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.     

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).     

Subcellular distribution of envoplakin and periplakin: insights into their role as precursors of the epidermal cornified envelope

Envoplakin and periplakin are two plakins that are precursors of the epidermal cornified envelope. We studied their distribution and interactions by transfection of primary human keratinocytes and other cells. Full-length periplakin localized to desmosomes, the interdesmosomal plasma membrane and intermediate filaments. Full length envoplakin also localized to desmosomes, but mainly accumulated in nuclear and cytoplasmic aggregates with associated intermediate filaments. The envoplakin rod domain was required for aggregation and the periplakin rod domain was necessary and sufficient to redistribute envoplakin to desmosomes and the cytoskeleton, confirming earlier predictions that the proteins can heterodimerize. The linker domain of each protein was required for intermediate filament association. Like the NH(2) terminus of desmoplakin, that of periplakin localized to desmosomes; however, in addition, the periplakin NH(2) terminus accumulated at cell surface microvilli in association with cortical actin. Endogenous periplakin was redistributed from microvilli when keratinocytes were treated with the actin disrupting drug Latrunculin B. We propose that whereas envoplakin and periplakin can localize independently to desmosomes, the distribution of envoplakin at the interdesmosomal plasma membrane depends on heterodimerization with periplakin and that the NH(2) terminus of periplakin therefore plays a key role in forming the scaffold on which the cornified envelope is assembled.     

Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin

Injury to epidermis and other stratified epithelia triggers profound but transient changes in the pattern of keratin expression. In postmitotic cells located at the wound edge, a strong induction of K6, K16, and K17 synthesis occurs at the expense of the keratins produced under the normal situation. The functional significance of these alterations in keratin expression is not known. Here, we report that overexpression of a wild-type human K16 gene in a tissue-specific fashion in transgenic mice causes aberrant keratinization of the hair follicle outer root sheath and proximal epidermis, and it leads to hyperproliferation and increased thickness of the living layers (acanthosis), as well as cornified layers (hyperkeratosis). The pathogenesis of lesions in transgenic mouse skin begins with a reorganization of keratin filaments in postmitotic keratinocytes, and it progresses in a transgene level-dependent fashion to include disruption of keratinocyte cytoarchitecture and structural alterations in desmosomes at the cell surface. No evidence of cell lysis could be found at the ultrastructural level. These results demonstrate that the disruption of the normal keratin profile caused by increased K16 expression interferes with the program of terminal differentiation in outer root sheath and epidermis. They further suggest that when present at sufficiently high intracellular levels, K16, along with K6 and K17, appear capable of inducing a reorganization of keratin filaments in the cytoplasm of skin epithelial cells.     

Chromosomal Localisation of the Human Envoplakin Gene (EVPL) to the Region of the Tylosis Oesophageal Cancer Gene (TOCG) on 17q25

Envoplakin is a membrane-associated precursor of the epidermal cornified envelope. Envoplakin is homologous to desmoplakin I and desmoplakin II (DPI/II), bullous pemphigoid antigen 1 (BPAG1), and plectin and is proposed to link desmosomes and keratin filaments to the cornified envelope. We describe the isolation of cosmids and yeast artificial chromosomes containing the complete human envoplakin gene (EVPL) and show, by analysis of somatic cell hybrids and chromosomalin situhybridisation, that the envoplakin gene, unlike the genes encoding BPAG1 and DPI/II, maps to 17q25 and is physically linked to D17S1603. This sequence-tagged site segregates with the autosomal dominant human disease focal nonepidermolytic palmoplantar keratosis (NEPKK; “tylosis”), which is associated with an increased risk of oesophageal cancer. The chromosomal localisation of the envoplakin gene, the homology of the encoded protein to keratin-binding proteins, and its expression in epidermal and oesophageal keratinocytes all raise the possibility that loss of envoplakin function could be responsible for this form of palmoplantar keratoderma.     

Tissue-specific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes

In contrast to the simplified keratin content of bovine, rabbit, and rat esophageal epithelium (composed mainly of a 57 and 46 or 51 kD keratin, depending on the animal species), human esophageal epithelium contained a quantitatively different array of keratin proteins, ranging in molecular weight from 37 to 61 kD. The pattern of keratin proteins from human esophageal epithelium differed qualitatively and quantitatively from that of human epidermis. Human esophageal epithelium lacked the 63, 65, and 67 kD keratins characteristic of human epidermis, consistent with the absence of a granular layer and an anucleate stratum corneum. Moreover, human esophageal epithelium contained a distinctive 61 kD keratin protein which was either not present or present in only small amounts in human epidermis and variable amounts of a 37 kD keratin. Whereas the 56, 59, and 67 kD keratins were the most abundant keratins in human epidermis, the 52, 57, and 61 kD keratins predominated in human esophageal epithelium. During in vitro cultivation, both human epidermal and esophageal keratinocytes produce colonies which are stratified, but the morphologic appearance of these cultured epithelia differs. Only cultured human epidermal keratinocytes contain keratohyalin granules in the outermost layers and a prominent 67 kD keratin on immunoprecipitation. Otherwise the keratin contents appear similar. In conclusion, human esophageal epithelium exhibited intertissue and interspecies differences in the pattern of keratin proteins. During in vitro cultivation, human esophageal keratinocytes retained some aspects of their distinctive program of differentiation.     

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.     

A new class of soluble basic protein precursors of the cornified envelope of mammalian epidermis

The cornified envelope hs been shown to be formed beneath the plasma membrane as a result of the cross-linking of soluble and membrane-associated precursor proteins by transglutaminase. We have obtained a monoclonal antibody which reacts with the periphery of cells in the upper layers of human epidermis by indirect immunofluorescence (IIF) following immunization of mice with cornified envelopes of cultured human keratinocytes. The antibody also stained the cell peripheries of bovine, rat and mouse epidermis as well as stratified epithelium. Neutral buffer extracts of human cultured keratinocytes and epidermis examined under denaturing conditions contained polypeptides of molecular weight 14 900 and 16 800 which reacted with the antibody, and an additional component of molecular weight 24 800 was found in cultured cells. The polypeptides were shown to have a pI of about 9.0. Under non-denaturing conditions the two lower-molecular-weight polypeptides had an apparent molecular weight of 30 000, while the 24 800 protein had one of 60 000. Incubation of the polypeptides under conditions that activate transglutaminase resulted in a disappearance of the polypeptides or the formation of cross-linked products. Basic polypeptides with somewhat different pI values and molecular weights were identified in neutral buffer extracts of bovine and rat epidermis. The HCE-2 antibody appears to identify a new class of basic protein precursors of mammalian cornified envelope.     

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.     

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.     

Desmoplakin controls microvilli length but not cell adhesion or keratin organization in the intestinal epithelium

Maintaining proper cell-cell adhesion in the intestine is essential for tissue homeostasis and barrier function. This adhesion is thought to be mediated by cell adhesion structures, including tight junctions, adherens junctions, and desmosomes, which concentrate in the apical junctional region. While clear roles for adherens and tight junctions have been established in simple epithelia, the function of desmosomes has not been addressed. In stratified epithelia, desmosomes impart mechanical strength to tissues by organizing and anchoring the keratin filament network. In this paper, we report that the desmosomal protein desmoplakin (DP) is not essential for cell adhesion in the intestinal epithelium. Surprisingly, when DP is lacking, keratin filament localization is also unperturbed, although keratin filaments no longer anchor at desmosomes. Unexpectedly, DP is important for proper microvillus structure. Our study highlights the tissue-specific functions of desmosomes and reveals that the canonical functions for these structures are not conserved in simple epithelium.     

Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.     

Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes

Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.     

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.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution- binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

Evidence that major 78-44-kD concanavalin A-binding glycopolypeptides in pig epidermis arise from the degradation of desmosomal glycoproteins during terminal differentiation

The major concanavalin A (Con A)-binding component in urea/deoxycholate/mercaptoethanol extracts from pig ear epidermis had an apparent Mr of 78 kD. In indirect immunofluorescence affinity- purified polyclonal antibodies against this glycopolypeptide strongly stained the surface of suprabasal cells in the epidermis of pig and human skin. Immunocytochemical labeling with gold-labeled second antibody localized this staining to externally disposed, trypsin- sensitive components of desmosomes. Western blotting showed that the 78- kD glycopolypeptide was immunologically related to several other Con A- binding components in pig epidermis. Immunoreactive components with Mr of 115 and 100 kD were membrane-bound, appeared to be susceptible to trypsin in intact epidermis, and were absent from the stratum corneum. Immunoreactive components of lower Mr (78-44 kD) were not membrane- bound, were resistant to trypsin in intact tissue, and were present predominantly in the keratinized layers of pig epidermis. The 115-44-kD glycopolypeptides were also recognized by antisera raised against desmoglein II/desmocollin glycoproteins isolated from bovine spinous layer desmosomes. In addition, these antisera reacted with 120- and 105- kD bands that were apparently not recognized by the anti-78-kD glycopolypeptide antiserum in immunoblotting. In immune precipitation the anti-78-kD glycopolypeptide and antidesmoglein II/desmocollin antisera precipitated comparable amounts of the radioiodinated 78-44-kD components. Both antisera also precipitated the 120- and 105-kD components although the anti-78-kD glycopolypeptide serum was less effective. Little reaction with the 115- and 105-kD components was observed in immune precipitation with either serum. Proteolytic peptide mapping confirmed that the various immunoreactive glycopolypeptides were biochemically as well as immunologically related. The results suggest that terminal differentiation in pig epidermis is accompanied by the orderly degradation of desmoglein II/desmocollin glycoproteins resulting in the accumulation of 78-44-kD glycopolypeptides in the stratum corneum. These glycopolypeptides may represent functionally important nonmembranous domains of cell-adhesion molecules in desmosomes.     

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.