contains several genes for type II keratins and also the gene for keratin 18, the type I keratin that is coexpressed with keratin 8. This location of both members of a keratin pair on a single chromosome is thus far unique among the keratin genes; it is consistent with the hypothesis that keratins 8 and 18 may be closer to an ancestral keratin gene than the keratins of more highly differentiated epithelia.     

Biochemical identification and tissue-specific expression patterns of keratins in the zebrafish Danio rerio

We have identified a number of type I and type II keratins in the zebrafish Danio rerio by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot-binding assay and immunoblotting. These keratins range from 56 kDa to 46 kDa in molecular mass and from pH 6.6 to pH 5.2 in isoelectric point. Type II zebrafish keratins exhibit significantly higher molecular masses (56–52 kDa) compared with the type I keratins (50–48 kDa), but the isoelectric points show no significant difference between the two keratin subclasses (type II: pH 6.0–5.5; type I: pH 6.1–5.2). According to their occurrence in various zebrafish tissues, the identified keratins can be classified into “E” (epidermal) and “S” (simple epithelial) proteins. A panel of monoclonal anti-keratin antibodies has been used for immunoblotting of zebrafish cytoskeletal preparations and immunofluorescence microscopy of frozen tissue sections. These antibodies have revealed differential cytoplasmic expression of keratins; this not only includes epithelia, but also a variety of mesenchymally derived cells and tissues. Thus, previously detected fundamental differences in keratin expression patterns between higher vertebrates and a salmonid, the rainbow trout Oncorhynchus mykiss, also apply between vertebrates and the zebrafish, a cyprinid. However, in spite of notable similarities, trout and zebrafish keratins differ from each other in many details. The present data provide a firm basis from which the application of keratins as cell differentiation markers in the well-established genetic model organism, the zebrafish, can be developed.