Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.     

Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes

Involucrin immunoreactivity was localized ultrastructurally with protein A--gold in epidermis and cultured keratinocytes embedded in Lowicryl K4M. In the skin, immunoreactivity was found predominantly in cells of the granular layer and inner stratum corneum. The label was associated primarily with amorphous cytoplasmic material and especially keratohyaline granules. Some labeling was observed at the cell periphery, but little with keratin filaments. Tissue samples examined without aldehyde fixation showed relatively greater labeling in the outer stratum corneum than fixed tissue. In cultured cells, the labeling was also associated primarily with cytoplasmic granular material and to a lesser extent with the cell periphery. Upon treatment with the ionophore X537A, keratin filaments were found in aggregated arrays and the plasma membranes became convoluted. That involucrin immunoreactivity persisted in the cytoplasm in cultured cells and in vivo after cross-linking occurs could account for considerable isopeptide bonding detected in epidermal keratin fractions and indicates that not all the involucrin participates in envelope formation.     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

Proliferation of human embryonic and fetal epidermal cells in organ culture

The morphology of human embryonic and fetal skin growth in organ culture at the air-medium interface was examined, and the labeling indices of the epidermal cells in such cultures were determined. The two-layered epidermis of embryonic specimens increased to five or six cell layers after 21 days in culture, and the periderm in such cultures changed from a flat cell type to one with many blebs. The organelles in the epidermal cells remained unchanged. Fetal epidermis, however, differentiated when grown in this organ culture system from three layers (basal, intermediate, and periderm) to an adult-type epidermis with basal, spinous, granular, and cornified cell layers. Keratohyalin granules, lamellar granules, and bundles of keratin filaments, organelles associated with epidermal cell differentiation, were observed in the suprabasal cells of such cultures. The periderm in these fetal cultures formed blebs early but was sloughed with the stratum corneum in older cultures. The rate of differentiation of the fetal epidermis in organ culture was related to the initial age of the specimen cultured, with the older specimens differentiating at a faster rate than the younger specimens. Labeling indices (LIs) of embryonic and fetal epidermis and periderm were determined. The LI for embryonic basal cells was 8.5% and for periderm was 8%. The fetal LIs were 7% for basal cells, 1% for intermediate cells, and 3% for periderm. The ability to maintain viable pieces of skin in organ culture affords a model for studying normal and abnormal human epidermal differentiation from fetal biopsies and for investigating proliferative diseases.     

Two-compartment model for rabbit skin organ culture

Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d. The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid.     

Induction of epidermal mucous metaplasia by culture of recombinants of undifferentiated epidermis and retinol-treated dermis in a chemically defined medium

Epidermal mucous metaplasia of cultured skin is known to be induced by excess retinol. Studies were made on whether retinol affects primarily the epidermis or the dermis during retinol-induced epidermal mucous metaplasia of 13-day-old chick embryonic skin in culture. When recombinants of 13-day-old normal epidermis and retinol-treated dermis were cultured for 7 days in chemically defined medium in the absence of retinol, hormones, and serum, they showed altered epidermal differentiation toward secretory epithelium (mucous metaplasia). Thus retinol acted primarily on dermal cells.     

Developmental changes in components of chick brain microtubule-associated protein-1 (MAP-1) and tau proteins

Microtubule proteins were purified from chick brains at various developmental stages from the 12-day embryo to adult. Three species of microtubule-associated protein-1 (MAP-1) and 5-7 molecular components of tau proteins were observed by SDS-polyacrylamide gel electrophoresis. The molecular compositions were observed to change during development of the chick brain.     

Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells

Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle's minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [3H]thymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ovo with the exception of alpha-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.     

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.     

The specification of feather and scale protein synthesis in epidermal-dermal recombinations

Keratin proteins synthesized by dorsal or tarsometatarsal embryonic chick epidermis in heterotopic and heterospecific epidermal-dermal recombinants were analyzed by polyacrylamide gel electrophoresis and were compared to those produced by normal nondissociated dorsal and tarsometatarsal embryonic skin, as well as to those produced by control homotopic recombinants. Recombinant skins were grafted on the chick chorioallantoic membrane and grown for 8 or 11 days. Recombinants comprising dorsal feather-forming dermis formed feathers, irrespective of the origin of the epidermis. The electrophoretic band patterns of the keratins extracted from these feathers were of typical feather type. Conversely recombinants comprising tarsometatarsal scale-forming dermis formed scales, irrespective of the origin of the epidermis. The band patterns of the keratins extracted from the epidermis of these scales were of typical scale type. Heterospecific recombinants comprising chick dorsal feather-forming epidermis and mouse plantar dermis gave rise to six footpads arranged in a typical mouse pattern. In these recombinants, the chick epidermis produced keratins, the band pattern of which was of typical chick scale type. These results demonstrate that the dermis not only induces the formation of cutaneous appendages in confirmity with its regional origin, but also triggers off in the epidermis the biosynthesis of either of two different keratin types, in accordance with the regional type (feather, scale, or pad) of cutaneous appendages induced. The possible relationship between region-specific morphogenesis and cytodifferentiation is discussed in comparison with results obtained in other kinds of epithelial-mesenchymal interactions.     

Changes in Soybean Agglutinin (SBA) and Peanut Agglutinin (PNA) Binding Pattern in the Epidermis of the Developing Chick Embryo

Lectin binding pattern in the developing chick embryonic epidermis was studied using peroxidase labeling method. The epidermis of the 13-day-old embryo is in an undifferentiated state. Little binding of soybean agglutinin (SBA), specific for N-acetyl-D-galactosamine, and peanut agglutinin (PNA), specific for β-D-galactose, was seen in such epidermal cells. As the epidermis developed toward keratinization, the cell membrane of the differentiating flattened cells was positively stained with SBA and PNA. The positive staining was also seen in the supranuclear region of the cells located between the flattened cells and the basal cells. The basal cells remained unstained in all the stages of development. Similar staining pattern with SBA and PNA was seen in the cultured skin explants during the epidermal differentiation in vitro. These observations show that the SBA- and PNA-reactive glycoconjugates accumulate during the epidermal cell differentiation, suggesting their important roles in the maintenance of the ordered structure of the epidermis.     

In vitro differentiation of chicken embryo skin cells transformed by Rous sarcoma virus

The epidermal cells isolated from 14-day chicken embryo shank skin epidermis were infected in vitro with Rous sarcoma virus (RSV). Within a few weeks, rapidly growing colonies of epithelial cells appeared among the sea of transformed fibroblastic cells. When isolated and subcultured, these cells were found to possess typical markers of skin epidermis. The presence of major keratin and typical epithelial cell type morphology strongly suggested that these cells were transformed epidermal cells retaining their differentiated characteristics but having the capacity to propagate in cell culture. If RSV tsNY68, an RSV mutant having a temperature lesion in the src gene, was used, similar transformed epidermal cells were obtained at 36 degrees C (permissive temperature). At the nonpermissive temperature (41 degrees C) the growth rate of these cells decreased and additional keratin species appeared. At 41 degrees C the cells were flattened and lost the refractivity in their peripheries. All the keratins which are synthesized at the nonpermissive temperature were present in normal differentiated shank skin of 19-day old chick embryo. These cells also had "cornified envelop," indicating extensive differentiation. Viral production was as efficient as transformed fibroblasts during the rapid growth phase, while it declined significantly after the cells reached confluency, exhibiting the differentiated characteristics. Since no normal epidermal cells could be cultured under our experimental conditions, these results represent examples in which the src gene is essential for propagation of differentiated cells in cell culture while it abolishes only a part of differentiated characteristics.     

Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis

The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein.     

KERATIN SYNTHESIS DURING DEVELOPMENT OF THE EMBRYONIC CHICK FEATHER

The synthesis of keratin proteins during development of the embryonic chick feather was studied by quantitative gel electrophoresis of the reduced and carboxymethylated proteins. The results demonstrated a coordinated synthesis of the major keratin proteins, during and after the onset of keratin synthesis. The results from gel electrophoresis correlated well with electron microscope visualization or keratin fibrils in the developing feathers. Autoradiography at the electron microscope level indicated that the feather cells lose the ability to synthesize DNA before keratin synthesis begins, but retain the ability to synthesize RNA after keratin synthesis begins.     

Putative histidin-rich proteins in the epidermis of lizards

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.     

Changes in sterol metabolism in the skin of developing chick embryo and alterations in the presence of an anticholesterolemic agent and a chemical carcinogen

Changes in sterol metabolism in the skin of chick embryo during its development were studied with embryonal chick skin and with the cultured skin tissues. Changes in sterol metabolism of the skin of chick embryo began to appear at day 17, as observed by the accumulation of dihydrolanosterol, and the ratio of dihydrolanostrol:cholesterol increased thereafter until hatching. A similar change in sterol metabolism was also observed with the cultured skin tissue of chick embryo, although the stages of development seem to have been delayed by 3 days. The active sterol metabolism of the cultured skin tissue was also confirmed by studies of incorporation of [2-14C]acetate into sterols. 20,25-Diazacholesterol almost completely inhibited the incorporation of [2-14C]acetate into C27 sterols, whereas a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide, inhibited the incorporation of [2-14C]acetate into lathosterol but not that into cholesterol.     

Differential extraction of keratin subunits and filaments from normal human epidermis

We have investigated keratin interactions in vivo by sequentially extracting water-insoluble proteins from normal human epidermis with increasing concentrations of urea (2, 4, 6, and 9.5 M) and examining each extract by one- and two-dimensional gel electrophoresis, immunoblot analysis using monoclonal anti-keratin antibodies, and EM. The viable layers of normal human epidermis contain keratins K1, K2, K5, K10/11, K14, and K15, which are sequentially expressed during the course of epidermal differentiation. Only keratins K5, K14, and K15, which are synthesized by epidermal basal cells, were solubilized in 2 M urea. Extraction of keratins K1, K2, and K10/11, which are expressed only in differentiating suprabasal cells, required 4-6 M urea. Negative staining of the 2-M urea extract revealed predominantly keratin filament subunits, whereas abundant intermediate-sized filaments were observed in the 4-urea and 6-M urea extracts. These results indicate that in normal human epidermis, keratins K5, K14, and K15 are more soluble than the differentiation-specific keratins K1, K2, and K10/11. This finding suggests that native keratin filaments of different polypeptide composition have differing properties, despite their similar morphology. Furthermore, the observation of stable filaments in 4 and 6 M urea suggests that epidermal keratins K1, K2, and K10/11, which ultimately form the bulk of the protective, nonviable stratum corneum, may comprise filaments that are unusually resistant to denaturation.     

The proteins of the embryonic chick epidermis : II. During culture in serum-containing medium with and without added vitamin A

The proteins of the 12-day embryonic chick anterior metatarsal epidermis have been studied during growth in vitro in a serum containing medium with and without added vitamin A (5 IU/ml). The keratinization observed in the serum-containing medium alone was thus shown to be defective since only two of the proteins associated with keratinization during development in ovo were synthesized by the cultured epidermis, whereas the major group of 9 keratin protein bands was almost completely absent. The possible structural origins of these keratin protein bands is discussed in the light of these findings.In the medium containing vitamin A, synthesis of the two keratin proteins observed in the control epidermis was prevented and instead the band pattern obtained from the retinol-treated epithelium remained very similar to that of the 12-day epidermal starting material. Certain bands were increased in intensity in the presence of vitamin A, however, and in particular, the major band of the 12-day epidermis, which appears to be peridermal in origin, was present in increased amounts.     

Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation

Differentiation of the epidermis during embryonic rabbit development was found to be accompanied by dramatic changes in keratin proteins. Immunofluorescent labeling with keratin antiserum revealed that the undifferentiated epithelium of 12-d embryos was already committed to making keratin proteins. At 18 d of embryogenesis, the epithelium contained keratin proteins in the molecular weight range of 40,000-59,000. The stratification of the epithelium into two cell layers at 20 d of development coincided with the appearance of a 65-kdalton keratin. When a thick stratum corneum developed at 29 d, several additional keratins became prominent, most notably the large keratins (61- and 64-kdalton) and a 54-kdalton keratin. In addition, the 40-kdalton keratin, which had been present in earlier embryonic epidermis, disappeared. Newborn epidermis resembled that of a 29-d embryonic epidermis, with the exception of the appearance or increase in concentration of two more keratin species (46- and 50-kdalton). In vitro culturing of keratinocytes from 12- and 14-d embryonic skin demonstrated that these cells contained essentially the same keratin profiles as the undifferentiated epithelium of 18-d embryos (40-59 kdalton). Keratinocytes grown from older embryos contained increased amounts of keratin, similar to the in vivo situation, but did not synthesize the high molecular weight keratins. The changes observed during embryonic epidermal differentiation appear to be recapitulated during the sequential maturation steps of adult epidermis.     

The proteins of the embryonic chick epidermis : I. During the normal development in ovo

As a preliminary to a study of the proteins of the embryonic chick epidermis when grown in vitro under various culture conditions, the proteins of the anterior metatarsal epidermis, from 11 days of embryonic life up to 9 days posthatching, have been studied. Carboxymethylated derivatives of the proteins extracted by a thiol reduction procedure have been analyzed by polyacrylamide gel electrophoresis. The results have shown that the differentiation of the epidermis is characterized by the appearance between days 14 and 17 of at least 11 major protein bands in the electrophoretic pattern. Two of these bands are of relatively high molecular weight protein and appear earlier than the remaining bands which form a group of closely related, low molecular weight protein species. The differentiation of the tissue also involves the disappearance from the electrophoretic pattern of all but one of the five major bands present in extracts of the 11/12-day epidermis. A study of the proteins derived from the isolated periderm of the 14-day chick embryo beak has suggested that one of the major bands in the 11/12-day metatarsal epidermal extracts may be a peridermal protein.