A novel type II cytokeratin, mK6irs, is expressed in the Huxley and Henle layers of the mouse inner root sheath

Hair follicle differentiation involves the expression of both epithelial-type keratins or cytokeratins and hair keratins as well as hair keratin-associated proteins. In this study, a cDNA clone encoding a cytokeratin family member was isolated using RNA differential display techniques. The predicted amino acid sequence derived from this clone, revealed a homology with a number of cytokeratins, not only in the central alpha-helical regions but also in the conserved portions of the amino and carboxy terminal domains, indicating that this protein represents a new member of the mouse type II cytokeratin family. Northern blot analysis showed expression in mouse skin, but not in other tissues, including tongue, esophagus, and forestomach. One- and two-dimensional western blot analysis showed that this new cytokeratin was 57 kDa in size and ran slightly below the area of cytokeratin 5, which corresponded to that of the cytokeratin 6 family members. Both RNA in situ hybridization and immunohistochemical studies of mouse anagen hair follicles demonstrated expression of this cytokeratin in the inner root sheath hair cone during anagen III and in the Henle and Huxley layers of the inner root sheath during anagen VI. The expression of the new cytokeratin began in the hair bulb and progressed up to the height of the keratogenous zone. Taken together the sum of the data analyzed, we have termed this novel cytokeratin mK6irs (mouse gene nomenclature k2-6g) to indicate both its similar mobility with K6 in two-dimensional gels and its specific expression in the inner root sheath of the hair follicle.     

hKAP1.6 and hKAP1.7, two novel human high sulfur keratin-associated proteins are expressed in the hair follicle cortex

Hair fiber differentiation involves the expression of both hair keratin intermediate filament proteins and their associated proteins, termed keratin-associated proteins. In this study, cDNA clones encoding two novel keratin-associated proteins were isolated from human hair follicle mRNA. The predicted amino acid sequence derived from these clones revealed that these proteins represent members of the human keratin-associated protein 1 family. They show strong sequence homology to two previously described keratin-associated protein 1 family members hKAP1.1 A and hKAP1.1B. We have called these new proteins hKAP1.6 and hKAP1.7, respectively. RNA in situ hybridization studies of human anagen hair follicles using a conserved probe for these four keratin-associated protein 1 members demonstrated the expression of this group in the differentiated portions of the hair cortex.     

Dynamic aspects of protein deimination in developing mouse epidermis

The cornified layer of mammalian epidermis contains deiminated keratins and filaggrin whose arginine residues are partly converted to citrulline residues by peptidylarginine deiminase (EC 3.5.3.15). We have attempted to study dynamic aspects of protein deimination using late embryonic to early postnatal mouse skin. The epidermis was separated from the dermis by brief immersion of skin into a weakly alkaline ammonium chloride solution. The total homogenate of the epidermis was subjected to western blotting analyses for quantitative densitometry of major keratins, deiminated proteins and immunoreactive filaggrin. We found marked increases in both deiminated keratins and deiminated filaggrin from the 18th day of gestation to 2 h after birth followed by rapid decreases to minimum levels at 6 h and subsequent gradual increases surpassing the earlier levels by 72 h after birth. Such variations were associated with consistent changes of the intensity of deiminated proteins stained immunocytochemically. These results suggest that the protein deimination might play a role in dealing with the drastic environmental change after birth. Furthermore, we found compartmentalization of both total and deiminated filaggrins into soluble and particulate fractions. The soluble compartment contained relatively more deiminated filaggrin than the particulate fraction.     

Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype–genotype correlation

Keratins are obligate heterodimer proteins that form the intermediate filament cytoskeleton of all epithelial cells. Keratins are tissue and differentiation specific and are expressed in pairs of types I and II proteins. The spectrum of inherited human keratin diseases has steadily increased since the causative role of mutations in the basal keratinocyte keratins 5 and 14 in epidermolysis bullosa simplex (EBS) was first reported in 1991. At the time of writing, mutations in 15 epithelial keratins and two trichocyte keratins have been associated with human diseases which include EBS, bullous congenital ichthyosiform erythroderma, epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, diffuse and focal non-epidermolytic palmoplantar keratoderma, pachyonychia congenita and monilethrix. Mutations in extracutaneous keratins have been reported in oral white sponge naevus and Meesmann's corneal dystrophy. New subtleties of phenotype-genotype correlation are emerging within the keratin diseases with widely varying clinical presentations attributable to similar mutations within the same keratin. Mutations in keratin-associated proteins have recently been reported for the first time. This article reviews clinical, ultrastructural and molecular aspects of all the keratin diseases described to date and delineates potential future areas of research in this field.     

Identification of citrulline residues in the V subdomains of keratin K1 derived from the cornified layer of newborn mouse epidermis

Citrulline residues are detected in keratins and filaggrin in the cornified layers of mammalian epidermis. Such citrulline residues are formed by the enzymatic deimination of arginine residues by peptidylarginine deiminase (EC 3.5.3.15). Major deiminated keratins are thought to be partially degraded/disulfide-cross-linked keratin K1 based on the immunoblotting profiles. In order to obtain more definitive evidence of the deimination of keratin K1 and also to investigate its functional significance, we attempted to identify its preferred acting sites of peptidylarginine deiminase. A partially degraded keratin K1 fraction obtained from the cornified layer of newborn mouse epidermis was subjected to limited proteolytic cleavages, and the resulting deiminated peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or reverse-phase high-performance liquid chromatography for N-terminal sequencing and/or amino acid analysis. At least two sites were identified, one in the V1 and the other in the V2 subdomains of keratin K1. An undecapeptide sequence covering the latter shows about 70% homology with an undecapeptide sequence in the V2 subdomain of human K1, a presumptive site of deimination. We speculated that the deimination of arginine residues in these subdomains might modulate their interactions with epidermal proteins other than keratins and filaggrin during the terminal stage of epidermal differentiation.     

Characterization of human keratin-associated protein 1 family members

Keratin-associated proteins are involved in the formation of the cross-linked network of the keratin-intermediate filament proteins that support hair fibers. In recent years, several keratin-associated protein genes have been identified and become an attractive topic in hair research. More recently, we isolated two cDNA encoding novel members of the human keratin-associated protein 1 family (human keratin-associated protein 1.6 and human keratin-associated protein 1.7), and described their expression in the hair follicle by RNA in situ hybridization. A comparison of human keratin-associated protein 1.6 and human keratin-associated protein 1.7 with other human keratin-associated protein 1 members revealed that keratin-associated protein 1 proteins are fundamentally composed of five distinct domains, and that they can be classified primarily by a striking variation in double cysteine-containing pentapeptide repeats in the repetitive I domain. The sum of the data analyzed suggests that human keratin-associated protein 1 family genes may have arisen mainly through gene duplication of the cysteine-repeat motifs during evolution.     

Regional variations in cytokeratin expression in palmo-plantar epidermis

The cytokeratin composition of palmo-plantar epidermis from sites with different degrees of mechanically induced thickening of the stratum corneum was analysed. The urea-soluble proteins of the stratum corneum were analysed by two-dimensional electrophoresis. Viable epidermal layers were analysed by immunofluorescence microscopy with polyclonal and monoclonal antibodies. A mouse monoclonal antibody specific for cytokeratin no. 9 was prepared for the study. Significant amounts of low molecular weight cytokeratins were found in suprabasal layers at sites with the most pronounced thickening of the stratum corneum. This was taken as evidence that palmo-plantar epidermis responds to mechanical stress with hyperproliferation. At sites where stratum corneum thickness is most increased this hyperproliferation appears to involve two different populations of cells--one capable of expressing high molecular weight, differentiation-related cytokeratins in the suprabasal epidermal layers, and one population that does not express these cytokeratins. At sites with intermediate epidermal hyperplasticity the high molecular weight cytokeratins were predominant in all suprabasal cells.     

Expression of common cytoplasmic antigens by epithelial cells of thymus and epidermis

Summary The expression of common cytoplasmic antigens by thymic epithelial cells and keratinocytes was analyzed by immunolabelling on cryostat sections of human and animal thymus and epidermis. Experimental sera to human epidermal keratin subunits of molecular weight (MW) 67 K, 63 K and 55 K were used, as well as human sera with antibodies to epidermal cytoplasmic antigens (KCA), reacting either with U-Cyt antigens expressed by cells from the upper compartment of the epidermis or with BLC antigens in cells from the basal layer. It was demonstrated that keratin subunits of MW 67 K and 63 K were detected only in the upper layers of human epidermis, whereas 55 K was present in all epidermal layers. On rabbit lip a labelling was obtained throughout the epidermis by the three immune sera. On thymus sections a cytoplasmic staining of epithelial cells was observed with the three immune sera, suggesting an antigenic similarity between cytokeratins of thymic epithelial cells and keratins of the upper layers of human epidermis. Two human sera with U-Cyt antibodies and one with BCL antibodies labelled human thymic epithelial cells but were negative with mice thymus. Ten other sera with KCA were negative on both thymus. These results show that the expression of keratins and cytoplasmic antigens, defined by human KCA, differed in immunofluorescence on normal human epidermis, rabbit lip and thymic epithelial cells. They suggests a heterogeneity among human keratinocyte cytoplasmic antigens defined by KCA and the presence in man of common antigenic constituents different from keratins in epidermal cells and thymic epithelial cells.Supported by INSERM CRL 812045 and UER Grange Blanche     

An asparagine to threonine substitution in the 1A domain of keratin 1: a novel mutation that causes epidermolytic hyperkeratosis

Epidermolytic hyperkeratosis (EHK) is a congenital, autosomal dominant disorder of cornification characterized by hyperkeratosis and blister formation. The clinical manifestations are heterogeneous, with respect to the extent of body surface involvement, palmar and plantar hyperkeratosis and the presence of erythroderma. Point mutations in the genes encoding the suprabasal-specific keratins, keratins 1 and 10 have been identified in EHK patients. The inappropriate amino acid substitutions cause a collapse of the keratin filament network, resulting in cytolysis of the involved keratinocytes. We report a severe case of EHK with a single base pair mutation that causes a threonine for asparagine substitution in residue 8 (N8T) of the 1A region of the keratin 1 protein. This is the region involved in molecular overlaps between neighboring keratin heterodimers. These findings suggest that even conservative amino acid substitutions in overlap regions can cause tonofilament clumping.     

Surface adaptations of the vertebrate epidermis to friction.

Epidermal surfaces in representative vertebrates specialized for lowered or increased friction were studied with the scanning electron microscope. Microvillous and microridged patterns predominate in aquatic vertebrates. In squamate reptiles, the complex and varied ornamentation of the Oberh?utchen functions both in adhesive modifications and in modulating surface reflectivity. Frictional surfaces in birds and mammals are characterized more by anatomical than by cytologic specializations, the detailing of surface cells being mostly a function of turnover rate.     

Polymorphic keratins in human epidermis

Human epidermal keratins from many different individuals were identified and compared by both high-resolution 1- and 2-dimensional gel electrophoresis and immunoblotting. While the polypeptide patterns obtained for keratin-enriched cytoskeletal preparations could be considered typical of normal interfollicular epidermis, they also disclosed variations, among the individuals, concerning some of the constituent protein subunits. Three sets of interindividually varying keratins could be distinguished owing to their distinct, though small, differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels and their similar or identical charge characteristics upon nonequilibrium pH gradient electrophoresis: the basic keratins 1a and 1b as well as 5a and 5b and the acidic keratins 10a and 10b. Of each set either a doublet, showing a marked 1:1 ratio of polypeptides, or the one or the other variant protein was detected together with keratin 14, which did not display any variation in a series of 148 individual tissue samples tested. Thus, the keratin composition of human epidermis could be summarized in the formula: (1a v 1b) + (5a v 5b) + (10a v 10b) + 14. The systematic appearance of the variants suggested that each protein within a set is the product of an independent allele. In support of this hypothesis we have found that the same variant is expressed in other epithelia of a given individual. Moreover, the frequency of any of the keratins in our sampling concurred with the frequency predicted by the Hardy-Weinberg relation for the distribution of alleles in a population, as did the frequency distribution of particular keratin patterns.     

A comparative study of the immunologic properties of hoof and nail fibrous proteins

We have studied the localization of epidermal (soft) and nail and hoof (hard) fibrous keratins in the various anatomic regions of bovine hoof and human nail. Indirect immunofluorescence was performed on frozen sections of various parts of hoof and nail with antibodies demonstrated to be specific for hard and soft fibrous keratins by the Ouchterlony technique. The antibody to hard fibrous keratin reacted with the upper region of hoof bed and matrix tissue but not perihoof epidermis. The antibody to soft fibrous keratin reacted with hoof bed, matrix tissue, and perihoof epidermis. Electrophoretic analysis of the fibrous proteins of hoof bed indicated they contained both soft and hard fibrous keratins while matrix tissue contained only hard keratins. Immunoblot analysis of matrix fibrous proteins indicated that most of the polypeptides reacted with both antibodies. These results indicate that the antibody to soft fibrous keratin cross-reacts with hard fibrous keratin but the antibody to hard fibrous keratin appears to be specific. Immunologic studies, therefore, must be correlated with electrophoretic studies in order to define the localization of the various types of fibrous keratins. Similar results were obtained with human nail.     

Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins

The type I keratin 19 is unusual in its tissue distribution in that under normal circumstances it does not seem to be restricted, as the other keratins are, to expression in either stratified or simple epithelia. In addition to the previously reported distribution of keratin 19 in human tissues, we have observed keratin 19 in epidermal basal cells, in a defined region of the hair follicle, and in nipple epidermis. We noticed that expression of keratin 19 appears to be especially characteristic of regions of labile or variable cellular differentiation as indicated by the presence of multiple keratin phenotypes in close proximity to each other. Using a monoclonal antibody recognizing keratin 19 (LP2K) to screen a human placenta cDNA expression library, we have isolated, cloned, and sequenced cDNA coding for full-length human keratin 19, as confirmed by its reactivity with several other known anti-keratin 19 monoclonal antibodies and by the near identity of its sequence with that of the bovine keratin 19 homologue. This similarity extends to both proteins being truncated at the C-terminal end to only 13 amino acids beyond the rod domain. Although the amino acid homology over the N-terminal and helical rod domains is particularly high, the human and bovine proteins diverge substantially over the short C-terminal domain, which suggests that this region has no conserved function. Comparison with other type I keratins indicates that the closest evolutionary neighbors of keratin 19 are keratinocyte keratins, probably 13 and 14, and not the simple epithelial keratin 18. Assessing the histochemistry and sequence data together, we propose that the cell may use this apparently deficient keratin as a "neutral" keratin. While unimpaired in its ability to polymerize (keeping the cell integrated into the epithelial sheet via filament-desmosome networks), keratin 19 expression does not irrevocably commit a cell to any one of the local differentiation options. Such predicted differentiational flexibility may also imply vulnerability to transformation.     

Ageing processes influence keratin and KAP expression in human hair follicles

In many cultures, a youthful look is strictly linked to strong and healthy hair. Source of the hair fibre is the hair follicle, a highly specialized skin appendage. Biological alterations because of intrinsic or extrinsic stimuli can destabilize this perfectly organized system, thus effecting hair growth or metabolism. Also, ageing could be characterized as a disturbance in this well-balanced machinery. Albeit the predominant symptom of hair ageing, greying, is addressed in a plurality of research activities, further age-related changes, e.g. related to hair structure, remain obscure. Therefore, we characterized hair follicles of two volunteer panels (below 25 years, above 50 years) on the molecular level, especially focussing on alterations influencing gene expression of keratins and keratin-associated proteins. We showed that concordantly to other biological systems the hair follicle undergoes several modifications during the ageing process associated among others with a significant decline in these structural proteins. Providing strategies to fight against these age-related changes is a challenge for hair science.     

Identification of new components of the cornified envelope of human and bovine epidermis

Antibodies raised in rabbits to purified cornified envelopes (CEs) of cultured human keratinocytes reacted in a peripheral fashion with the granular and spinous layers of human, cow, rat, and mouse epidermis. This reaction could not be abolished by absorption of the antibody with purified human involucrin to which the antibody reacted by immunoblot, thus indicating the presence of an additional antigenic determinant(s). Antibodies raised to CEs of human epidermis stained the cytoplasm of epidermal cells and gave a strong reaction to cytokeratins and a weak one to involucrin, indicating that in tissue the keratins are also cross-linked. The antibody prepared to bovine CEs reacted with keratins, but when absorbed with prekeratin it gave a peripheral staining pattern with epidermis and reacted strongly with a 126 kD component of the neutral buffer extract of cow snout epidermis and weakly with 205 kD and 85 kD ones. This antibody reacted with human involucrin by immunoblot while an antibody to involucrin stained 143 kD, 119 kD, 113 kD, and 107 kD polypeptides in the bovine extract. These latter 3 bands were shown to be substrates of transglutaminase. Further, a monoclonal antibody to bovine CEs reacted with the 119 kD and 113 kD bands and gave a peripheral staining pattern in the epidermis. Proof that the 126 kD protein was a precursor of the envelope was obtained by preparing an antibody to it and demonstrating peripheral staining of epidermal cells. These results point out the value of preparing antibodies to CE as an additional approach to studying the composition of CEs and demonstrate previously undescribed components in human and bovine tissue.     

Human hair keratin-associated proteins

Hair keratin-associated proteins (KAP) are a major component of the hair fiber, and play crucial roles in forming a strong hair shaft through a cross-linked network with keratin intermediate filaments (KIF), which are produced from hair keratins. Recently, the study of human KAP has advanced significantly. So far, five clusters of human KAP genes have been characterized, leading to the identification of more than 80 individual human KAP genes. In situ hybridization studies have demonstrated sequential and spatial expression patterns of these KAP members in differential portions of the hair fiber cortex and cuticle. Furthermore, several human KAP genes have size polymorphisms that are mainly because of variable numbers of cysteine-rich repeat segments, and the patterns of some of these size variants are distinct between different human populations.