Characterization of sciellin, a precursor to the cornified envelope of human keratinocytes

The cornified envelope, located beneath the plasma membrane of terminally differentiated keratinocytes, is formed as protein precursors are cross-linked by a membrane associated transglutaminase. This report characterizes a new precursor to the cornified envelope. A monoclonal antibody derived from mice immunized with cornified envelopes of human cultured keratinocytes stained the periphery of more differentiated cells in epidermis and other stratified squamous epithelia including hair and nails. The epitope was widely conserved among mammals as determined by immunohistochemical and Western analysis. Immunoelectron microscopy localized the epitope to the cell periphery in the upper stratum spinosum and granulosum of epidermis. In the hair follicle, the epitope was present in the internal root sheath and in the infundibulum, the innermost aspect of the external root sheath. The antibody recognized a protein of relative mobility (M(r)) 82,000, pI 7.8. The protein was a transglutaminase substrate as shown by a dansylcadaverine incorporation assay. Purified cornified envelopes absorbed the reactivity of the antibody to the partially purified protein and cleavage of envelopes by cyanogen bromide resulted in release of immunoreactive fragments. The protein was soluble only in denaturing buffers such as 8 M urea or 2% sodium dodecyl-sulfate (SDS). Partial solubility could be achieved in 50 mM TRIS pH 8.3 plus 0.3 M NaCl (high salt buffer); the presence of a reducing agent did not affect solubility. Extraction of cultured keratinocytes in 8 M urea and subsequent dialysis against 50 mM TRIS pH 8.3 buffer resulted in precipitation of the protein with the keratin filaments. Dialysis against high salt buffer prevented precipitation of the protein. The unique solubility properties of this protein suggest that it aggregates with itself and/or with keratin filaments. The possible role of the protein in cornified envelope assembly is discussed. We have named this protein Sciellin (from the old english "sciell" for shell).     

Isopeptide bond formation in epidermis

Summary Proteins which are major substrates of epidermal transglutaminases can be identified in cultured keratinocytes of human, cow, and new-born rat.Cow and human keratinocytes both contain substrate proteins which are 30000 to 50000 daltons in size but dissociable in SDS to 12000 daltons or less. In both species these proteins correspond to in vivo synthesized proteins which are probable precursors of cornified envelope. Human keratinocytes synthesize a 125000 dalton protein which is also a precursor of cornified envelope both in cells and tissue. By SDS electrophoresis two 100000 dalton substrate proteins are seen in cow keratinocyte extracts and a 23000 dalton substrate protein is seen in rat keratinocyte extracts. Minor substrates of transglutaminase are seen in human keratinocytes, and one has been isolated by preparative electrophoresis. Major structural proteins of epidermis which are in vitro substrates of epidermal transglutaminase include the keratins and the stratum corneum basic protein.     

Isolation of two immunologically related transglutaminase substrates from cultured human keratinocytes

Transglutaminase substrates A and B were identified in soluble extracts of cultured keratinocytes and human epidermis by their reactions with dansyl cadaverine in the presence of Ca++ ion. Substrate B was present in substantial amounts in both extracts whereas A, easily seen in cell extracts, was decreased and sometimes not detected in tissue extracts. Substrates A and B from cultured cells were separated by Sephadex G-75 chromatography and isolated by preparative sodium dodecyl sulfate (SDS) gel electrophoresis by which A had a mol wt of 125,000 and B had a mol wt of 12,000. Amino acid analysis of A, B, and cornified envelope were similar but not identical. The isopeptide bond is not a significant structural feature of A inasmuch as its content is less than 0.25 bonds/molecule. Antibodies raised to A cross-reacted with B and vice versa and A showed partial identity to B when reacted with anti-B. Anti-A reacted with epidermis being adsorbed by the edges of cornifying cells but only weakly by cells of the Malpighian layer. Anti-B also reacted with cornifying, but its reaction was more intense with the cytoplasm of Malpighian cells. Substrate A appears to be incorporated into cornified envelope immediately after its appearance in cells of the granular layer and seems similar to a protein isolated by a different method. Substrate B, convertible by transglutaminase to higher molecular weight species, may also participate in cornified envelope assembly and shares some structural similarities to A.     

Isolation of two immunologically related transglutaminase substrates from cultured human keratinocytes

Summary Transglutaminase substrates A and B were identified in soluble extracts of cultured keratinocytes and human epidermis by their reactions with dansyl cadaverine in the presence of Ca⁺⁺ ion. Substrate B was present in substantial amounts in both extracts whereas A, easily seen in cell extracts, was decreased and sometimes not detected in tissue extracts. Substrates A and B from cultured cells were separated by Sephadex G-75 chromatography and isolated by preparative sodium dodecyl sulfate (SDS) gel electrophoresis by which A had a mol wt of 125,000 and B had a mol wt of 12,000. Amino acid analysis of A, B, and cornified envelope were similar but not identical. The isopeptide bond is not a significant structural feature of A inasmuch as its content is less than 0.25 bonds/molecule. Antibodies raised to A cross-reacted with B and vice versa and A showed partial identity to B when reacted with anti-B. Anti-A reacted with epidermis being adsorbed by the edges of cornifying cells but only weakly by cells of the Malpighian layer. Anti-B also reacted with cornifying, but its reaction was more intense with the cytoplasm of Malpighian cells. Substrate A appears to be incorporated into cornified envelope immediately after its appearance in cells of the granular layer and seems similar to a protein isolated by a different method. Substrate B, convertible by transglutaminase to higher molecular weight species, may also participate in cornified envelope assembly and shares some structural similarities to A. This work was supported by Grant AM-06838 from the National Institutes of Health.     

Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5- kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210- kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin- binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."     

The pancornulins: a group of basic low molecular weight proteins in mammalian epidermis and epithelium that may function as cornified envelope precursors

1. A monoclonal antibody (HCE-2) to human epidermal and epithelial cornified envelopes identified a group of soluble basic protein precursors. 2. Using HCE-2, envelope-like staining was observed in the epidermis and stratified squamous epithelium of a number of mammalian species. 3. Basic polypeptides reactive to HCE-2 varied in size and number among the different animals. 4. In those species studied, HCE-2-reactive peptides were substrates for transglutaminase and protease treatment of cornified envelopes released HCE-2-reactive degradation products. 5. These results suggest a new family of proteins in mammalian epidermis that may function as cornified envelope precursors.     

Differentiation of human scalp hair follicle keratinocytes in culture

The morphology of human scalp hair follicle keratinocytes, cultured on the bovine eye lens capsule, is studied by light and electron microscopy. The hair follicle keratinocytes in the stratified cultures are characterized by the presence of numerous tonofilaments, desmosomes and lysosomes and by the presence of glycogen accumulations. The cells in the upper layers develop a cornified envelope. Moreover, an incomplete basal lamina is found between the capsule and the basal cells. However, some features of epidermal keratinocytes in vivo, such as keratohyalin granules and stratum corneum formation, are absent. Analysis of the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis also reveals differences between the cultured hair follicle cells and epidermis, whilst the patterns of cultured cells and hair follicle sheaths are similar. The morphological and protein biosynthetic aspects of terminal differentiation of the keratinocytes in vitro are correlated. These results are discussed in the light of the findings with cultured epidermal keratinocytes, reported in the literature.     

Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes

The cytochrome P450 CYP2B19 is a keratinocyte-specific arachidonic acid epoxygenase expressed in the granular cell layer of mouse epidermis. In cultured keratinocytes, CYP2B19 mRNAs are up-regulated coordinately with those of profilaggrin, another granular cell-specific marker. We investigated effects of the CYP2B19 metabolites 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on keratinocyte transglutaminase activities and cornified cell envelope formation. Keratinocytes were differentiated in vitro in the presence of biotinylated cadaverine. Transglutaminases cross-linked this substrate into endogenous proteins in situ; an enzyme-linked immunosorbent assay was used to quantify the biotinylated proteins. Exogenously added or endogenously formed 14,15-EET increased transglutaminase cross-linking activities in cultured human and mouse epidermal keratinocytes in a modified in situ assay. Transglutaminase activities increased approximately 8-fold (p < or = 0.02 versus mock control) in human keratinocytes transduced with adenovirus particles expressing a 14S,15R-EET epoxygenase (P450 BM3v). The physiological transglutaminase substrate involucrin was preferentially biotinylated in situ, determined by immunoblotting and mass spectrometry. P450 BM3v-induced transglutaminase activation was associated with increased 14,15-EET formation (p = 0.002) and spontaneous cell cornification (p < or = 0.001). Preferential involucrin biotinylation and the increased cornified cell envelope formation provided evidence that transglutaminases mediated the P450 BM3v-induced cross-linking activities. These results support a physiological role for 14,15-EET epoxygenases in regulating epidermal cornification, and they have important implications for epidermal barrier functions in vivo.     

Periplakin, a Novel Component of Cornified Envelopes and Desmosomes That Belongs to the Plakin Family and Forms Complexes with Envoplakin

The cornified envelope is a layer of transglutaminase cross-linked protein that is assembled under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We have determined the cDNA sequence of one of the proteins that becomes incorporated into the cornified envelope of cultured epidermal keratinocytes, a protein with an apparent molecular mass of 195 kD that is encoded by a mRNA with an estimated size of 6.3 kb. The protein is expressed in keratinizing and nonkeratinizing stratified squamous epithelia and in a number of other epithelia. Expression of the protein is upregulated during the terminal differentiation of epidermal keratinocytes in vivo and in culture. Immunogold electron microscopy was used to demonstrate an association of the 195-kD protein with the desmosomal plaque and with keratin filaments in the differentiated layers of the epidermis. Sequence analysis showed that the 195-kD protein is a member of the plakin family of proteins, to which envoplakin, desmoplakin, bullous pemphigoid antigen 1, and plectin belong. Envoplakin and the 195-kD protein coimmunoprecipitate. Analysis of their rod domain sequences suggests that the formation of both homodimers and heterodimers would be energetically favorable. Confocal immunofluorescent microscopy of cultured epidermal keratinocytes revealed that envoplakin and the 195-kD protein form a network radiating from desmosomes, and we speculate that the two proteins may provide a scaffolding onto which the cornified envelope is assembled. We propose to name the 195-kD protein periplakin.     

A new method for quantifying keratinocyte differentiation using immunofluorescent staining of involucrin and cytofluorography

Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation.     

Comparative tumor morphogenesis of two human colon adenocarcinoma cell clones xenografted in the immunosuppressed newborn rat

Abstract. Involucrin is a precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of the epidermis and other stratifying squamous epithelia. To study involucrin gene expression and the function of involucrin, we expressed a 6 kb DNA fragment of the human involucrin gene, containing approximately 2.5 kb of upstream sequence and 0.5 kb of downstream sequence, in transgenic mice. The transgene produces a 68 kDa protein that is detected by a human involucrin-specific antibody, and is expressed in a tissuespecific and differentiation-appropriate manner (i.e., expression is confined to the suprabasal layers of the epidermis, extocervix, trachea, esophagus and conjunctiva).
Soluble involucrin levels are two to four times higher in transgenic epidermal keratinocytes compared to human foreskin keratinocytes. Newborn heterozygous animals have a normal birth weight and a normal appearing epidermis and hair growth begins at 4 to 5 days of age (i.e., the same time as hair growth in non-transgenic animals). In a subpopulation of the newborn homozygous animals birth weight is reduced, the epidermis is scaly and hair growth begins late, at around 9 to 10 days of age. In addition, the hair tends to stand erect on both heterozygous and homozygous adult animals giving the appearance of diffuse alopecia.
Immunofluorescent and electron microscopy localize involucrin in the hair follicle and cornified envelope, respectively. These results suggest that overexpression of involucrin may cause abnormalities in hair follicle structure/function and cornified envelope structure. These animals provide a new model for the study of cornified envelope structure and function.     

Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.

Late in the terminal differentiation of epidermis and cultured epidermal cells, a protein envelope located beneath the plasma membrane becomes cross-linked by cellular transglutaminase. The process of cross-linking can be initiated in cultured epidermal cells by agents affecting cell membrane permeability--nonionic detergents, high salt concentrations and ionophores. These agents initiate the cross-linking process by making calcium ions available to the transglutaminase. A soluble precursor of the cross-linked envelope has been identified in crude extracts of cultured epidermal cells by its ability to incorporate labeled amines through the action of transglutaminase. The protein has been purified to homogeneity by gel filtration and chromatography on columns of DEAE-cellulose and hydroxyapatite. Comprising an estimated 5--10% of the soluble cell proteins, it has a molecular weight of about 92,000, is isoelectric at pH 4.5 +/- 0.3 and has an unusual amino acid composition (46% Glx residues). It is chemically and immunochemically unrelated to keratins. The following evidence confirms that the protein becomes incorporated into cross-linked envelopes: first, washed cross-linked envelopes bind antibody to the purified protein, as shown by indirect immunofluorescence; second, absorption of the antiserum with washed envelopes removes all detectable antibodies to the purified protein; and third, the protein cannot be extracted from keratinocytes after their envelopes have become cross-linked. Examination of sections of epidermis by immunofluorescence, using antiserum to the purified protein, reveals that in addition to the stratum corneum, the living cells of the outer half of the spinous layer react strongly. The envelope precursor is present in the cytoplasm, but becomes concentrated at the cell periphery, where it will be cross-linked later, when the cells have passed through the granular layer. The protein is also concentrated in a peripheral location in cultured epidermal cells.     

Epidermal and hair follicle transglutaminases. Partial characterization of soluble enzymes in newborn mouse skin

Treatment of skins of newborn mice with the neutral protease Dispase in order to separate dermis and epidermis causes pronounced changes in the levels of transglutaminase activity in the epidermis. Two soluble transglutaminases, one anionic enzyme and one cationic enzyme, of Mr approximately 90,000 and approximately 50,000, respectively, are extracted from epidermis; and the activities of both enzymes increase as a function of the time of Dispase treatment of skin. When the anionic Mr approximately 90,000 enzyme is incubated with Dispase after its chromatographic isolation from epidermal extracts, it is converted to a lower molecular weight enzyme. Hair follicles isolated from dermis prepared by a 12-h Dispase treatment of the skin of newborn mice contain two soluble cationic transglutaminases, one of which is indistinguishable from that of epidermis and the other which is not seen in epidermis. Both of these hair follicle enzymes are of Mr approximately 50,000 and appear to exist in monomeric form. They have been partially purified. Based upon these findings, we suggest that transglutaminase processing and control occur during normal differentiation of keratinocytes in epidermis and of hair follicle epidermal cells in dermis and that production of the proper forms of the enzyme may be essential to the formation of mature cornified envelopes and hair shafts, respectively.     

Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.     

Characterization of polypeptide chains of epidermal prekeratin and reconstituted filaments

Prekeratin was isolated from bovine snout epidermis with 0.1 M citric acid/sodium citrate buffer, pH 2.6 (buffer A). Filaments, 6.0-9.0 nm wide, were produced by dialysis against low ionic strength buffer A or by dissociating prekeratin in 8 M urea solution followed by dialysis against 0.005 M Tris-HCl buffer, pH 8.0. The polypeptide composition of both prekeratin and filaments was studied by four different SDS-polyacrylamide gel electrophoresis methods. The best resolution was obtained by Laemmli's technique in which both prekeratin and filaments were separated into three major and seven distinct minor bands of polypeptides. The major ones comprise approx. 70% of total polypeptides and their estimated molecular weights are 68 000, 54 000, and 50 000. The molecular weight of minor ones is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000. It is proposed that the major polypeptides form the backbone structure of epidermal filaments and the minor polypeptides play a role in its stabilization.     

Immuno-cross reactivity of transglutaminase and cornification marker proteins in the epidermis of vertebrates suggests common processes of soft cornification across species

In differentiating mammalian keratinocytes proteins are linked to the plasma membrane by epidermal transglutaminases through N-epsilon-(gamma-glutamyl)-lysine isopeptide bonds to form the cornified cell envelope. The presence of transglutaminases and their protein substrates in the epidermis of nonmammalian vertebrates is not known. The present study analyses the presence and localization of the above proteins in the epidermis using immuno-cross reactivity across different classes of amniotes. After immunoblotting, some protein bands appear labelled for loricrin, sciellin, and transglutaminase in most species. These proteins are scarce to absent in the epidermis of aquatic species (goldfish and newt) where a stratum corneum is absent or very thin. The molecular weight of transglutaminase immunoreactive bands generally varies between 40 to 62 kDa, with the most represented bands at 52-57 kDa in most species. The more intense loricrin- and sciellin-immunoreactive bands are seen at 50-55-62 kDa, but are weak or absent in aquatic vertebrates. Loricrine-like immunoreactivity is present in the epidermis where alpha-(soft)-keratinization occurs. Isopeptide bonds are mainly associated to bands in the range of 50-62 kDa. In vertebrates where hard-keratin is expressed (the beta-keratin corneous layer of sauropsids and in feathers) or in hair cortex of mammals, no loricrin-like, transglutaminase-, and isopeptide-bond-immunoreactivities are seen. Immunoblotting however shows loricrin-, sciellin-, and trasnsglutaminase-positive bands in the corneous layers containing beta-keratin. Histologically, the epidermis of most amniotes shows variable transglutaminase immunoreactivity, but isopeptide-bond and sciellin immunoreactivities are weak or undetactable in most species. The limitations of immunohistochemical methods are discussed and compared with results from immunoblotting. In reptilian epidermis transglutaminase is mainly localized in 0.15-0.3 microm dense granules or diffuse in transitional alpha-keratogenic cells. In beta-keratogenic cells few small dense granules show a weak immunolabeling. Transglutaminase is present in nuclei of terminal differentiating alpha- and beta-keratinocytes, as in those of mature inner and outer root sheath. The present study suggests that keratinization based on loricrin, sciellin and transglutaminase was probably present in the stratum corneoum of basic amniotes in the Carboniferous. These proteins were mainly maintained in alpha-keratogenic layers of amniotes but decreased in beta-keratogenic layers of sauropsids (reptiles and birds). The study suggests that similar proteins for the formation of the cornified cell envelope are present in alpha-keratinocytes across vertebrates but not in beta-keratinocytes.     

Targeted ablation of the murine involucrin gene

Involucrin is synthesized in abundance during terminal differentiation of keratinocytes. Involucrin is a substrate for transglutaminase and one of the precursors of the cross-linked envelopes present in the corneocytes of the epidermis and other stratified squamous epithelia. These envelopes make an important contribution to the physical resistance of the epidermis. We have generated mice lacking involucrin from embryonic stem cells whose involucrin gene had been ablated by homologous recombination. These mice developed normally, possessed apparently normal epidermis and hair follicles, and made cornified envelopes that could not be distinguished from those of wild-type mice. No compensatory increase of mRNA for other envelope precursors was observed.     

Many faces of periplakin: domain-specific antibodies detect the protein throughout the epidermis,explaining the multiple protein-protein interactions

Periplakin was first identified as a precursor of the cornified cell envelope, residing in desmosomes and in interdesmosomal plasma membrane of differentiated keratinocytes in epidermis. However, most antibodies used so far have been raised against the C-terminal peptide of periplakin, the region which is heavily involved in protein-protein interactions. In order to avoid the lack of signal due to the epitope masking, other regions of periplakin were selected as antigens. Indeed, periplakin is present throughout the epidermis, but while the head domain is accessible for the antibody recognition throughout the epidermis, the rod domain is readily accessible only in the basal cell layer and needs to be unmasked by detergent treatment in the upper layers of epidermis. The tail domain, which is known to be heavily involved in the protein-protein interactions, specifically binding to the intermediate filament proteins, was masked in the basal cell layer, but was readily detectable in the outer layers of epidermis, where periplakin assembles to the cornified cell envelope. The in vitro assay revealed that periplakin tail serves as a substrate for the tissue-type transglutaminase, TGase 2. The cross-linking may potentially render the tail unaccessible for antibody recognition. A tailless periplakin, unable to function as a cytolinker, results from the proteolytic processing of periplakin by caspase 6, the caspase executing apoptosis through the processing of intermediate filament proteins. It is concluded that multiple localization and protein-protein interactions indicate a functional role for periplakin and its domains throughout the epithelial differentiation.This work was supported by RO1 33588 from NIH, NIAMS     

The cornified envelope: a model of cell death in the skin

The epidermis functions as a barrier against the environment by means of several layers of terminally differentiated, dead keratinocytes - the cornified layer, which forms the endpoint of epidermal differentiation and death. The cornified envelope replaces the plasma membrane of differentiating keratinocytes and consists of keratins that are enclosed within an insoluble amalgam of proteins, which are crosslinked by transglutaminases and surrounded by a lipid envelope. New insights into the molecular mechanisms and the physiological endpoints of cornification are increasing our understanding of the pathological defects of this unique form of programmed cell death, which is associated with barrier malfunctions and ichthyosis.