Inhibition by epidermal growth factor (EGF) of epidermal DNA synthesis in cultured chick embryonic skin pretreated with retinol and/or hydrocortisone: specific increment in EGF binding activity in both retinol- and hydrocortisone-pretreated epidermis without correlation to EGF-mediated inhibition of cell growth.

When tarsometatarsal skin of 13-day-old chick embryos that had been cultured in medium containing 5% delipidized FCS with or without retinol (20 microM) and/or hydrocortisone (20 nM) for 1 day was cultured in a chemically defined medium without either the hormone or retinol for 1 day, epidermal DNA synthesis of hydrocortisone- and/or retinol-pretreated skin was inhibited when compared to that of control skin. The addition of epidermal growth factor (EGF, 10 ng/ml) to retinol- or hydrocortisone-pretreated skin further inhibited the epidermal DNA synthesis. Epidermal DNA synthesis in retinol- and hydrocortisone-pretreated skin was more strongly inhibited than in retinol- or hydrocortisone-pretreated skin, but was not further inhibited by EGF. In epidermis which was induced to differentiation toward keratinization by hydrocortisone or mucous metaplasia by retinol, EGF inhibited DNA synthesis. The extent of [125I]-EGF binding to the epidermis of retinol- and hydrocortisone-pretreated skin was 160-180% that in control skin, with no change in affinity. Hence there is no correlation between EGF-binding and the mitogenic activity of EGF.     

Tgm2/Gh, Gbx1 and TGF-beta are involved in retinoic acid-induced transdifferentiation from epidermis to mucosal epithelium

We previously demonstrated that retinoic acid (RA) induces epidermis to transdifferentiate to mucosal epithelium with goblet cells in chick embryonic cultured skin. To characterize the molecular mechanism of this transdifferentiation process, we used rat embryonic cultured skin and immunohistochemistry to confirm that RA-induced epidermal transdifferentiation accompanies the expression of markers of esophagus epithelium. Because Gbx1, TG2/Gh (transglutaminase2) and TGF-beta2 are reported individually to be induced by RA in cultures of chick embryonic skin, mouse epidermal cells and human hair follicles respectively, here, we investigated whether cooperative interplay of Gbx1, TG2/Gh and TGF-beta2 is required for the transdifferentiation of epidermal cells to mucosal cells. We have shown that expression of Gbx1, TG2/Gh and TGF-beta proteins were all upregulated in RA-induced transdifferentiated skin and that the former two were expressed in the epidermis, while TGF-beta was expressed in the dermis. Inhibitors of the TGF-beta signal pathway partially inhibited transdifferentiation. Overexpression of both hTG2/Gh and mGbx1 together in the epidermis by electroporation resulted in cuboidal cells in the upper cell layers of the epidermis without keratinized layers, although epidermal keratinization was observed in skin by overexpression of either of them. Labeling DNA with BrdU indicated that RA directly transdifferentiated transient amplifying epidermal cells, not stem cells, to mucosal cells. This study showed that coexpression of TG/2 and Gbx1 in the epidermis was required for esophagus-like mucosal transdifferentiation, and that increase in TGF-beta2 expression by RA in the dermis was essential to induce transdifferentiation through epithelial-mesenchymal interaction.     

Induction of the alpha-type keratinization by hydrocortisone in embryonic chick skins grown in a chemically defined medium: An electron microscopic study

Previous biochemical analyses showed the differential accumulation of the epidermal structural protein, which yielded S-carboxymethylated epidermal protein A (SCMEpA), in the hydrocortisone-induced in vitro keratinization of 13-day embryonic chick tarsometatarsal skin growing in a chemically defined medium (Sugimoto et al., 1974). Fine structural features of such an in vitro keratinization process were studied by electron microscopy in the present work.After 2 days of culture with hydrocortisone (0.02 or 0.2 μM), development of the tonofilament bundles occurred to some extent, but the keratinized layer was not formed. Keratinization was observed after 4 days of culture with hydrocortisone (0.02 or 0.2 μM). Desmosomes and tonofilament bundles were prominent in the cytoplasm of the basal and intermediate cell layers of the epidermis. Keratohyalin granules and lipid droplets appeared in the upper layer. Degradation of cellular organelles such as nuclei and mitochondria then proceeded, leaving only filament bundles and electron-dense amorphous masses in the cytoplasm. Thickened cellular envelopes, which are characteristic of keratinized cells, were also observed. These features are characteristic of alpha-type keratinization which is common for other body surfaces. Beta-type keratinization, typical of normal embryonic scales, was not observed even after 6 days of culture with hydrocortisone. Keratinization of embryonic subperiderm of beta-type did not occur either. These ultrastructural observations clearly showed that hydrocortisone induced the alpha-type keratinization. It was also suggested that SCMEpA was closely related to alpha-type keratinization.     

Epidermal protein metabolism directed toward keratinization by hydrocortisone in the chick embryonic skin growing in a chemically defined medium.

The metabolism of the epidermal structural and nonstructural proteins was studied in hydrocortisone-induced in vitro keratinization of 13-day chick embryonic skin growing in a chemically defined medium. The protein metabolism of the epidermis was examined by determining the amounts of radioactivity incorporated into the fractions of reduced, S-carboxymethylated epidermal proteins (SCMEp) which were separated by polyacrylamide gel electrophoresis. A group of high molecular weight, glycine-rich derivatives of the epidermal fibrous protein called SCMEpA were found to be actively synthesized in the hydrocortisone-treated epidermis alone, while a group of undefined protein derivatives called SCMEpX was shown to be synthesized exclusively in the nontreated epidermis. Chase-culture of the prelabeled explants revealed that hydrocortisone accelerated the degradation of general proteins including SCMEpX while SCMEpA remained metabolically stable throughout the culture. Actinomycin D did not significantly affect the hydrocortisone-induced synthesis of SCMEpA but greatly inhibited that of SCMEpX of the nontreated epidermis, suggesting the induction by the steroid of relatively stable mRNA for SCMEpA. From these findings, it is concluded that hydrocortisone directed the cultured epidermis toward keratinization through acceleration of the synthesis of epidermal structural proteins and degradation of other proteins.     

Expression of Hex homeobox gene during skin development: Increase in epidermal cell proliferation by transfecting the Hex to the dermis

A number of homeobox genes have been found to be expressed in skin and its appendages, such as scale and feather, and appear to be candidates for the regulation of the development of these tissues. We report that the proline-rich divergent homeobox gene Hex is expressed during development of chick embryonic skin and its appendages (scale and feather). In situ hybridization analysis revealed that, during development of the skin, a transient expression of the Hex gene was observed. While the expression of Hex in the dermis was closely correlated with proliferation activity of epidermal basal cells, that in the epidermis was related to a suppression of epidermal differentiation. When dermal fibroblasts were transfected with Hex, stimulation of both DNA synthesis and proliferation of the epidermal cells followed by two-fold scale ridge elongation and increase in epidermal area was observed during culture of the skin, whereas epidemal keratinization was not affected. This is the first study to demonstrate that Hex is expressed during development of the skin and its appendages and that its expression in the dermal cells regulates epidermal cell proliferation through epithelial mesenchymal interaction.     

The Effect of Hydrocortisone on the Epidermal Proliferation in an Organ Culture of Chick Embryonic Skin

Hydrocortisone is regarded as an initiator of keratinization in embryonic skin. The present investigation dealt with the effect of hydrocortisone on the proliferation of epidermal cells during early development: Cell kinetic analyses using ³H-thymidine autoradiography were applied to a skin organ culture prepared from a 13-day chick embryo.
Hydrocortisone at a concentration between 0.01 and 1.0 μg/ml was effective in initiating a morphological change leading to the epidermal keratinization in vitro and caused a marked decrease in the mitotic and labeling indices of epidermal basal cells, the decrease being maximum at 2 days of culture previous to the morphological change.
During continuous labeling with ³H-thymidine, the number of labeled basal cells reached 100% within 2 days in the control and 4 days in the culture treated with hydrocortisone. This confirmed that the growth fraction of epidermal basal cells was 1.0 even after the administration of hydrocortisone.
The duration of each cell cycle phase at 2 days of culture was determined by percent labeled mitoses and double-labeling analyses. It was concluded that hydrocortisone extended the generation time of epidermal basal cells at this time point about three fold over the control. This extension was mainly due to the elongation of the G ₁ phase.     

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.     

Hoxb13 up-regulates transglutaminase activity and drives terminal differentiation in an epidermal organotypic model

Hox genes act to differentiate and pattern embryonic structures by promoting the proliferation of specific cell types. An exception is Hoxb13, which functions as a proapoptotic and antiproliferative protein during development of the caudal spinal cord and tail vertebrae and has also been implicated in adult cutaneous wound repair. The adult epidermis, which expresses several Hox genes including Hoxb13, is continually renewed in a program of growth arrest, differentiation, and a specialized form of apoptosis (cornification). Yet little is known about the function(s) of these genes in skin. Based on its role during embryogenesis, Hoxb13 is an attractive candidate to be involved in the regulation of epidermal differentiation. Here, we demonstrate that Hoxb13 overexpression in an adult organotypic epidermal model recapitulates actions of Hoxb13 reported in embryonic development. Epidermal cell proliferation is decreased, apoptosis increased, and excessive terminal differentiation observed, as characterized by enhanced transglutaminase activity and excessive cornified envelope formation. Overexpression of Hoxb13 also produces abnormal phenotypes in the epidermal tissue that resemble certain pathological features of dysplastic skin diseases. Our results suggest that Hoxb13 functions to promote epidermal differentiation, a critical process for skin regeneration and for the maintenance of normal barrier function.     

TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells.

In the epidermis of skin, a fine balance exists between proliferating progenitor cells and terminally differentiating cells. We examined the effects of TGF-beta s and retinoic acid (RA) on controlling this balance in normal and malignant human epidermal keratinocytes cultured under conditions where most morphological and biochemical features of epidermis in vivo are retained. Our results revealed marked and pleiotropic effects of both TGF-beta and RA on keratinocytes. In contrast to retinoids, TGF-beta s acted on mitotically active basal cells to retard cell proliferation. Although withdrawal from the cell cycle is a necessary prerequisite for commitment to terminal differentiation, TGF-beta s inhibited normal keratinization in suprabasal cells and promoted the type of differentiation commonly associated with wound-healing and epidermal hyperproliferation. The actions of TGF-beta s and RA on normal keratinization were synergistic, whereas those on abnormal differentiation associated with hyperproliferation were antagonistic. These observations underscore the notion that environmental changes can act separately on proliferating and differentiating cells within the population. Under the conditions used here, the action of TGF-beta s on human keratinocytes was dominant over RA, and TGF-beta s did not seem to be induced as a consequence of RA treatment. This finding is consistent with the fact that RA accelerated, rather than inhibited, proliferation in raft cultures. Collectively, our data suggest that the effects of both factors on epidermal growth and differentiation are multifaceted and the extent to which their action is coupled in keratinocytes may vary under different conditions and/or in different species.     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

Activation of keratinization of keratinocytes from fetal rat skin with N-(O-linoleoyl) omega-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) as a marker of epidermis

To elucidate the functional significance of sphingolipids altered in the epidermal differentiation, we examined, the effects of sphingolipids on the activity of transglutaminase and the formation of cornified envelopes in the keratinocytes from fetal rat skin. N-(O-linoleoyl) omega-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) that was characteristically contained in the mammalian epidermis, as well as nonhydroxy fatty acid-containing GalCer and GlcCer, significantly enhanced the activity and the formation, but no or rather inhibited activity was observed with ceramides, GalCer with alpha-hydroxy fatty acid, saponified lipokeratinogenoside, etc. This indicates that skin-characteristic lipokeratinogenoside functions to regulate the transglutaminase for the formation of cornified envelopes in the process of keratinization.     

Stimulation by Bt2cAMP of epidermal mucous metaplasia in retinol-pretreated chick embryonic cultured skin, and its inhibition by herbimycin A, an inhibitor for protein-tyrosine kinase.

Epidermal mucous metaplasia of cultured 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing retinol (20 microM) for only 8-24 h and then in a chemically defined medium without vitamins or serum for 6 days. In the induction of mucous metaplasia, retinol primarily affects the dermal cells and a signal(s) induced in the dermis by excess retinol alters epidermal differentiation toward secretory epithelium. In this work we found that Bt2cAMP (2 mM) stimulated mucous metaplasia severalfold when added to retinol-pretreated skin but inhibited epidermal mucous metaplasia when added together with retinol. Forskolin (100 microM), an activator of adenylate cyclase, also stimulated mucous metaplasia when added to retinol-pretreated skin. On the other hand, transduction in the epidermal cells of a signal(s) induced in dermal cells by excess retinol was inhibited by herbimycin A (500 ng/ml), an inhibitor of protein-tyrosine kinases, and TPA (0.1 microM), an activator of protein kinase C. Hence these findings indicated that cAMP stimulated signal-induced mucous metaplasia, and that transduction of the signal(s) in the epidermal cells required protein-tyrosine kinase and was inhibited by protein kinase C.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Short term retinol treatment in vitro induces stable transdifferentiation of chick epidermal cells into mucus-secreting cells

Summary Epidermal mucous metaplasia of cultured skin can be induced by treatment with excess retinol for several days (Fell 1957). In the induction of mucous metaplasia, retinol primarily affects the dermal cells and retinol-pretreated dermis can alter epidermal differentiation towards secretory epithelium (Obinata et al. 1987). In this work, we found that mucous metaplasia could be induced by culturing 13-day-old chick embryonic tarsometatarsal skin in medium containing retinol (20 M) for only 8–24 h, followed by culture in a chemically defined medium (BGJb) without retinol or serum for 6 days. The application of cycloheximide together with retinol during the first 8 h of culture inhibited epidermal mucous metaplasia during subsequent culture for 6 days in BGJb, indicating that induction of a signal(s) in the dermis by excess retinol requires protein synthesis. However, the presence of 20 nM hydrocortisone (Takata et al. 1981) throughout the culture period did not inhibit retinol-induced epidermal mucous metaplasia of the epidermis. This indicates that a brief treatment of the skin with excess retinol determines the direction of epithelial differentiation toward secretory epithelium; this is a simpler in vitro system for the induction of epidermal mucous metaplasia than those established before. Offprint requests to: A. Obinata     

Expression of the HB9 homeobox gene concomitant with proliferation accompanying epidermal stratification during development of chick embryonic tarsometatarsal skin

A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271–283). During scale ridge elongation at 12–15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.     

Reconstruction of the skin in three-dimensional collagen gel matrix culture

Summary The skin comprises three layers: epidermis, dermis, and hypodermis. We report here on a skin, reconstructed in vitro, that is composed of all three layers. The topmost layer, epidermis, was exposed to air by a new method. The exposure induced an extensive proliferation, and differentiation, i.e. keratinization was eventually observed in the cultured epidermal cells. Skin thus cultured will be a useful graft of transplantation and provide an ideal model system in which to study diseases of the skin.     

Epidermal cell proliferation and terminal differentiation in skin organ culture after topical exposure to sodium dodecyl sulphate

Summary Epidermal cell proliferation and differentiation were investigated in vitro after exposure to the anionic surfactant sodium dodecyl sulfate (SDS). Human skin organ cultures were exposed topically to various concentrations of SDS for 22 h, after which the irritant was removed. Cell proliferation was measured immunohistochemically by incorporation of bromodeoxyuridine (BrdU) into the DNA of cells during S-phase, while the expression of transglutaminase and involucrin were used as markers of differentiation. Cell proliferation was moderately increased at concentrations of SDS that did not affect the histomorphology (0.1% and 0.2% SDS). A marked increase of cell proliferation was observed 22 to 44 h after removal of SDS at a concentration (0.4%) that induced slight cellular damage. Exposure of human skin organ cultures to a toxic concentration of SDS (1.0%) led to decreased cell proliferation. Transglutaminase and involucrin were expressed in the more basal layers of the epidermis after exposure to 0.4% or 1.0% SDS. Moreover, intra-epidermal sweat gland ducts were positive for transglutaminase at these irritant concentrations. These in vitro data demonstrate that SDS-induced alterations of epidermal cell kinetics, as described in vivo are at least partly due to local mechanisms and do not require the influx of infiltrate cells. However, we were unable to relate the altered cell kinetics to the release of interleukin-1α or interleukin-6. Furthermore, supplementation of the culture medium with 12-hydroxyeicosantetraenoic acid did not affect epidermal cell proliferation. Rabbit skin cultures appeared more sensitive to SDS than human skin. At nontoxic doses, the irritant induced an increase of epidermal cell proliferation, similar to that observed in human skin discs.     

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.     

Regulatory effects of epidermal growth factor and retinol on the glucocorticoid receptor level in cultured chick embryonic skin

When undifferentiated skin from 13-day-old chick embryos was cultured in a chemically defined medium, glucocorticoid specifically decreased the dexamethasone-binding activity of the epidermal cytosol after 1 day of culture, 3 days before it induced formation of a cornified layer over the intermediate cells of the epidermis. The binding activity reappeared after removal of the steroid from the medium. This reappearance was inhibited by epidermal growth factor (EGF, 100 ng/ml). The Addition of 2 microM retinol resulted in a 3-fold increase in specific dexamethasone binding in the epidermal cytosol within 12 h with no change in the binding affinity. The inhibition of glucocorticoid-induced keratinization by retinol is due a to mechanism other than inactivation of the glucocorticoid receptor.     

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.