An ultra-high sulfur keratin gene is expressed specifically during hair growth

To study the regulation of the hair cycle in the mouse, we have isolated and characterized a gene for ultra high sulfur keratin that is expressed specifically during the active hair growth cycle. The gene (gUHSK-704Eco) was isolated as a member of a gene cluster on a recombinant phage with a DNA insert of 18 kb that was isolated by screening a murine genomic library at low stringency with a synthetic oligonucleotide derived from a sheep high sulfur keratin gene (Powell, Nucleic Acids Res. 1983 11, 5327). The murine ultra-high sulfur keratin gene has no intervening sequence; the 558 nucleotide of the coding region specify 186 amino acids, of which 70 (37%) are cysteine. A Cys-Cys-Gln-Pro repeat is found 12 times within the coding region. RNA dot blots show that the ultra-high sulfur keratin gene is expressed during the hair cycle concomitant with the anterior-posterior temporal pattern of the normal murine hair cycle.     

Type II epithelial keratin 6hf (K6hf) is expressed in the companion layer, matrix, and medulla in anagen-stage hair follicles

More than half of the known keratin genes (n approximately 50) are expressed in the hair follicle. An in-depth knowledge of their differential expression in this organ will help us to understand the mechanisms of its formation and cycling, and the etiology of inherited hair disorders. Keratin 6hf is a type II keratin recently shown to occur in the companion layer. We cloned the mouse ortholog and characterized its expression in skin and oral mucosa. The mK6hf gene is 9.1 kb long and located in the cluster of type II keratin genes on mouse chromosome 15, between the keratin 6 (mK6alpha/mK6beta) and hair keratin genes. In situ hybridization and protein immunolocalization showed that, in addition to the companion layer, mK6hf is expressed in the upper matrix and medulla of the anagen-stage hair. This distribution is seen for all types of mouse hairs and medullated human hairs. The distribution of keratin 6hf protein in the hair shaft mirrors that of keratin 17, and the observation of reduced levels of keratin 6hf in keratin 17 null hair argues for a direct interaction between them. mK6hf is also expressed in the nail bed epithelium and fungiform papillae of dorsal tongue epithelium. Our findings provide an additional marker for the hair matrix and medulla, and suggest that the cellular precursors for the medulla, cortex, and cuticle compartments are already spatially segregated within the hair matrix. They also have obvious implications for the epithelial alterations associated with defects in keratin 6 genes.     

Cloning and characterization of a mouse type I hair keratin cDNA

A cDNA library was prepared from poly(A)-containing C57BL/6J mouse hair-root-enriched mRNA. The library was screened using a radiolabeled nucleic acid probe prepared from a sheep wool, Type I keratin cDNA clone (SWK2). Clone MHKA-1 was shown to contain a mouse hair, Type I keratin cDNA insert by positive hybridization-selection translation assay, and by the corresponding deduced amino acid sequence. The size of the cDNA insert is 1585 bp (excluding homopolymer tails) and on the basis of Northern blot analysis it corresponds to a mRNA of approximately 1.6 Kb. The cloned cDNA sequence includes the entire coding region for a protein of 416 amino acids (including the initiation methionine). Comparison of the deduced amino acid sequence with that of sheep wool, Type I keratin (8c1) reveals an overall corresponding sequence identity of 87%. In contrast, the MHKA-1 protein is significantly less similar to non-hair Type I keratins. An additional 3 amino acids (adjacent proline residues) not present in the wool protein have been identified near the middle of the carboxy-terminal end of the mouse protein. MHKA-1 is the first of a series of mouse hair follicle cDNA clones to be identified and characterized that will enable us to study follicular regulatory mechanisms and the interrelationships among the proteins in the mammalian hair follicle.     

Transient expression of mouse hair keratins in transfected HeLa cells: interactions between "hard" and "soft" keratins.

Although it has been shown previously that an acidic (type I) "soft" keratin can interact with many basic (type II) "soft" keratins to form 10-nm intermediate filaments, it has been unclear whether "soft" keratins are compatible with the "hard" keratins typically found in hair and nail. To address this issue and to generate more structural information about hard keratins, we have isolated and sequenced a cDNA clone that encodes a mouse hair basic keratin (b4). Our sequence data revealed new information regarding the structural conservation of hard keratins as a group, being significantly different from soft keratins. Using expression vectors containing appropriate cDNA inserts, we studied the expression of this basic (b4) as well as an acidic (a1) mouse hair keratin in HeLa cells. The expression of these alien hair keratins in the transfected cells was surveyed using a panel of monoclonal and polyclonal antibodies. Our results indicated that the basic and acidic hair keratin readily incorporated into the existing endogenous soft keratin network of HeLa cells. Overproduction of hair keratin, however, occasionally led to the formation of cytoplasmic aggregates containing both hard and soft keratins. These data suggest that although small amounts of newly synthesized hair keratins can incorporate into the "scaffolding" of the preformed soft keratin filament network, possibly through dynamic subunit exchange, overproduction of hard keratins can lead to the partial collapse of the soft keratin network. These observations, along with the deduced amino acid sequence data, support and extend the concept that hard and soft keratins, although closely related, are divergent enough to justify their being divided into two separate subgroups.     

Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge

Putative epithelial stem cells in the hair follicle bulge are thought to play pivotal roles in the homeostasis, aging, and carcinogenesis of the cutaneous epithelium. Elucidating the role of bulge cells in these processes has been hampered by the lack of gene promoters that target this area with specificity. Here we describe the isolation of the mouse keratin 15 (K15) promoter and demonstrate its utility for preferentially targeting hair follicle bulge cells in adult K15/lacZ transgenic mice. We found that patterns of K15 expression and promoter activity changed with age and correlated with levels of differentiation within the cutaneous epithelium; less differentiated keratinocytes in the epidermis of the neonatal mouse and in the bulge area of the adult mouse preferentially expressed K15. These findings demonstrate the utility of the K15 promoter for targeting epithelial stem cells in the hair follicle bulge and set the stage for elucidating the role of bulge cells in skin biology.     

Mapping of monilethrix to the type II keratin gene cluster at chromosome 12q13 in three new families, including one with variable expressivity

Monilethrix is an autosomal dominant disorder chiefly affecting hair. The degree of hair dystrophy is highly variable, as is the presence of additional features, such as follicular keratoses. In three British families of monilethrix, linkage has recently been reported to the type II keratin gene cluster at chromosome 12q13, and it has been suggested that the disease is due to a defect in the hard keratins of hair and nail. If monilethrix is a keratin disorder, we would predict that some pedigrees might map to the type I keratin gene cluster on 17q where hard keratin genes are also found. We have now studied clinically and by linkage analysis three new and unrelated pedigrees from England, Scotland and Spain, the first of which showed a variant phenotype. In this family the disease was expressed in four of 12 cases only as a follicular keratosis of the neck, elbows and knees, and without clinical or historical evidence of hair anomalies: non-penetrance in an obligate carrier was also observed. In all three families, we have established linkage to a series of microsatellite markers at the type II locus at 12q13 (Z_max=6.34 at 0=0.00 for D12S368) and have excluded linkage from the type I keratin gene cluster on 17q. It remains probable that monilethrix is a disorder of hard keratins, but at present there is no evidence that it is due to defects in type I keratins.     

Differential expression of type I hair keratins.

The hair follicle provides an excellent system in which to study growth and differentiation. Hair keratins are useful tissue-specific molecular markers for these events. By comparing a second mouse Type I hair keratin cDNA clone, MHKA-2, with our previously described MHKA-1, we have been able to contrast the nucleotide sequences and corresponding deduced amino acid sequences of the smallest (mHa4) and the largest (mHa1) major Type I hair keratins. Both nucleotide sequences and both deduced amino acid sequences share high identity but have distinct segments suitable for generation of specific molecular probes. Comparison of amino acid sequences adjacent to the central helical domains has demonstrated homologous subdomains, designated H1 and H2, in the Type I hair keratin nonhelical termini. Although there is only 56% amino acid identity in the carboxy-terminal nonhelical domains, a common sequence, T-------CGPC----R, has been identified in this domain, suggesting a possible common functional role for this portion of the molecule. In addition, it appears that mHa4 may differ in part from mHa1 by deletion of a segment between the H2 subdomain and the conserved sequence. Staining of mouse and human hair follicles with AmHa1, a monospecific polyclonal antibody for mHa1, and AE13, an antibody specific for all Type I hair keratins, suggests differential expression of individual Type I hair keratins in both species. This supports our hypothesis that distinct functional requirements are satisfied by the multiplicity of hair keratins.     

Keratin 16 expression defines a subset of epithelial cells during skin morphogenesis and the hair cycle

The morphogenesis of skin epithelia and adult hair follicle cycling both require integrated signaling between the epithelium and underlying mesenchyme. Because of their unique regulation, keratin intermediate filaments represent useful markers for the analysis of determination and differentiation processes in complex epithelia, such as the skin. In this study, we analyzed the distribution of mouse type I keratin 16 during skin morphogenesis, in the adult hair cycle, and in challenged epidermis. In mature hair follicles, we find keratin 16 along with its type II keratin partner keratin 6 in the companion layer of the outer root sheath during anagen and in the club hair sheath during catagen and telogen. During embryonic development, the distribution of keratin 16 is uncoupled from its presumed polymerization partner, keratin 6. Keratin 16 initially localizes within early hair germs, but rapidly shifts to a subset of cells at the interface of basal and suprabasal cells above and around the hair germ. The presence of keratin 16 at the transition between mitotically active and differentiating cells is recapitulated in primary keratinocytes cultured in vitro and in phorbol 12-myristate 13-acetate-treated back skin in vivo. We propose that keratin 16 marks cells in an intermediate state of cellular properties in which keratinocytes retain the flexibility required for activities such as cell migration and even mitosis but are resilient enough to provide the structural integrity required of the early suprabasal layers in the context of development, adult hair cycling, and wound repair.     

Ber-EP4 antigen is a marker for a cell population related to the secondary hair germ

Ber-EP4 is an antibody to a cell membrane glycoprotein of unknown function. In the skin, Ber-EP4 immunoreactivity has been reported to be localized in structures composed of basaloid epithelial cells, i.e. fetal epithelial germ cells, basal cell carcinoma, and trichoepithelioma as well as eccrine or apocrine ducts. In this study, we further characterized the follicular expression of Ber-EP4 immunoreactivity at different stages of the hair cycle of human terminal hair follicles. In addition, to clarify the location of Ber-EP4(+) cells, we compared the Ber-EP4 immunoreactivity with the expression of keratin 15 and keratin 19. Positive staining by Ber-EP4 was found in the lower part of the epithelial strand of late catagen hair follicles, in the secondary hair germ of telogen hair follicles, and in the matrix of early anagen hair follicles but not in any parts of mature anagen hair follicles. In contrast, the follicular expression of keratin 15 detected by using LHK15 antibody was restricted to two distinct parts of anagen hair follicles, i.e. the outer root sheath above the hair bulb and that of the isthmus including the bulge area, and to the outer root sheath of late catagen and telogen hair follicles. The follicular expressions of keratin 19 and that of keratin 15 were apparently superimposed, whereas keratin 15 expression was more extended. The immunoreactivity of LHK15 antibody and antikeratin 19 antibody against the secondary hair germ of telogen follicles was negative or dim. Our results suggest that Ber-EP4 reacts with the secondary hair germ and possibly a cell population related to the secondary hair germ but not with the presumptive stem cell population as revealed immunohistochemically either by the keratin 15 or keratin 19 expression.     

A novel mouse type I intermediate filament gene, keratin 17n (K17n), exhibits preferred expression in nail tissue

Inactivating the type I keratin 17 gene (mK17) causes severe but reversible hair loss in a strain-dependent fashion in mouse (McGowan et al, Genes Dev. 16:1412, 2002). Missense mutations in human K17 give rise to two dominantly inherited disorders apparented to ectodermal dysplasias, pachyonychia congenita (PC), and steatocystoma multiplex (SM). In contrast to the null phenotype in mouse, marked lesions are seen in the nail and nail bed and sebaceous glands of PC and SM patients, respectively. In an effort to understand the lack of nail involvement in mK17 null mice, we discovered that the gene located immediately 5' upstream from mK17 is functional and encodes a type I keratin protein highly analogous to mK17. mRNA and protein localization studies show that the expression of this novel gene is highly restricted and most prevalent in the nail bed and matrix, leading to its designation as mK17n (n stands for nail). Weak expression of mK17n also occurs in vibrissae follicles, in filiform and fungiform papillae of oral mucosa. These findings have direct implications for the mK17 null phenotype. Depending on the existence of a human ortholog or a functional equivalent, our findings may also provide a molecular explanation for several unusual aspects of hK17-based diseases.     

Characterization of mouse profilaggrin: evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N- terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal antibody raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.     

Prediction of a coding sequence for a novel type II keratin from N-terminal sequences of mouse epidermal proteins site-specifically deiminated in embryonic development

BACKGROUND: Epidermal keratinization involves various post-translational modifications including the deimination of arginine residues. Major deiminated proteins are derived from keratin K1. Two preferred deimination sites were identified in the V subdomain of mouse K1. An antibody against one of the deiminated peptide sequences (ACP) recognized deiminated mouse and human K1, and stained the cornified layers of human and infant mouse epidermis. ACP also stained the outermost layer of mouse embryonic epidermis. Western blotting revealed minor proteins showing strong ACP-positive signals in the mouse embryonic epidermal extract in which deiminated K1 derivatives were hardly detected. OBJECTIVE: To characterize ACP-positive proteins expressed in mouse embryonic epidermis. METHODS: ACP-positive proteins were isolated by preparative gel electrophoresis for N-terminal sequencing followed by blast searches for matching sequences in the protein and nucleotide database. RESULTS: We obtained N-terminal sequences of two ACP-positive proteins. A cDNA clone in the est_mouse database has an open reading frame for 202 amino acid residues containing both sequenced peptides. The deduced sequence shows typical features of the N-terminal portion of type II keratins. A virtually identical sequence to this reading frame is present in a genomic contig of chromosome 15 on which keratin type II genes are clustered. Sequential searches for overlapping cDNA clones in the est_mouse database along with similar searches in the genomic contig formulated a hypothetical cDNA sequence encoding a putative protein of 572 amino acid residues tentatively called K1-emb. CONCLUSION: We predicted a sequence of novel type II keratin site-specifically deiminated in embryonic mouse epidermis.     

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.     

A Missense Mutation within the Helix Initiation Motif of the Keratin K71 Gene Underlies Autosomal Dominant Woolly Hair/Hypotrichosis

Woolly hair (WH) is an abnormal variant of tightly curled hair, which is frequently associated with hypotrichosis. Non-syndromic forms of WH can show either autosomal-dominant WH (ADWH) or autosomal-recessive WH (ARWH) inheritance patterns. ARWH has recently been shown to be caused by mutations in either the lysophosphatidic acid receptor 6 (LPAR6) or lipase H (LIPH) gene. More recently, a mutation in the keratin K74 (KRT74) gene has been reported to underlie ADWH. Importantly, all of these genes are abundantly expressed in the inner root sheath (IRS) of human hair follicles. Besides these findings, the molecular mechanisms underlying hereditary WH have not been fully disclosed. In this study, we identified a Japanese family with ADWH and associated hypotrichosis. After exclusion of known causative genes, we discovered the heterozygous mutation c.422T>G (p.Phe141Cys) within the helix initiation motif of the IRS-specific keratin K71 (KRT71) gene in affected family members. We demonstrated that the mutant K71 protein led to disruption of keratin intermediate filament formation in cultured cells. To our knowledge, it is previously unreported that the KRT71 mutation is associated with a hereditary hair disorder in humans. Our findings further underscore the crucial role of the IRS-specific keratins in hair follicle development and hair growth in humans.