Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.

Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.     

Relation of protein synthesis and transglutaminase activity to formation of the cross-linked envelope during terminal differentiation of the cultured human epidermal keratinocyte

When serially cultivated human epidermal keratinocytes are placed in suspension culture they stop growing and form, beneath the plasma membrane, an insoluble envelope consisting of protein cross-linked by ε- (γ-glutamyl)lysine. The formation of envelopes in suspended cells is preceded by a sharp decline in the rate of protein synthesis, and most envelopes appear only after the average rate of protein synthesis has fallen to a very low level. If protein synthesis is reduced over 98 percent with cycloheximide or emetine at the time that surface-grown cells are placed in suspension culture, cross-linked envelopes form in most of the cells. This shows that the precursor of the envelope and the cross-linking enzyme are already in the cytoplasm in most cells of growing surface cultures. The process of envelope formation by suspension cultures is actually accelerated by the inhibitors of protein synthesis; an increased number of cells with cross-linked envelopes is observable within 4-6 h after the addition of cycloheximide. The inhibitor also induces a large fraction of the cells of surface cultures to form enveloped within a few days. These findings suggest that arrest of protein synthesis leads to activation of the cross-linking process. Agents known to inhibit transglutaminase-mediated protein cross-linking-putrescine, iodoacetamide, and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA)- also prevent envelope formation. Though the activity of the cross-linking transglutaminase depends on the presence of cellular Ca++, we have not been able to activate the cross-linking process by high external Ca++ concentration or ionophores.     

Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope.

Both keratohyalin granules (KHG) and cornified envelopes were stained histochemically in an indirect immunofluorescent study by antiphosphorylated cystatin alpha antibody, indicating that phosphorylated cystatin alpha is a component of the cornified envelope proteins. When phosphorylated cystatin alpha (P-cystatin alpha) was incubated with epidermal transglutaminase (TGase) and Ca2+ ions, polymerized protein was produced by formation of epsilon-(gamma-glutamyl)lysine cross-linking peptide bonds between lysine residues of cystatin alpha and glutamine residues of suitable protein(s) in the enzyme preparation. However, phosphorylated and non-phosphorylated cystatins were polymerized to similar extents by the TGase. Immunofluorescent and immunoelectron microscopic observations revealed that P-cystatin alpha could be detected in vivo in the KHG and cornified envelopes. Treatment of sphingosine, a specific inhibitor of protein kinase C, markedly suppressed the incorporation of cystatin alpha into KHG. Thus phosphorylation of cystatin alpha by protein kinase C may play an important role in targeting cystatin alpha into KHG.     

Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation.

Human epidermal cells grown in culture synthesize abundant keratins. These keratins are similar to those of stratum corneum of human epidermal callus in their insolubility in dilute aqueous buffers, their molecular weight range of 40,000 to 60,000, their immunolgical reactivity, and their ability to assemble into 80 A tonofilaments in vitro; but there are differences in the molecular weights of some of the proteins, the number of components, and their charge heterogeneity, related at least in part to phosphorylation. About 30% of all the proteins of living cultured keratinocytes consists of keratins, compared with over 85% of stratum corneum. All the keratins of human stratum corneum were found to be cross-linked by intermolecular disulfide bonds while most keratins of the living cells were not. As the cells mature in Methocel-stabilized suspension culture, their keratins become increasingly disulfide cross-linked. When uncross-linked tonofilaments of living keratinocytes are dissolved in 8 M urea and the filaments reconstituted in vitro their keratins become disulfide cross-linked under aerobic conditions and consequently insoluble in solutions of 8 M urea or sodium dodecyl sulfate. The results indicate that the uncross-linked state of the keratins in living cells is due to the reducing intracellular environment and not to a precursor state related to the primary structure of the proteins. The disulfide cross-links stabilizing the keratin filaments must be distinguished from the epsilon-(gamma-glutamyl)lysine cross-links stabilizing the cornified cell envelope.     

Sphingolipid activator proteins are required for epidermal permeability barrier formation

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.     

Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action

Physiological deletion of cells ensues programmed death which involves formation of apoptotic bodies with fragmented DNA. Here we report that apoptotic hepatocytes are insoluble in detergents, urea, guanidine hydrochloride, reducing agents and thereby can be isolated from rat liver following collagenase treatment. They are wrinkled, spherical structures similar to cornified envelopes of epidermis by phase-contrast microscopy and show irregular, globular morphology by scanning-electron microscopy. Part of their DNA content is cleaved into nucleosomal and oligonucleosomal fragments. Their insolubility, like that of the cornified envelope, is evoked by epsilon-(gamma-glutamyl)lysine and N1,N8-bis(gamma-glutamyl)spermidine protein cross-linking bonds formed by transglutaminase.     

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.     

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.     

Terminal differentiation of cultured human epidermal cells.

Three aspects of terminal differentiation of the epidermal keratinocyte have been studied in cell culture—the development of detergent-insoluble cytoplasmic filaments, the formation of a cornified cell envelope and the destruction of the cell nucleus.In the presence of lethally irradiated 3T3 cells, single human epidermal keratinocytes grow into stratified colonies. After the colonies become confluent, the culture enters a steady state in which the upper cells are shed from the surface of the cell layer like stratum corneum cells in vivo and are replaced by the proliferation of dividing cells in the basal layer. The cells shed into the medium are flattened and elongated squames, and are insoluble in solutions of sodium dodecylsulfate. Since the squames usually detach before their nuclei are digested, the cultures behave like some wet-surfaced, stratified squamous epithelia in that they possess little or no anucleate stratum corneum. The rates of proliferation and squame detachment in confluent cultures are increased by the presence of epidermal growth factor.Most of the squames harvested from the medium are permeable to trypan blue. The permeable squames may or may not have a visible nucleus, but squames not permeable to trypan blue nearly always possess a nucleus. When freshly detached squames containing nuclei are incubated in medium containing serum, their nuclei are digested and disappear within a few days. On the other hand, if the squames are washed and incubated in serum-free medium, their nuclei are not digested. This suggests that the permeable cell membrane permits a serum component essential for nuclear digestion to enter the cytoplasm.When growing colonies of epidermal keratinocytes are disaggregated and the cells suspended in medium containing methyl cellulose, they cannot multiply, but within a few days the cells become permeable to trypan blue and insoluble in sodium dodecylsulfate. This insolubility is due to disulfide linking of the proteins of the abundant cytoplasmic filaments, for the filaments are dissolved when β-mercaptoethanol is added as well, leaving the emptied cornified cell envelopes. Nuclear digestion follows some days later. In the absence of serum, cells become permeable and develop detergent-insoluble filaments and a cornified envelope, but, as in the case of spontaneously detached squames of surface cultures, their nuclei are not destroyed. Purified plasminogen supports nuclear destruction, whereas serum depleted of plasminogen does not.Earlier studies on intact skin have suggested that chemical gradients between epidermis and dermis might be responsible for the differentiation of the epidermal cells. In surface culture, basal cells multiply and nonbasal cells undergo terminal differentiation, even though all the cells are bathed in the same medium and the terminally differentiating cells have, if anything, better access to the medium than do the basal cells. Differentiation also begins in virtually all singly suspended cells uniformly exposed to the medium. The program of differentiation is therefore independent of the orientation of any chemical gradients in the cellular environment. Cell-cell contacts are not required for the development of detergent insolubility, the formation of the cornified envelope or the process of nuclear digestion, although they are essential for the formation of flattened squames. Unlike proliferation, which is strongly dependent upon fibroblast products, terminal differentiation proceeds in the absence of fibroblast support.     

Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation.     

ε-(γ-Glutamyl)lysine Cross-link in the Terminal Differentiation of Mammalian Epidermis

Purified horny cell membrane of the epidermis of newborn rat was prepared by discontinuous sucrose density gradient ultracentrifugation after 0.1 n sodium hydroxide treatment. ε-(γ-Glutamyl)-lysine cross-link was identified mainly in the horny cell membranes of the epidermis of newborn rat, cow snout, and human stratum corneum. When, [¹⁴C]-lysine was incubated with minced epidermis of newborn rat, [¹⁴C]-lysine labeled ε-(γ-glutamyl)lysine cross-link was identified in purified horny cell membrane. Results indicate that the protein newly synthesized in the granular layer might play a role in the formation of thickened cell membrane in the late stage of the epidermal differentiation.     

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).     

Epidermal differentiation: the role of proteases and their inhibitors

Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency infurless mice, targeted ablation of the serine protease Matriptase/MTSP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent findings on the role of cystatin M/E and legumain as a functional dyad in skin and hair follicle cornification, a paradigm example of the regulatory functions exerted by epidermal proteases, will be discussed.     

Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice

The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope.     

AKT-dependent HspB1 (Hsp27) activity in epidermal differentiation

AKT activity has been reported in the epidermis associated with keratinocyte survival and differentiation. We show in developing skin that Akt activity associates first with post-proliferative, para-basal keratinocytes and later with terminally differentiated keratinocytes that are forming the fetal stratum corneum. In adult epidermis the dominant Akt activity is in these highly differentiated granular keratinocytes, involved in stratum corneum assembly. Stratum corneum is crucial for protective barrier activity, and its formation involves complex and poorly understood processes such as nuclear dissolution, keratin filament aggregation, and assembly of a multiprotein cell cornified envelope. A key protein in these processes is filaggrin. We show that one target of Akt in granular keratinocytes is HspB1 (heat shock protein 27). Loss of epidermal HspB1 caused hyperkeratinization and misprocessing of filaggrin. Akt-mediated HspB1 phosphorylation promotes a transient interaction with filaggrin and intracellular redistribution of HspB1. This is the first demonstration of a specific interaction between HspB1 and a stratum corneum protein and indicates that HspB1 has chaperone activity during stratum corneum formation. This work demonstrates a new role for Akt in epidermis.     

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.     

The human loricrin gene.

Loricrin is the major protein component of the cornified cell envelope of terminally differentiated mammalian epidermal (stratum corneum) cells. Using a specific human cDNA clone, we have isolated and characterized the human loricrin gene. We show that it has a very simple structure of a single intron of 1188 base pairs (bp) in the 5'-untranslated region; there are no introns in coding sequences. By use of rodent-human somatic cell hybrids, followed by in situ hybridization with a biotin-labeled genomic DNA clone, the single-copy gene maps to chromosome location 1q21. Polymerase chain reaction analyses of genomic DNAs from different individuals show that human loricrin consists of two allelic size variants, due to sequence variations in its second glycine loop domain, and these variants segregate in the human population by normal Mendelian mechanisms. Furthermore, there are multiple sequence variants within these two size class alleles due to various deletions of 12 bp (4 amino acids) in the major loop of this glycine loop domain. By use of a specific loricrin antibody, we show by immunogold electron microscopy that loricrin initially appears in the granular layer of human epidermis and forms composite keratohyalin granules with profilaggrin, but localizes to the cell periphery (cell envelope) of fully differentiated stratum corneum cells.     

Sodium-N-butyrate induces cytoskeletal rearrangements and formation of cornified envelopes in cultured adult human keratinocytes

The technique developed in our laboratory allows us to culture multilayered, stratified sheets of human keratinocytes, which can be used to cover the burn wounds of patients. Organization of cells in these cultures resembles stratum germinativum and stratum spinosum but there are only a few fully keratinized cells and the stratum corneum is not developed. Since the fully differentiated sheets may offer additional advantages as epidermal transplants, attempts were made to enhance the degree of differentiation in vitro. In the present study sodium-N-butyrate (NaB) was used as a differentiating agent and its effect on the cell cycle and cytoarchitecture of epidermal cells was investigated. Incubation of keratinocytes in the presence of 2.5 mM NaB induced the appearance of enucleated cornified envelopes, covering approximately 70-80% of the surface of the cultures. Their appearance correlated with a decrease in expression of keratin K13, previously shown to be inhibited during terminal differentiation of human keratinocytes. An increase in transglutaminase transferase activity was also observed. The induction of cornified layers also correlated with an increase in the amount of microfilament (MF)-associated actin. NaB also induced changes in the cell cycle distribution of the keratinocyte cultures. A decrease in the proportion of S and G1B phase cells was paralleled by an increase in G1A cells, maximally expressed 30-48 h following addition of the inducer. Interestingly, NaB also induced a cell arrest in G2 phase. These cell cycle perturbations preceded the onset of keratinocyte differentiation. The results indicate that the enhanced differentiation of human keratinocytes in the presence of NaB may serve as a means to produce epidermal sheets with improved properties for transplantation in a clinical setting. It also serves as an in vitro model system to study the interrelationships between biochemical events and cell cycle changes accompanying differentiation.     

Immunohistochemical analysis of stratum corneum components in oral squamous epithelia

The generation of a stratum corneum in squamous epithelia involves marked changes in morphology and in the expression of cell products. We have examined the expression of some of the components involved in this process in oral squamous epithelia with different terminal differentiation patterns by use of immunofluorescent techniques. Involucrin and transglutaminase are involved in formation of cornified envelopes consistently seen in the stratum corneum. Both components were present in keratinized oral epithelia (palatal epithelium and hyperkeratinized buccal epithelium). The nonkeratinized normal buccal epithelium stained positive as well. Filaggrin, a protein derived from a precursor present in keratohyalin granules, is proposed to aggregate keratin filaments in the cornified layer. Although the staining differed markedly in quantity, this component was likewise detected in both keratinized and nonkeratinized epithelia. The staining patterns for different keratin polypeptides, however, showed qualitative differences between the different epithelia. Thus, it seems that the keratin composition shows differentiation-specific characteristics, whereas the presence of other important components needed to generate a stratum corneum is not as closely related to the terminal differentiation pattern of oral epithelia.