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.     

Reversible inhibition by DMSO of hydrocortisone-induced keratinization of chick embryonic skin

Hydrocortisone, at a physiological concentration of 10^?8 M, induces keratinization of chick embryonic tarsometatarsal skin in a chemically defined medium in 4 days [1]. The presence of 1–4% DMSO with hydrocortisone reversibly prevented this keratinization. DMSO suppressed the appearance of epidermal structural protein, which was preferentially induced by hydrocortisone. It also suppressed hydrocortisone-induced epidermal transglutaminase activity; which was presumably responsible for polymerization and decrease in solubility of epidermal protein in keratinization, and it suppressed increase of epidermal protein. When DMSO was added to differentiated skin or added concomitantly with a higher concentration of hydrocortisone, epidermal transglutaminase activity was suppressed. Electron microscopic studies showed that hydrocortisone induced tonofilament bundles and keratinized cells with cellular envelopes, which are all characterestic of α-type keratinization of chick embryonic skin [2], and that DMSO inhibited hydrocortisone induced keratinization and kept the epidermis in an undifferentiated state. Moreover, DMSO inhibited epidermal DNA synthesis and increase in thickness of the epidermis during culture of hydrocortisone-treated skin, indicating that it suppressed cell proliferation as well as cell differentiation. DMSO by itself at 1 or 2 % did not affect epidermal cell differentiation, but suppressed cell proliferation when compared with untreated control.     

TMEM45A Is Dispensable for Epidermal Morphogenesis,Keratinization and Barrier Formation

TMEM45A gene encodes an initially uncharacterized predicted transmembrane protein. We previously showed that this gene is highly expressed in keratinocytes where its expression correlates with keratinization, suggesting a role in normal epidermal physiology. To test this hypothesis, we generated TMEM45A knockout mice and found that these mice develop without any evident phenotype. The morphology of the epidermis assessed by histology and by labelling differentiation markers in immunofluorescence was not altered. Toluidine blue permeability assay showed that the epidermal barrier develops normally during embryonic development. We also showed that depletion of TMEM45A in human keratinocytes does not alter their potential to form in vitro 3D-reconstructed epidermis. Indeed, epidermis with normal morphogenesis were generated from TMEM45A-silenced keratinocytes. Their expression of differentiation markers quantified by RT-qPCR and evidenced by immunofluorescence labelling as well as their barrier function estimated by Lucifer yellow permeability were similar to the control epidermis. In summary, TMEM45A gene expression is dispensable for epidermal morphogenesis, keratinization and barrier formation. If this protein plays a role in the epidermis, its experimental depletion can possibly be compensated by other proteins in the two experimental models analyzed in this study.     

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.     

Turnover of chloroplast and cytoplasmic ribosomes during gametogenesis in Chlamydomonas reinhardi

A number of novel observations on ribosomal metabolism were made during gametic differentiation of Chlamydomonas reinhardi. Throughout the gametogenic process the amount of chloroplast and cytoplasmic ribosomes decreased steadily. The kinetics and extent of such decreases were different for each of the two ribosomal species. Comparable rRNA degradation accompanied this ribosome degradation. Concurrent with the substantial ribosome degradation was the synthesis of rRNA, ribosomal proteins and the assembly of new chloroplast and cytoplasmic ribosomes throughout gametogenesis. The newly synthesized chloroplast ribosomes exhibited distinctively faster turnover than their cytoplasmic counterpart. Cytoplasmic ribosomes, pulse-labeled in early gametogenic stages, retained label until differentiation was nearly complete even though a net decrease in the level of cytoplasmic ribosomes continued, indicating that the newly synthesized cytoplasmic ribosomes were preferentially retained during differentiation. Hence the regulation of ribosome metabolism during gametogenesis contrasts with the conservation of ribosomes obtained during vegetative growth of C. reinhardi and other organisms. This unique pattern of ribosome metabolism suggests that new ribosome synthesis is necessary during gametogenesis and that some specific structural or functional difference relating to the development stage of the life cycle might exist between degraded and newly synthesized ribosomes.     

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.     

Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture

We studied the influence of hydrocortisone (HC) on hyaluronan (HA) metabolism in explants of human skin, a model retaining normal three-dimensional architecture of dermal connective tissue and dynamic growth and stratification of epidermal keratinocytes. The synthesis of hyaluronan and proteoglycans (PGs), and DNA, were determined with ³H-glucosamine and ³H-thymidine labelings, respectively. The total content and histological distribution of hyaluronan was studied utilizing a biotinylated aggrecan-link protein complex. A low concentration of HC (10^?9 M) stimulated the incorporation of ³H-glucosamine into hyaluronan in epidermis by 23% and reduced the disappearance rate of hyaluronan by 25% in chase experiments, resulting in a 74% increase in total hyaluronan (per epidermal dry weight) after a 5-day culture in 10^?9 M HC. On the other hand, a high concentration of HC (10^?5 M) reduced both synthesis (-42%) and degradation (-46%) of epidermal hyaluronan during 24 h labeling and chase periods. The cumulative effect of a 5-day treatment was a 24% decrease of total epidermal hyaluronan. The high dose (10^?5 M) also reduced keratinocyte DNA synthesis and epidermal thickness. In dermis, only the high (10^?5 M) concentration of HC was effective, inhibiting the incorporation of ³H-glucosamine into hyaluronan by 28%. No significant influences on total hyaluronan content or the disappearance rate of hyaluronan in dermal tissue was found. All HC concentrations lacked significant effects on newly synthesized PGs in epidermal and dermal tissues, but reduced the labeled PGs diffusing into culture medium. A low physiological concentration of HC thus maintains active synthesis and high concentration of hyaluronan in epidermal tissue, while high pharmacological doses of HC slows hyaluronan turnover and reduces its content in epidermis, an effect correlated with enhanced terminal differentiation, reduced proliferation rate and reduced number of vital keratinocyte layers. © 1995 Wiley-Liss, Inc.     

Effect of hydrocortisone on skin development in the chick embryo: ultrastructural, immunohistological, and biochemical analysis

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.     

A Rapid,Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.     

Changes in Protein Synthesis upon Cytokinin-Mediated Adventitious Bud Induction and during Seedling Development in Norway Spruce, Picea abies

A pulse-treatment of embryos of Picea abies (L.) Karst with cytokinin efficiently and reproducibly induces the coordinate de novo formation of bud primordia from subepidermal cells. The cytokinin treatment also affects the germinative development of the embryo; chloroplast maturation is delayed, and cell elongation is completely suppressed. We have analyzed the protein patterns in developing spruce embryos with the aim of identifying proteins which are differentially synthesized during early bud-differentiation and germination. In addition to a set of major seed storage proteins and a large set of constitutively synthesized proteins, we distinguish two sets of proteins that showed different patterns of synthesis in relation to germination. One was synthesized at high rates during germination, and the second set during post-germinative seedling development. Twenty-two proteins were differentially synthesized in the bud-induced versus the germinating embryos. Interestingly, all 22 belonged to either the germination phase-abundant or the seedling protein sets, whereas the constitutively synthesized proteins were unaffected by the treatment. Proteins synthesized exclusively in bud-induced embryos were not found. In total, the bud-induction treatment caused a maintenance of a protein synthesis pattern typical for the germination phase in the nontreated embryos, and the de novo formation of buds was not preceded by a major change in gene expression in the tissue.     

The induction of protein X in DNA repair and cell division mutants of Escherichia coli

Certain temperature-sensitive Escherichia coli cell division mutants and DNA repair mutants were treated in several ways to alter DNA synthesis or cell division. The bacteria were pulsed with [³⁵S]methionine; then membrane proteins were prepared and examined using sodium dodecyl sulfate/polyacrylamide slab gels. Autoradiography was performed on the slab gels so that the rate of synthesis of protein X could be determined by microdensitometry.Several changes in the rate of synthesis of the 40,000 molecular weight protein X were found in the different mutants. The wild-type (rec⁺ and lex⁺) strains synthesized protein X in response to DNA synthesis inhibition. However, neither recA^? strains nor lex^? strains synthesized protein X.Both the filament forming, temperature-sensitive mutants tif^? and tsl^? (which was derived from lex^?) synthesized protein X when DNA synthesis was inhibited, but at rates different from the wild-type strains. Moreover, these strains also produced protein X at their non-permissive temperature, even though DNA synthesis was not inhibited. In the tif^? mutant, the rate of synthesis of protein X was influenced by the addition of nucleic acid precursors.A double mutant tsl^?recA^? produced protein X when DNA synthesis was inhibited, or at the non-permissive temperature (although DNA synthesis was normal). This was the only strain carrying a recA^? mutation capable of synthesizing protein X.From these results it is suggested that the genes lex, recA and tif comprise a system that controls DNA repair and limits DNA degradation by the recBC nuclease. The inducer of this control system might be a DNA degradation product.     

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on newly synthesized proteins in mouse epidermis

We have analyzed the effects of treatment of mouse epidermis with the potent tumor promoter TPA on the profile of newly synthesized proteins. TPA was applied to the skin of the intact mouse, and either 3 or 24 hr later skin fragments were pulse-labeled in vitro with ³⁵S-methionine for 4 hr. The epidermal proteins were extracted and separated by two-dimensional gel electrophoresis. Over 200 individual proteins were resolved in acidic gels. At least 10 of these showed major (by a factor of 5 or more) increases or decreases in response to TPA; eight of these appear to be keratin proteins. Two-dimensional gel profiles of basic proteins synthesized by mouse epidermis resolved over 100 individual proteins. Only one of these showed a significant change in response to TPA. This 41 kd protein increased more than 100-fold within 24 hr after the application of TPA. Treatment of mouse skin with mezerein, a plant diterpene structurally related to TPA, produces an almost identical change in the pattern of proteins produced. Four agents that induce hyperplasia but are not potent tumor promoters, ethylphenylpropiolate, acetic acid, turpentine oil and the Ca⁺⁺ ionophore A23187, modulate the synthesis of only three of the keratin proteins. Thus the changes in protein profiles induced by TPA and mezerein are not simply the consequence of hyperplasia. In addition, application to mouse skin of a glucocorticoid that is a potent inhibitor of tumor promotion inhibits most of the changes in protein profiles induced by TPA. Taken together, these results indicate that TPA and mezerein induce early and marked changes in the profile of specific epidermal proteins. It seems likely that some of these changes are directly related to the process of tumor promotion.     

Keratinization in the epidermis of amphibians and the lungfish: comparison with amniote keratinization

Keratinization in the epidermis of amphibians and the lungfish has been studied by electron microscopy, autoradiography and immunocytochemistry to determine whether histidine-rich proteins, filaggrin and loricrin are present. In the lungfish and amphibian tadpoles, anti-keratin antibodies (AE1 and AE3) stain the whole epidermis but not the AE2 antibody, a marker for keratinization. In adult epidermis, the AE2 antibody mainly stains keratinized layers, AE1 mainly stained basal cells, less suprabasal cells and no pre-keratinized and keratinized layers, and AE3 stains all epidermal layers. This staining pattern resembles that of amniote epidermis. Little tritiated histidine is taken up in toad epidermis at 4-6 h post-injection but 24 h after injection the radioactivity is most concentrated in the replacement layer beneath the corneus. This indicates that protein synthesis takes place in the epidermis but, due to the metabolic conversion that takes place in 24 h, it is unlikely that histidine-rich proteins are formed. Neither filaggrin-like nor loricrine-like immunoreactivities are present in amphibian and lungfish epidermis. This indicates absence of histidine-rich matrix proteins and corneous cell envelope proteins and only mucus is present among keratin filaments. Filaggrine-like and loricrin-like proteins are characteristic of amniotes epidermis and might have originated in basic amniotes (cotylosaurs).     

Collagen and elastin synthesis in the developing chick aorta

Thoracic aortas from 8- to 18-day embryonic chicks were incubated in vitro for 30 min with [³H]glycine and the newly synthesized, labeled proteins were subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The gels were fractionated and the incorporation of label into procollagen (125,000 M_r) and tropoelastin (70,000 M_r) was estimated by summation of the radioactivity found in the appropriate regions of the gel. The analyses showed that at Day 8 approximately 14% of the incorporated [³H]glycine was found in procollagen and 22% in tropoelastin. In the following 6 days of development, there was a significant decline in the relative incorporation into procollagen and an increase into tropoelastin so that at Days 14–18 less than 10% of the label was found in collagen and 40% was now found in tropoelastin. Since glucocorticoids have been shown to alter the rate of synthesis of other proteins in the developing chick, 150 μg of hydrocortisone was injected into 8-day eggs and 24 h later the aortas were incubated and treated as described above. The pattern of protein synthesis exhibited by the hormone-treated aortas resembled that of 14- to 18-day embryos. Furthermore, incubation of 8-day aortas with 10^?8m hydrocortisone for 24 h produced a significant increase in the rate of elastin synthesis relative to that of other proteins. These results demonstrate that collagen and elastin synthesis vary during development of the chick aorta and they suggest that glucocorticoids may be involved in the control of their synthesis.     

Cell-to-cell ionic communication stimulated by 20-hydroxyecdysone occurs in the absence of protein synthesis and gap junction growth

When the larval epidermis of the beetle Tenebrio molitor is exposed to 20-hydroxyecdysone for 18–24 hr in vitro, the intercellular resistance to ion movement drops by about 30%. This drop in intercellular resistance is prevented when epidermal RNA synthesis is inhibited. It does not, however, appear to depend on de novo protein synthesis. Cycloheximide and emetine do not prevent the ecdysteroid from elevating junctional conductance. These drugs may even synergize with, or mimic, the action of the hormone on ionic coupling. A thin-section analysis of junctional ultrastructure showed that the absolute amount of gap junction at the epidermal cell interfaces does not alter after exposure to either 20-hydroxyecdysone or inhibitors of protein synthesis. These findings suggest that the low-resistance pathways in the beetle epidermis are not maximally conductive in the unstimulated state.     

20-Hydroxyecdysone control of the synthesis of putative calcium-binding proteins in the epidermis of Tenebrio molitor

The altered pattern of synthesis of putative calcium-binding proteins (pCaBPs) in the mid-instar epidermis following exposure to 20-hydroxyecdysone (20HE) in vitro was followed through the incorporation of [³⁵S]methionine into newly synthesized polypeptides. pCaBPs were separated from other epidermal polypeptides by Ca²⁺-dependent hydrophobic interaction chromatography, followed by polyacrylamide gel electrophoresis and fluorography. The dominant effect of 20HE is to depress pCaBP synthesis. Of the 17 newly-synthesized pCaBPs consistently detected in total cell lysates, the synthesis of ten was depressed strongly and that of the remaining seven was unaltered by exposure to 20HE. Most newly-synthesized pCaBPs identified were found in the cytosolic fraction of the epidermis. One pCaBP was identified as Tenebrio calmodulin based on its altered electrophoretic mobility in the absence of calcium ions, its isoelectric focusing point, its binding to phenyl-Sepharose and phenothiazine and its binding to antibodies against purified mammalian calmodulin. The synthetic rate of this pCaBP was not affected by 20HE. The distribution of another pCaBP (32 kDa) shifted from the cytosolic to the microsomal fraction on adding Ca²⁺ (or reversed by adding EGTA) to the cell extract before fractionation. The synthesis of this protein was depressed by 20HE. These findings suggest that 20HE influences epidermal behaviour, at least in part, through its ability to modulate the synthetic rate of several pCaBPs.     

Abnormal keratinization in the pupoid fetus (pf/pf) mutant mouse epidermis

During its development the epidermis of the pf/pf mutant mouse is invaded by cells from the underlying dermis. These invading cells establish a network of cells including fibroblasts, endothelial cells, and nerve fibers, throughout the epidermis. Subsequent to these events the keratohyalin protein, filaggrin, is drastically reduced and keratinization fails to occur. Heterotypic tissue recombinations indicate that the pf gene is not expressed in the skin. After simply grafting whole mutant dorsal skin, filaggrin synthesis is initiated and an orderly process of epidermal differentiation is achieved. These results suggest that the pf gene acts systemically and that the failure of epidermal differentiation in the mutant occurs secondary to abnormal epidermal organization.     

Protein Synthesis during Neural and Epidermal Differentiation in Cynops Embryo

Two-dimensional gel electrophoresis was used to analyze protein synthesis in relation to neural and epidermal differentiation in Cynops pyrrhogaster embryo. Various regions of embryos at different developmental stages, from late morula to early neurula stages, were excised, radiolabelled with ³⁵S-methionine, and the pattern of protein synthesis were compared. The following four types of protein spots were observed: (1) six proteins synthesized characteristically in the epidermal region of the embryo after gastrulation, (2) two proteins synthesized in both epidermal and endodermal regions, but not in other regions, after gastrulation, (3) a protein first detected at early blastula stage, of which expression was nearly constant in presumptive epidermis region but declined in the other regions, (4) the candidate for neural plate specific protein synthesized at a very high level in ectoderm explants treated with concanavalin A, a substance which evokes neural induction.     

Immunofluorescent studies of epidermal protein during UV induced carcinogenesis.

Previous studies with agar diffusion technique demonstrated that antibodies produced in rabbits by injection of urea extractable proteins of rat cornfied cells cross react with proteins extracted from normal epidermis of hairless mice using the same technique. In the present study we investigated by indirect immunofluorescence microscopy the immunoreactivity of epidermal proteins in normal and ultraviolet light (UVB) induced hyperplasia and malignant transformation. Reactivity to the antibody was seen over the entire epidermis of nontreated skin and hypertrophied epidermis which occurred at 6-8 weeks after initiation of UVB irradiation. However, the reactivity diminished when malignant changes took place in the epidermal cells. Almost complete disappearance of the immunoresponse was observed in squamous cell carcinoma produced by further UVB radiation. These results suggest that the reactivity of this urea extractable protein serves as an additional immunologic marker for normal epidermal cells. Alterations in the immunoreactivity parallels UVB induced carcinogenesis.