Cornified Cell Envelope Research Articles

Structure and Transcriptional Regulation of the Human Cystatin A Gene: THE 12-O-TETRADECANOYLPHORBOL-13-ACETATE (TPA) RESPONSIVE ELEMENT-2 SITE (−272 TO −278) ON CYSTATIN A GENE IS CRITICAL FOR TPA-DEPENDENT REGULATION

Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to -2595 in the 5'-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5' upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between -272 and -278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-alpha expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5'-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.

Structural-mechanical integration of keratin intermediate filaments with cell peripheral structures in the cornified epidermal keratinocyte.

Previous articleNext article No AccessINTERMEDIATE FILAMENTS, HEMIDESMOSOMES, AND DESMOSOMESStructural-Mechanical Integration of Keratin Intermediate Filaments With Cell Peripheral Structures in the Cornified Epidermal KeratinocyteP. M. SteinertP. M. Steinert Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 194, Number 3June 1998 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.2307/1543115 Views: 7Total views on this site Copyright © 1998 by Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.

Biochemical Evidence That Small Proline-rich Proteins and Trichohyalin Function in Epithelia by Modulation of the Biomechanical Properties of Their Cornified Cell Envelopes

The cornified cell envelope (CE) is a specialized structure involved in barrier function in stratified squamous epithelia, and is assembled by transglutaminase cross-linking of several proteins. Murine forestomach epithelium undergoes particularly rigorous mechanical trauma, and these CEs contain the highest known content of small proline-rich proteins (SPRs). Sequencing analyses of these CEs revealed that SPRs function as cross-bridgers by joining other proteins by use of multiple adjacent glutamines and lysines on only the amino and carboxyl termini and in functionally non-polar ways. Forestomach CEs also use trichohyalin as a novel cross-bridging protein. We performed mathematical modeling of amino acid compositions of the CEs of mouse and human epidermis of different body sites. Although the sum of loricrin + SPRs was conserved, the amount of SPRs varied in relation to the presumed physical requirements of the tissues. Our data suggest that SPRs could serve as modifiers of a composite CE material composed of mostly loricrin; we propose that increasing amounts of cross-bridging SPRs modify the structure of the CE, just as cross-linking proteins strengthen other types of tissues. In this way, different epithelia may use varying amounts of the cross-bridging SPRs to alter the biomechanical properties of the tissue in accordance with specific physical requirements and functions.

Transglutaminase 1 Mutations in Lamellar Ichthyosis: LOSS OF ACTIVITY DUE TO FAILURE OF ACTIVATION BY PROTEOLYTIC PROCESSING

Lamellar ichthyosis is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. It is caused by mutations in the TGM1 gene that encodes the transglutaminase 1 (TGase 1) enzyme, which is critical for the assembly of the cornified cell envelope in terminally differentiating keratinocytes. TGase 1 is a complex enzyme existing as both cytosolic and membrane-bound forms. Moreover, TGase 1 is proteolytically processed, and the major functionally active form consists of a membrane-bound 67/33/10-kDa complex with a myristoylated and palmitoylated amino-terminal 10-kDa membrane anchorage fragment. To understand better how point mutations, deletions, and truncations found in lamellar ichthyosis disease affect the structure and function of TGase 1, we have expressed in baculovirus and keratinocytes a number of reported TGase 1 mutants. The structural implications of these mutations were examined using a homology-derived three-dimensional model of TGase 1 generated from the known x-ray structure of the related coagulation factor XIIIa enzyme. The present studies demonstrate that loss of TGase 1 activity is not restricted to mutations that directly affect the enzymatic activity. We report a new class of mutations that impair the subsequent post-synthetic processing of the protein into its highly active functional forms.

Cornified cell envelope assembly: a model based on electron microscopic determinations of thickness and projected density.

In stratifying squamous epithelia, the cornified cell envelope (CE), a peripheral layer of crosslinked protein, is assembled sequentially from precursor proteins initially dispersed in the cytoplasm. Its major component is loricrin (37 kDa in mouse), which contributes from approx. 60% to >80% of the protein mass in different tissues. Despite its importance to the mechanical resilience and impenetrability of these tissues, detailed information has not been obtained on CE structure, even on such basic properties as its thickness or uniformity across a given CE or from tissue to tissue. To address this issue, we have studied CEs isolated from three murine epithelia, namely epidermis, forestomach and footpad, by electron microscopy of metal-shadowed specimens and scanning transmission electron microscopy (STEM) of unstained specimens. The former data reveal that the cytoplasmic surface is smoothly textured whereas the extracellular surface is corrugated, and that the average thickness is 15.3+/-1.2 nm, and strikingly uniform. Measurements of mass-per-unit-area from the STEM images yielded values of approx. 7.0+/-0.8 kDa/nm2, which were remarkably consistent over all three tissues. These data imply that the mature CE has a uniquely defined thickness. To explain its uniformity, we postulate that loricrin forms a molecular monolayer, not a variable number of multiple layers. In this scenario, the packing density is one loricrin monomer per 7 nm2, and loricrin should have an elongated shape, 2.5-3.0 nm wide by approx. 11 nm long. Moreover, we anticipate that any inter-tissue variations in the mechanical properties of CEs should depend more on protein composition and cross-linking pattern than on the thickness of the protein layer deposited.

A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.

We have addressed the question of how keratin intermediate filaments are associated with the cell envelope at the periphery of cornified epidermal cells. Many peptides from human epidermal cell envelopes containing isopeptide crosslinks inserted by transglutaminases in vivo have been characterized. A major subset involves the type II keratin chains keratin 1, 2e, 5, or 6 crosslinked to several protein partners through a lysine residue located in a conserved region of the V1 subdomain of their head domains. This sequence specificity was confirmed in in vitro crosslinking experiments. Previously the causative mutation in a family with diffuse nonepidermolytic palmar-plantar keratoderma was shown to be the loss in one allele of the same lysine residue of the keratin 1 chain. Ultrastructural studies of affected palm epidermis have revealed abnormalities in the organization of keratin filaments subjacent to the cell envelope and in the shape of the cornified cells. Together, these data suggest a mechanism for the coordination of cornified cell structure by permanent covalent attachment of the keratin intermediate filament cytoskeleton to the cell envelope by transglutaminase crosslinking. Furthermore, these studies identify the essential role of a conserved lysine residue on the head domains of type II keratins in the supramolecular organization of keratin filaments in cells.

Complex ceramides are bound to involucrin, envoplakin, desmoplakin, periplakin and perhaps other proteins of human foreskin epidermal cornified cell envelopes

Complex ceramides are bound to involucrin, envoplakin, desmoplakin, periplakin and perhaps other proteins of human foreskin epidermal cornified cell envelopes

Small proline rich proteins serve to modulate the biomechanical properties of epithelial cornified cell envelopes

Small proline rich proteins serve to modulate the biomechanical properties of epithelial cornified cell envelopes

Abnormal cornified cell envelope formation in mutilating palmoplantar keratoderma unrelated to epidermal differentiation complex

Abnormal cornified cell envelope formation in mutilating palmoplantar keratoderma unrelated to epidermal differentiation complex

The α and η Isoforms of Protein Kinase C Stimulate Transcription of Human Involucrin Gene

Involucrin is one of the precursor proteins of the cornified cell envelope that is formed beneath the cell membrane during terminal differentiation of keratinocytes. 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a potent protein kinase C (PKC) activator, induces terminal differentiation of keratinocytes. We previously demonstrated that involucrin promoter activity is stimulated by TPA in cultured fetal rat skin keratinocytes. PKC is a large family of proteins and keratinocytes containing five PKC isozymes: alpha, delta, epsilon, eta, and zeta. In order to determine the role of the PKC isozyme(s) on involucrin gene expression, we constructed the chloramphenicol acetyl transferase (CAT)-involucrin promoter expression vector by connecting the 5'-upstream region of the human involucrin gene containing the untranslated first exon to the CAT reporter gene. The CAT-involucrin promoter expression vector was transfected with various PKC isozyme expression vectors into SV40-transformed human keratinocytes (SVHK cells). Transfection of the CAT-involucrin promoter expression vector with PKC-alpha or PKC-eta expression vectors resulted in a significant increase in the TPA-dependent involucrin promoter activity. The PKC inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride, inhibited the promoter activity stimulated by TPA. Transfection of PKC-delta, -epsilon, and -zeta had no effect on the involucrin-promoter activity. Although the promoter activity was stimulated by transfection of PKC-gamma, TPA did not enhance the promoter activity in the PKC-gamma-transfected SVHK cells. Previously we showed three AP-1 binding sites (AP1-1, -2, and -3) on the involucrin promoter region. Both the basal and the TPA-stimulated involucrin promoter activities were suppressed by deleting the AP1-1 site (-119 to -113) that is the most proximal to the transcription start site. The deletion of AP1-2 (-297 to -303) or AP1-3 (-447 to -453) did not affect the involucrin promoter activity. Gel retardation analyses disclosed that TPA stimulated the specific DNA binding of the nuclear protein(s) of control, PKC-alpha, or PKC-eta-transfected SVHK cells, but not of PKC-gamma-transfected cells. Addition of anti-c-Jun and anti-c-Fos antibodies decreased the specific protein-DNA complex band with a concomitant appearance of supershifted bands. These results indicate that PKC, specifically PKC-alpha and PKC-eta, mediates the TPA-dependent activation of involucrin gene expression of SVHK cells. PKC-gamma, which is not present in keratinocytes, also induces involucrin gene expression in a TPA-independent manner, when introduced into SVHK cells.

Structural organization of cornified cell envelopes and alterations in inherited skin disorders.

The cornified cell envelope is a highly insoluble and extremely tough structure formed beneath the cell membrane during terminal differentiation of keratinocytes. Its main function is to provide human skin with a protective barrier against the environment. Sequential cross-linking of several integral components catalyzed by transglutaminases leads to a gradual increase in the thickness of the envelope and underscores its rigidity. Key structural players in this cross-linking process include involucrin, loricrin, SPRRs, elafin, cystatin A, S100 family proteins, and some desmosomal proteins. The recent identification of genetic skin diseases with mutations in the genes encoding some of these proteins, including transglutaminase 1 and loricrin, has disclosed that abnormal cornified cell envelope synthesis is significantly involved in the pathophysiology of certain inherited keratodermas and reflects perturbations in the complex, yet highly orderly process of cornified cell envelope formation in normal skin biology.

Small Proline-Rich Proteins Are Cross-Bridging Proteins in the Cornified Cell Envelopes of Stratified Squamous Epithelia

The cornified cell envelope (CE) is a specialized structure which contributes barrier function to stratified squamous epithelial cells. It is composed of an amalgam of several structural proteins that are rendered insoluble by isopeptide bond crosslinking by transglutaminases. One set of the structural proteins present in CEs of most such epithelia are the small proline rich (SPR) proteins, which are a family of about 12 related structural proteins. We have recovered a large number of peptides containing isopeptide crosslinks, including 236 involving SPR proteins, following proteolysis of CEs isolated from foreskin epidermal tissue and cultured epidermal keratinocytes. Analysis of this database has provided novel information on their function. First, we found that SPRs became crosslinked to many other structural proteins within the CE. Second, multiple glutamine and lysine residues located only on the amino- and carboxy-termini of the SPR proteins were involved in crosslinking, so that the two ends are functionally equivalent. Third, the SPRs functioned as cross-bridging proteins, by directly adjoining other CE structural proteins. In the specialized case of the epidermal CE, the SPRs cross-bridged between loricrin. In cultured keratinocytes which make little loricrin and serve as a model for internal stratified squamous epithelia, the SPRs formed extensive cross-bridges among themselves. Thus SPRs are ubiquitous cross-bridging proteins whose differential expression patterns apparently reflect specific barrier requirements of different epithelia.

Characterization and Purification of Human Corneodesmosin, an Epidermal Basic Glycoprotein Associated with Corneocyte-specific Modified Desmosomes

Using monoclonal antibodies, we identified a new protein in mammalian epidermis, which we called corneodesmosin. It is located in the extracellular part of the modified desmosomes in the cornified layer of the tissue, and its proteolysis (from 52-56 to 33 kDa) is thought to be a major prerequisite of desquamation. We have now further characterized human corneodesmosin. Proteolysis of purified cornified cell envelopes produced immunoreactive fragments, confirming the covalent linkage of the protein to these structures. Sequential extraction of epidermal proteins indicated that the 52-56-kDa precursor form of the protein exists in two distinct pools, one extracted with a nondenaturing hypotonic buffer, and the other with urea. Two-dimensional gel analysis and reactivity with phosphoserine-specific antibodies showed that it is a basic phosphoprotein. Deglycosylation experiments, reactivity with lectins, and chromatography on concanavalin A-Sepharose indicated that corneodesmosin is N-glycosylated. Partial sequences, 10 and 15 amino acids long, of the purified 52-56-kDa corneodesmosin showed identity with sequences predicted from a previously cloned gene, proved to be expressed in the epidermis and designated S. This indicates that corneodesmosin is probably encoded by the S gene, the function of which was unknown until now. A model of corneodesmosin maturation during cornification is proposed.

Spatial Distributions of Sulfur-Rich Proteins in Cornifying Epithelia

We have used energy-filtered electron microscopy and electron energy loss spectroscopy (EELS) to characterize the distributions of sulfur-rich proteins in granular layer cells of squamous stratifying epithelia and their redistribution in the cornified layer, with particular attention to assembly of the cornified cell envelope (CE). Our measurements providequantitativeinformation that complements highly specific butqualitativedata from immunocytochemistry. Spatial distributions of sulfur, phosphorus, and nitrogen were mapped in unstained thin sections of mouse epidermis and forestomach, using a postcolumn energy filter. Nitrogen images were indicative of total protein while phosphorus images provided a control to validate the algorithms used to calculate the elemental maps. Sulfur was found at high levels in round L-granules in the granulocyte cytoplasm and in the cornified CE, correlating with the presence of the protein, loricrin (≈7% Cys/Met residues). EELS confirmed these observations quantitatively: either L-granules consist exclusively of loricrin or any additional components must have an equally high net sulfur content. These data also confirm the large fraction (≈75%) of loricrin in the CE, as inferred from modeling of its amino acid composition. We also observed extracellular deposits between cornified squames in fetal mouse epidermis that we callperipheral granules.Their sulfur content is at least as high as that of L-granules but they do not label with anti-loricrin antibodies, suggesting the presence of another sulfur-rich protein.

The Molecular Pathology of Progressive Symmetric Erythrokeratoderma: A Frameshift Mutation in the Loricrin Gene and Perturbations in the Cornified Cell Envelope

The erythrokeratodermas (EKs) are a group of disorders characterized by erythematous plaques associated with variable features that include palmoplantar keratoderma. One type of EK is known as "progressive symmetric erythrokeratoderma" (PSEK). We studied members of a family of Japanese origin in which the index case with PSEK had had well-demarcated nonmigratory erythematous plaques on her extremities since birth. Sequence determination of the loricrin gene revealed an insertion of a C following nucleotide 709. The mutation results in a frameshift that changes the terminal 91 amino acids in the wild-type polypeptide into missense amino acids and adds 65 additional residues. This further implicates loricrin defects in the pathogenesis of disorders with palmoplantar keratoderma and pseudoainhum.

174 Cornified cell envelope (CCE) precursor proteins and transglutaminase (TGase) 1 are coordinately expressed in the sites of hair canal morphogenesis during human hair follicle development

174 Cornified cell envelope (CCE) precursor proteins and transglutaminase (TGase) 1 are coordinately expressed in the sites of hair canal morphogenesis during human hair follicle development

Cloning of Human Keratolinin cDNA: Keratolinin Is Identical with a Cysteine Proteinase Inhibitor, Cysteine A, and Is Regulated by Ca2+, TPA, and cAMP

Keratolinin has been described as one of the precursor proteins of cornified cell envelope of keratinocytes. Using rabbit polyclonal anti-human keratolinin antibody, we isolated a cDNA clone of human keratolinin gene from a human Agt11 cDNA expression library that was constructed by random priming from poly(A)+RNA extracted from cultured normal human keratinocytes. Screening by rabbit anti-human keratolinin antibody detected one positive clone (HKL-1). The recombinant 12.5-kDa protein constructed from the clone reacted specifically with the anti-human keratolinin antibody. DNA sequence analysis revealed that HKL-1 clone was 448 bp long, and its putative amino acid sequence was identical with that of a human cysteine proteinase inhibitor, cystatin A. Western blot analysis showed that the commercially available recombinant cystatin A also reacted specifically with the anti-human keratolinin antibody. Northern blot analysis indicated that HKL-1 clone hybridizes with mRNA of about 0.5 kb, consistent with the size of the HKL-1 clone. The keratolinin mRNA was highly expressed in cultured human keratinocytes in high Ca2+ (1 mM); in low Ca2+ (0.05 mM), the keratolinin mRNA expression was significantly lower. Using SV40-transformed human keratinocytes (SVHK cells), we further analyzed the regulation of keratolinin mRNA. In low Ca2+ (0.05 mM), keratolinin mRNA in SVHK cells was marginally detectable. Upon shift to 1 mM calcium, keratolinin mRNA was markedly increased. The upregulation of keratolinin mRNA was also observed by the treatment of SVHK cells with 10 ng TPA per ml or 100 microM forskolin under low calcium conditions (0.05 mM). Our results indicate that keratolinin is identical with cystatin A, a cysteine proteinase inhibitor, and its expression is positively regulated by Ca2+, TPA, and forskolin.

Involvement of c-JUN in the regulation of terminal differentiation genes in normal and malignant keratinocytes.

In stratifying cultures of human keratinocytes, expression of the proto-oncoprotein c-JUN and the small proline rich 2 (SPRR2) protein, a precursor of the cornified cell envelope, are inversely related. Whereas c-JUN is typically found in basal proliferating cells, SPRR2 is restricted to suprabasal differentiating layers. Malignant keratinocytes (derived from squamous cell carcinoma, SCC) have reduced sprr2 expression, consistent with their low potential to differentiate, and express c-jun at higher levels than normal keratinocytes. A direct relation between c-jun and sprr2 expression was shown in several ways: transient ectopic expression of c-jun inhibits sprr2a promoter activity in normal differentiating cells, whereas in malignant keratinocytes a dominant negative c-jun mutant restored at least partially both the low promoter activity and the expression of endogenous sprr2. These effects are mediated via a 134 bp promoter fragment which does not include the sprr2a AP-1 binding site. Interestingly, in an SCC cell line, constitutively expressing the dominant c-jun mutant, expression of the terminal differentiation marker involucrin is also strongly increased, suggesting that c-JUN is a general modulator of keratinocyte terminal differentiation rather than only affecting the expression of sprr2.

Novel Transglutaminase Inhibitors Reduce the Cornified Cell Envelope Formation

Transglutaminase (TGase) is a calcium-dependent enzyme which catalyzes the iso-peptide cross-link between peptide-bound glutamine and lysinein vivo.Though the cross-link is developed as a barrier function in the skin system, overexpression of this could invoke skin hyperkeratosis in psoriasis and roughness in aged skin. In former research, many strong irreversible TGase inhibitors failed application because of high cytotoxicity. We selected one peptide after primary screening of six synthetic peptides designed from domains of known TGase substrates. Then we attempted to reduce the size and finally obtained two tetrameric peptides. When we treated keratinocyte with these TGase inhibitors under calcium-induced differentiation, the formation of a cornified cell envelope (CE) was decreased to the same level of CE under proliferating conditions without cytotoxic effect. Therefore, we propose that these TGase inhibitors may be useful for solving the physiological hypercross-linking problems for pharmaceutical or cosmetic purposes.

Expression of the SPRRCornification Genes Is Differentially Affected by Carcinogenic Transformation

The small proline rich protein ( SPRR) genes constitute a family of conserved genes which are part of the human epidermal differentiation complex (EDC) on chromosome 1q21 and code for precursor proteins of the cornified cell envelope. The expression of these genes is strictly linked to keratinocyte terminal differentiation both in vivoand in vitro.Here we show that cultured cell lines derived from squamous cell carcinoma (SCC) show significantly lower levels of SPRRexpression than normal human keratinocytes. However, the residual SPRRexpression in SCC lines appears to be both gene and cell line specific. Expression of SPRR2appears to correlate well with the residual ability of these cells to differentiate. However, the kinetics of SPRR2expression, following treatment with calcium, an inducer of keratinocyte differentiation, are typical for each cell line and differ substantially from the ones found in normal cells. In most cell lines a rapid transient expression of SPRR2contrasts with a slow induction leading to a high sustained level of expression in normal cells. This pattern of expression is typical for SPRR2and not observed for the other SPRRgenes or involucrin. Our analysis indicates that the expression of various keratinocyte terminal differentiation markers, even when involved in the same biological process (cornification), can be differentially affected by carcinogenic transformation.