Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin

A novel mouse cDNA named hornerin was isolated by RNA differential display applied to developing mouse skin. Hornerin, which has 2,496 amino acids, comprises EF-hand domains at the N terminus followed by a spacer sequence and a large repetitive domain, indicating that hornerin is a novel member of the "fused gene"-type cornified envelope precursor protein family. The repetitive domain of hornerin was found to be rich in glycine, serine, and glutamine. Hornerin was expressed in the tongue, esophagus, forestomach, and skin among the adult mouse tissues examined, all of them cornifying stratified epithelium. In the embryonic mouse skin, hornerin mRNA was first detected on gestational day 15.5 in the epidermis coincidentally with the formation of a granular layer. In accordance with this, hornerin was detected in the granular and cornified layers of the mature epidermis. In the granular cells of the epidermis, the hornerin protein was detected in keratohyalin granules together with profilaggrin. Furthermore, Western blot analysis of the mouse skin showed that the hornerin protein was cleaved during the process of epidermal differentiation, indicating possible posttranslational proteolytic processing as is observed in profilaggrin. Differentiation of primary mouse epidermal keratinocytes with 0.12 mm Ca(2+) resulted in the induction of hornerin. These results indicate that hornerin is structurally as well as functionally most similar to profilaggrin among the family members and possibly plays pleiotropic roles, including a role in cornification.     

Expression of hornerin in stratified squamous epithelium in the mouse: a comparative analysis with profilaggrin.

We have recently identified a novel protein named hornerin, the structural features of which are most similar to those of profilaggrin, an essential protein for keratinization of epidermal tissues. In this study we examined the expression of hornerin compared with that of profilaggrin in various mouse tissues. Hornerin was expressed in the upper epidermis of newborn mouse skin, as was profilaggrin. In addition, both hornerin and profilaggrin were expressed in the tongue, esophagus, and forestomach. In all four tissues, immunostaining for hornerin and profilaggrin showed a granular pattern, and most of the signals for the two proteins were co-localized on keratohyalin granules. This was confirmed by double immunoelectron microscopy. Within keratohyalin granules, hornerin was detected more frequently in the periphery, whereas profilaggrin was equally distributed. A quantitative RT-PCR revealed that both genes were expressed at highest levels in the forestomach and at the next highest levels in skin. Profilaggrin mRNA was most abundant in the forestomach. In skin, the amount of hornerin mRNA was more than fourfold greater than the amount of profilaggrin mRNA. These results form the basis for a better understanding of possible overlapping and/or differential functions of hornerin and profilaggrin.     

Expression of filaggrin-2 protein in the epidermis of human skin diseases: A comparative analysis with filaggrin

Filaggrin-2 is a member of the S100 fused-type protein family, and the structural features and expression of filaggrin-2 are similar to those of profilaggrin, a protein essential for keratinization. In the present study, we investigated the expression profile of filaggrin-2 in patients with skin diseases using antibodies against the repetitive region of filaggrin-2. In tissue samples from patients with skin diseases which are associated with a decrease in filaggrin, including ichthyosis vulgaris, atopic dermatitis and psoriasis vulgaris, the expression level of filaggrin-2 was markedly decreased compared to that in normal skin samples. In contrast, the expression of filaggrin-2 increased in parallel with that of filaggrin in samples of tissue from patients with skin diseases associated with hyperkeratosis, such as lichen planus and epidermolytic ichthyosis. Interestingly, filaggrin-2 signals were observed in slightly higher layers of the epidermis in comparison to those of filaggrin. Similarly, the expression of filaggrin-2 proteins was induced slightly later than filaggrin in the cultured keratinocytes. These findings suggest that filaggrin-2 may play an overlapping role with filaggrin in epithelial cornification; however, it may also have a partially distinct role in the molecular processes of cornification.     

Zinc accumulation and metallothionein gene expression in the proliferating epidermis during wound healing in mouse skin

Metallothionein (MT), a low molecular weight metal-binding protein, has been related to zinc and copper metabolism, the acute-phase response, and cellular proliferation. In this study, we investigated changes in zinc metabolism and MT gene expression occurring in tissue damage and repair during wound healing in mouse skin. Northern blot analysis revealed that a significant increase of MT mRNA was observed in the liver for 18 h after wounding, and serum zinc downfall and hepatic zinc uptake were observed. In situ hybridization analysis showed that no significant expression of MT mRNA was detected within the first 9 h after wounding. However, it was expressed restrictively in the proliferating epidermis of the wound margin after 12 h. Zinc began to accumulate in wounded skin after MT gene expressed. Northern blotting and immunocytochemical staining revealed that MT has been synthesized actively during the growth phase compared with the stationary phase in normal human epidermal keratinocytes in vitro. Intracellular zinc accumulation was observed in the proliferating cells. We concluded that hepatic MT plays an important role as an acute phase protein against host damage, and epidermal MT contributes in the supply of zinc to wounded tissue and activates proliferation for the regeneration of epidermis. Accepted: 2 July 1999     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

Caspase-14 protects against epidermal UVB photodamage and water loss

Caspase-14 belongs to a conserved family of aspartate-specific proteinases. Its expression is restricted almost exclusively to the suprabasal layers of the epidermis and the hair follicles. Moreover, the proteolytic activation of caspase-14 is associated with stratum corneum formation, implicating caspase-14 in terminal keratinocyte differentiation and cornification. Here, we show that the skin of caspase-14-deficient mice was shiny and lichenified, indicating an altered stratum-corneum composition. Caspase-14-deficient epidermis contained significantly more alveolar keratohyalin F-granules, the profilaggrin stores. Accordingly, caspase-14-deficient epidermis is characterized by an altered profilaggrin processing pattern and we show that recombinant caspase-14 can directly cleave profilaggrin in vitro. Caspase-14-deficient epidermis is characterized by reduced skin-hydration levels and increased water loss. In view of the important role of filaggrin in the structure and moisturization of the skin, the knockout phenotype could be explained by an aberrant processing of filaggrin. Importantly, the skin of caspase-14-deficient mice was highly sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation, leading to increased levels of UVB-induced apoptosis. Removal of the stratum corneum indicate that caspase-14 controls the UVB scavenging capacity of the stratum corneum.     

Characterization of human cytokeratin 2, an epidermal cytoskeletal protein synthesized late during differentiation.

Among the more than 30 different human proteins of the cytokeratin (CK) group of intermediate filament (IF) proteins, the significance of the epidermal polypeptide CK 2 (Moll et al., 1982, Cell 31, 11-24) has been repeatedly questioned in the literature. Here, we show, by in vitro translation and protein gel electrophoresis, that human epidermis from various body sites does indeed contain relatively large amounts of mRNA encoding a distinct polypeptide comigrating with native epidermal CK 2. We also report the isolation of a cDNA clone encoding the complete sequence of CK 2, which is a type II CK different from--but related to--epidermal CKs 1 and 5 on the one hand and corneal CK 3 on the other. The mRNA of approximately 2.6 kb encodes a polypeptide of 645 amino acids and M(r) 65,852, in good agreement with the value of 65.5 kDa previously estimated from gel electrophoresis. This human CK, the largest so far known, displays several features typical of CKs of stratified epithelia, including numerous repeats of glycine-rich tetrapeptides in the head and tail domains. Northern blot and in situ hybridizations have shown that CK 2 is expressed strictly suprabasally, usually starting in the third or fourth cell layer of epidermis, and this was confirmed at the protein level by immunohistochemistry using CK 2-specific antibodies. The protein has been detected as a regular epidermal component in skin samples from different body sites, albeit as a minor CK in "soft skin" (e.g., breast nipple, penile shaft, axilla), but not in foreskin epithelium and in other epithelia, in squamous metaplasias and carcinomas, or in cultured cell lines derived therefrom. We propose that CK 2 is a late cytoskeletal IF addition synthesized during maturation of epidermal keratinocytes which probably contributes to terminal cornification.     

Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in Ichthyosis vulgaris

Filaggrin is a histidine-rich, basic protein whose name was first proposed based on its ability to aggregate intermediate filaments in vitro. Based on this in vitro observation, it has generally been assumed that filaggrin functions in vivo as a matrix protein which causes keratin filaments to become densely packed in the terminally differentiated cornified cells. Inconsistent with this view however, is the well-known observation that keratin aggregation appears to proceed normally in the affected epidermis of ichthyosis vulgaris patients despite a greatly reduced quantity of filaggrin. To address this issue, we used immuno-electron microscopy to localize filaggrin and its cross-reactive precursor, profilaggrin, in human and mouse epidermis, as well as in ichthyosis vulgaris epidermis. We found that the localization of filaggrin in lower cornified cells correlates precisely with the formation of aggregated keratin filaments, and the disappearance of filaggrin in upper cornified cells correlates precisely with the loosening of keratin filaments. Furthermore, we showed that, even in ichthyosis vulgaris, small amounts of filaggrin/profilaggrin are present as electron-dense deposits associated with keratin filaments in the granular cells, and that the localization of this small amount of antigen again correlates with the aggregation state of keratin filaments. These data strongly suggest that filaggrin is indeed involved in filament aggregation in vivo.     

Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation.

A new human 33-kDa serine protease was purified from human epidermis, and its cDNA was cloned from a keratinocyte library, from mRNA from a human keratinocyte line (HaCat) and from mRNA from human skin. Polyclonal antibodies specific for the new protein detected three groups of proteins in partially purified extracts of cornified eptihelium of human plantar skin. The three components are proposed to correspond to proenzyme, active enzyme, and proteolytically modified active enzyme. After N-deglycosylation, there was a decrease in apparent molecular mass of all detected components. Expression of the cloned cDNA in a eukaryotic virus-derived system yielded a recombinant protein that could be converted to an active protease by treatment with trypsin. Polymerase chain reaction analyses of cDNA from a number of human tissues showed high expression of the new enzyme in the skin and low expression in brain, placenta, and kidney. Homology searches yielded the highest score for porcine enamel matrix protease (55% amino acid sequence homology). High scores were also obtained for human and mouse neuropsin and for human stratum corneum chymotryptic enzyme. The function of this new protease, tentatively named stratum corneum tryptic enzyme, may be related to stratum corneum turnover and desquamation in the epidermis.     

Appearance of tenascin in healing skin of the mouse: possible involvement in seaming of wounded tissues

Distribution of the extracellular matrix glycoprotein tenascin during wound healing in mouse skin was studied immunohistochemically. Within 24 hours after wounding, and preceding the formation of granulation tissue, tenascin appeared in the basement membranes beneath epidermis and hair follicles adjacent to the wound edges and in the wounded edges of cutaneous muscle layer. Granulation tissue began to form in the wound space at about 1-2 days and was immediately covered by epidermis. Tenascin first appeared in the periphery of the granulation tissue beneath healing epidermis and around the wounded edges of cutaneous muscle layer. Then the tenascin-positive area extended into the inner region of granulation tissue. At about 5-7 days, all of the granulation tissue was intensely stained with anti-tenascin serum. Tenascin immunoreactivity decreased as granulation tissue was replaced with reconstructed dermal tissue at 7-14 days. In most cases, tenascin staining persisted longest in the dermis beneath the healing epidermis and at the juncture of healing edges of cutaneous muscle layer. It disappeared at about 10-14 days after wounding. These findings suggest that tenascin may play an important role in the seaming of wounded tissues.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair

Fetal wound healing is characterized by minimal inflammation and scarless repair. IL-6 stimulates inflammation in postnatal wound healing. We hypothesized that fetal skin has a diminished IL-6 response and that exogenous IL-6 will result in scar formation. Human adult or fetal skin was placed subcutaneously in SCID mice and incisionally wounded. Wounds were excised after 4, 12, 24 or 72 h for IL-6 mRNA quantification by RT-PCR. In other grafts, 5 microgram of IL-6 was injected at wounding and then harvested at 7 days for analysis of scar formation. IL-6 production was examined in primary cultures of human fetal or adult dermal fibroblasts incubated for 8 h with 0, 0.1, 1 or 10 ng/ml of PDGF-BB. IL-6 mRNA was detected 4 h after wounding in fetal and adult wounds, but by 12 h there was no IL-6 mRNA in the fetal wounds. Adult wounds had IL-6 mRNA persisting to 72 h. IL-6 administration to fetal wounds resulted in scar formation. Fetal fibroblasts produced less IL-6 protein and mRNA at all points examined (P<0.01 vs adult). Diminished production of inflammatory cytokines such as IL-6 may be responsible for the lack of inflammation seen during fetal wound healing. Diminished inflammation may provide a permissive environment for scarless wound healing.