Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ε-(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4-hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

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.     

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.     

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."     

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.     

Skin structure and cornification proteins in the soft-shelled turtle Trionyx spiniferus

In contrast to most chelonians, the fully aquatic soft-shelled turtles have a smooth, unscaled, and pliable shell. The skin of the shell, tail, limbs, and neck of juveniles of Trionyx spiniferus has been studied by ultrastructural, immunocytochemical, and immunoblotting methods. The epidermis of the carapace and plastron has a thick corneous layer composed of alpha-corneocytes surrounded by a cornified cell envelope. The softer epidermis is similar to that of the shell but the epidermis and corneous layer are much thinner. Pre-corneous cells in both soft and shell epidermis are rich in vesicles produced in the Golgi apparatus and smooth endoplasmic vesicles, and contain numerous dense-core mucus-like and vesicular (lamellar) bodies. Secreted material is present among corneocytes where it probably forms an extensive intercellular lipid-mucus waterproof barrier. The dermis is very thick and composed of several layers of collagen bundles that form a plywood-patterned dermis. This dermis constitutes a strong mechanical barrier that compensates for the low content in beta-keratin, and lack of cornified scutes and dermal bones. The growth of the shell mainly occurs along the lateral margins. Immunocytochemistry reveals the presence of some beta-keratin in soft and shell epidermis, and this is confirmed by immunoblotting where bands at 18 and 32-35 kDa are present. Other proteins of the cornified cell envelope (loricrin and sciellin) or associated to lipid trafficking (caveolin-1) are also detected by immunoblotting. Loricrin positive bands at 24 and 57 kDa are present while bands cross-reactive for sciellin are seen at 45 and 53 kDa. Caveolin-1 positive bands are seen at 21-22 kDa. The presence of these proteins indicates that the epidermis is both coriaceous and waterproof. These results suggest that the shell of Trionyx is tough enough to be as mechanically efficient as the hard shell of the other turtles. At the same time, a soft shell is lighter, its shape is more easily controlled by muscles, and it allows a more controlled closure and retraction of limbs and neck inside the shell. Thus, the shell makes the animal more streamlined for swimming.     

Identification and characterization of a novel component of the cornified envelope, cornifelin

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.     

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.     

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.     

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.     

Up-regulation of loricrin expression by cell adhesion molecule nectin-1 through Rap1-ERK signaling in keratinocytes

Nectin is an immunoglobulin-like cell-cell adhesion molecule, which plays essential roles in the initial step of formation of adherens junctions and tight junctions. We demonstrate here the role of nectin-1 in the epidermis using nectin-1-/- mice. Newborn nectin-1-/- pups showed shiny and slightly reddish skin; the amount of loricrin, one of the differentiation markers and also a major component of cornified cell envelopes, was markedly reduced in the epidermis of nectin-1-/- mice. The amounts of repetin and SPRRP, other components of cornified cell envelopes, were markedly elevated probably due to a compensatory mechanism to overcome the impaired expression of loricrin. However, cornified cells from nectin-1-/- mice were sensitive to mechanical stress. Moreover, Ca2+-induced activation of ERK through Rap1 and expression of loricrin were reduced in primary cultured nectin-1-/- keratinocytes; in turn, the inhibition of ERK activation reduced the amount of loricrin in wild-type keratinocytes. These results indicate that nectin-1 plays a key role in the expression of loricrin in the epidermis.     

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).     

Temperature-sensitive regulation of epidermal morphogenesis and the expression of cornified envelope precursors by EGF and TGFα

Epidermis reconstructed on de-epidermized dermis was used to investigate the effects of growth factors and culture temperature on epidermal morphogenesis and the expression of cornified envelope precursors. Cultures grown at 33°C or 37°C in the absence or presence of transforming growth factor alpha (TGFα), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), or insulin-like growth factor (IGF) show a similar morphology to that of native epidermis. Loricrin and SPRR2 are expressed in the stratum granulosum and SPRR3 is absent. Cultures grown in epidermal growth factor (EGF)-supplemented medium at 37°C have a normal morphology, whereas cultures grown at 33°C have a disorganized basal layer, no stratum granulosum, and nuclei are present in the stratum corneum. Loricrin is absent, and SPRR2 and SPRR3 expression extend into the spinous layers. Irrespective of the culture condition used, involucrin is aberrantly expressed in all suprabasal layers. EGF stimulated keratinocyte proliferation and migration to a greater degree than TGFα. Epidermis reconstructed on fibroblast-populated collagen gels at 33°C led to the same disturbances in keratinocyte differentiation as seen in cultures grown on de-epidermized dermis at 33°C in the presence of EGF, whereas parallel cultures grown at 37°C have a similar morphology to that of native epidermis.     

Targeted deletion of the sciellin gene resulted in normal development and maturation

Sciellin, together with other precursor proteins, was cross-linked by transglutaminase 1 to form the cornified envelope, an essential component of the physical barrier of the epidermis and stratified squamous epithelia. To more fully understand the function of sciellin in cornified envelope formation, we generated sciellin null mice. The mice appeared normal in their development and maturation and there were no structural features that distinguished them from littermate controls. Isolated cornified envelopes appeared normal in structure and were not more fragile to mechanical stress. There was no evidence of decreased barrier function or altered expression of other cornified envelope components. Transgenic mice expressing the repeat domain appeared to have a normal phenotype, like the null, and did not alter endogenous sciellin expression. We conclude that sciellin null mice had no structural anomalies and the transgenic mice did not act as a dominant-negative mutation.     

Characterization of Human Recombinant Transglutaminase 1 Purified from Baculovirus-infected Insect Cells

Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both μ- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.     

Characterization of human recombinant transglutaminase 1 purified from baculovirus-infected insect cells

Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both mu- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.     

Ultrastructural localization of caspase-14 in human epidermis.

Caspase-14 has been implicated in the formation of stratum corneum because of its specific expression and activation in terminally differentiating keratinocytes. However, its precise physiological role and its protein substrate are elusive. We studied the ultrastructural localization of caspase-14 in human epidermis to compare its distribution pattern with that of well-characterized differentiation markers. Immunogold cytochemistry confirmed that caspase-14 is nearly absent in basal and spinous layers. In the granular, layer nuclei and keratohyalin granules were labeled with increasing intensity towards the transitional layer. Particularly strong caspase-14 labeling was associated with areas known to be occupied by involucrin and loricrin, whereas F-granules, occupied by profilaggrin/filaggrin, were much less labeled. A high density of gold particles was also present at the forming cornified cell envelope, including desmosomes. In corneocytes, intense labeling was both cytoplasmic and associated with nuclear remnants and corneodesmosomes. These observations will allow focusing efforts of biochemical substrate screening on a subset of proteins localizing to distinct compartments of terminally differentiated keratinocytes.