Dexamethasone receptors in human epidermis

Epidermis was removed from skin using a dermatome. Specific, high affinity, low capacity binding proteins for [³H]-dexamethasone were identified in the cytosol having an apparent K_d of 14×10^-9 mol/1 with approximately 95 fmol of binding sites per mg protein.     

Simultaneous Evaluations of 1,25-Dihydroxyvitamin D3 Receptors in Fresh Layers of Epidermis and Dermis from Normal Rat Skin

The 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptors in fresh layers of rat epidermis and dermis were compared after separation of the two layers by treatment with dispase. After 18 h treatment with 500 U/ml dispase, the epidermal sheet was easily peeled from the dermis with forceps, without dissociation of keratinocytes. The cytotoxicity of dispase was very low. A specific binding protein for 1,25-(OH)₂D₃, with a sedimentation coefficient of 3.2 S on a sucrose density gradient, was demonstrated in both layers of fresh rat skin. Scatchard analyses of cytosol binding to the hormone gave affinity constants of 0.09 ± 0.06 (mean ± SE) nM and 0.38 ±0.19 nM and binding capacities of 17.5 ± 0.5 and 14.5 ± 3.0 fmol/mg protein for the epidermis and dermis, respectively. This procedure allows simultaneous determinations of the receptor statuses of the epidermis and dermis from the same skin sample under various conditions.     

A comparison of the glucocorticoid receptors in the dermis and epidermis of the rat

The binding of glucocorticoids and non-glucocorticoids to the cytosol receptor in rat dermis and epidermis was measured using an in vitro assay. Dose-response data showed the order of binding potency in both tissues to be: clobetasol propionate, fluocinolone acetonide, clobetasone buryrate, betamethasone-17-valerate, triamcinolone acetonide, RO 12 7024 and dexamethasone. Binding affinity was similar in epidermis and dermis for most steroids but some steroids showed relatively less binding in dermis. Differences in the dermal epidermal binding ratio might be useful in predicting the therapeutic ratio of new steroids.     

Correlation between various proteins of bovine snout epidermis

Summary.— A comparison is made of several properties of urea-extractable proteins which were isolated from mammalian epidermis. A 6 mol/l urea solution extracts from epidermis at least 3 proteins which are precipitated maximally at pH 6·3, 5·5 and 4·5. The urea 4·5 and 5·5 proteins are soluble in water at pH 7·5 to 8·5. The urea 6·3 proteins were rather insoluble in the absence of urea, but were partially solubilized by 0·05 N NaOH to yield protein which precipitated maximally at pH 5·5, the alkali-soluble 5·5 protein. Also, the —S—S— bonds of the urea 6·3 proteins were reduced with Clelands reagent (dithiothreitol) and the resultant free-SH groups were reacted with iodoacetate to yield soluble- and insoluble-S-carboxymethyl derivatives of these proteins. The urea 6·3 and alkali-soluble 5·5 proteins have molecular weights exceeding 20 × 10⁶, whereas the S-carboxymethyl proteins have a molecular weight of 2·2 × 10⁵. The urea 5·5 protein extracted from epidermis at room temperature, and a portion of the urea 5·5 protein extracted at 0°C, have a molecular weight of 5·0 × 10⁴. With antisera prepared in rabbits against the urea 5·5 and 6·3 proteins, the following proteins were found to have common antigenic determinants: urea 4·5, 5·5 and 6·3 proteins, alkali-soluble 5·5 protein, and the S-carboxymethyl-soluble proteins.     

Human neonatal keratinocytes have very high levels of cellular vitamin A-binding proteins

Since cellular retinol- and retinoic acid-binding proteins (CRBP and CRABP) mediate the effects of vitamin A on epidermal differentiation, the levels of these binding proteins were measured in the epidermal and dermal layers of newborn, human foreskin as well as in primary cultures of keratinocytes and fibroblasts from these layers. Ligand binding assays with saturating concentrations of all trans-[3H]retinol or of all trans-[11-3H]retinoic acid were used to quantitate amounts of binding proteins in cytosols prepared from these skin layers or cultured cells. The epidermal levels of CRABP and CRBP (60.9 +/- 14.4 and 7.3 +/- 1.7 pmol per mg cytosol protein, respectively) were markedly higher than that reported for adult epidermis but were comparable to levels in keratinocytes cultured from neonatal foreskin epidermis (61.8 +/- 7.8 and 10.7 +/- 2.5, respectively). The levels of CRABP were much lower in the foreskin dermis than in the epidermis and the levels measured in the fibroblasts cultured from this dermis were consistent with the dermal levels. However, CRBP levels in cultured dermal fibroblasts were very low and could not account for the dermal CRBP levels, suggesting that another dermal cell type has high levels of CRBP.     

Qualitative and quantitative analysis of cytosol retinoid binding proteins in human skin

The distribution of the cytosol retinol and retinoic acid binding proteins are known to vary greatly within the different layers of the eye, a retinoid target organ. We have analyzed the cytosol retinoid binding from adult human skin, another retinoid target organ, and examined the relative contribution of the epidermis and dermis to the total retinoid binding. The mean specific activity of [3H]retinol (0.52 +/- 0.06 pmol/mg protein) and [3H]retinoic acid (3.20 +/- 0.45 pmol/mg protein) binding to cytosol preparations from different specimens of adult human skin was determined. On the average these skins bound 7-fold more retinoic acid than retinol. When skin was treated with EDTA and separated into epidermal and dermal fractions, [3H]retinol and [3H]retinoic acid binding was found in the cytosol derived from epidermis (0.36 +/- 0.03 pmol/mg protein, 3.69 +/- 0.13 pmol/mg protein, respectively) but not from dermis. To confirm that the absence of dermal binding was not due to loss during the EDTA separation, we assayed skin keratomed at 0.1, 0.2, and 0.3 mm. The skin obtained at 0.1 mm was upper epidermis and exhibited binding for both retinol and retinoid acid. The 0.2 mm skin, which added lower epidermis but little dermal contamination, had higher specific activities for both retinol and retinoic acid binding. The 0.3 mm skin which added primarily dermis, had lower specific activities for binding both retinoids. This is consistent with the concept that the epidermis is responsible for the majority of retinoid binding in adult human skin obtained from the lower limb.     

Presence of somatostatin in normal human epidermis

Somatostatin (SOM) is a ubiquitous peptide which is responsible for the inhibition of numerous biological functions. SOM is described as an antiproliferative molecule and an inhibitor of exocrine or endocrine secretion from a variety of tissues, including pancreas, gastrointestinal tract, central and peripheral nervous system. Mediation of SOM effects can be indirect or direct, respectively, through other molecules or receptors on target cells. We have searched for the presence of SOM in the epidermis using immunofluorescence, confocal laser scanning microscopy, radioimmunoassay, and chromatography. Immunofluorescence and confocal laser scanning microscopy studies were performed using rabbit antiserum anti-SOM and mouse monoclonal antibody directed to CD1a Langerhans cell (LC) marker disclosed with fluorescein or tetramethylrhodamine isothiocyanate conjugates. SOM was extracted from whole skin or epidermal cell suspension or LC-enriched suspensions and analysed by radioimmunoassay. We used an antiserum which was reactive for the 6–11 portion of native SOM. Chromatographic columns were performed on extracts from whole skin. The epidermis was SOM immunoreactive. LC were immunoreactive for SOM and the staining was membranous. SOM was extracted from the whole skin at about 0·13±0·02 fmol/mg of tissue (mean±SEM). The SOM concentration in epidermal cell suspensions was 1·5±0·9 fmol/10⁶ cells. Data obtained with LC-enriched suspensions showed large variations between donors. Extracts from skin showed one peak with an elution profile like that of 14 amino acid SOM. This study demonstrates that 14 amino acid SOM is expressed in normal human epidermis.     

Ibuprofen epidermal levels after topical application in vitro: effect of formulation, application time, dose variation and occlusion

The influence of vehicle (two oil-in-water emulsions and two gels, each containing 10% or 5% ibuprofen) application time (0·5,1,2 h), applied dose (1·5,3,6. 2 mg/cm²), time (24 h after an 0·5h application time), and occlusion on the epidermal concentration of ibuprofen was investigated. The drug concentration in the epidermis was measured by HPLC. All results were expressed as μg drug/mg epidermal protein. The application time had no influence on the epidermal drug concentration, whereas the two gel formulations produced concentrations approximately twice those obtained with the emulsions. A significant positive correlation was found between the applied dose and the epidermal concentration for each formulation. After 24 h the amount of drug remaining in the epidermis was low (reduced by factors of 10-20). Occlusion produced higher concentrations, but only with the 2-h application time. The methods are reliable, and useful in ranking vehicles according to their ability to release ibuprofen into the epidermis. Future investigations should explore the relationship between epidermal concentration and anti-inflammatory efficacy in vivo.     

The binding of topically applied glucocorticoids to rat skin

The epidermis of rat skin was shown to be the major site of accumulation of label after the topical application of [³H]-cortisol, [³H]-corticosterone and [³H]-triamcinolone acetonide. However, incubation of the skin in vitro in a way which ensured contact only between the dermal side of the skin and the steroid showed preferential accumulation of label from [³H]-cortisol and [³H]-corticosterone in the epidermis whereas label from [³H]-triamcinolone acetonide appeared evenly spread in the dermis and epidermis. The affinity of corticosterone to soluble proteins in skin homogenates was greater than that of cortisol as measured by displacement binding and correspondingly glucose uptake into the skin in vitro was inhibited for 18 h after a single application of corticosterone but was not inhibited by cortisol.     

Diffusion measurements in epidermal tissues with fluorescent recovery after photobleaching

Background/purpose: Pressure ulcers are areas of soft tissue breakdown, resulting from sustained mechanical loading of the skin and underlying tissues. Measuring biochemical markers that are released upon mechanical loading by the epidermis seems a promising method for objective risk assessment of the development of pressure ulcers. This risk assessment method will better determine the risk of a patient to develop pressure ulcers than the risk score lists currently used. So far, experimental studies have been performed that measure the tissue response in the culture supernatant. To elucidate the transport of the biochemical markers within the epidermis, the diffusion coefficient needs to be established. Methods: In the current study, fluorescent recovery after photobleaching (FRAP) is used to determine the diffusion coefficient of fluorescent‐labeled dextran molecules in human epidermis, porcine epidermis and engineered epidermal equivalents. These dextran molecules have a similar weight to the biochemical markers. Results: Similar diffusion coefficients were found for human and porcine epidermal samples (6.2 × 10⁻⁵±1.2 × 10⁻⁵ and 5.9 × 10⁻⁵±6.1 × 10⁻⁶ mm²/s, respectively), whereas the diffusion coefficient of the engineered epidermal equivalent was significantly lower (2.3 × 10⁻⁵±1.0 × 10⁻⁵ mm²/s). Conclusion: The diffusion could be measured in epidermal tissues using FRAP. In the future, the diffusion coefficients obtained in the current study will be used to study the difference between the transport in EpiDerm cultures and in human epidermis.     

Naringenin as an opener of mitochondrial potassium channels in dermal fibroblasts

Flavonoids belong to a large group of polyphenolic compounds that are widely present in plants. Certain flavonoids, including naringenin, have cytoprotective properties. Although the antioxidant effect has long been thought to be a crucial factor accounting for the cellular effects of flavonoids, mitochondrial channels have emerged recently as targets for cytoprotective strategies. In the present study, we characterized interactions between naringenin and the mitochondrial potassium (mitoBK_Ca and mitoK_ATP) channels recently described in dermal fibroblasts. With the use of the patch‐clamp technique and mitoplasts isolated from primary human dermal fibroblast cells, our study shows that naringenin in micromolar concentrations leads to an increase in mitoBK_Ca channel activity. The opening probability of the channel decreased from 0.97 in the control conditions (200 μmol/L Ca²⁺) to 0.06 at a low Ca²⁺ level (1 μmol/L) and increased to 0.85 after the application of 10 μmol/L naringenin. Additionally, the activity of the mitoK_ATP channel increased following the application of 10 μmol/L naringenin. To investigate the effects of naringenin on mitochondrial function, the oxygen consumption of dermal fibroblast cells was measured in potassium‐containing media. The addition of naringenin significantly and dose‐dependently increased the respiratory rate from 5.8 ± 0.2 to 14.0 ± 0.6 nmol O₂ × min^?1 × mg protein^?1. Additionally, a Raman spectroscopy analysis of skin penetration indicated that the naringenin was distributed in all skin layers, including the epidermis and dermis. In this study, we demonstrated that a flavonoid, naringenin, can activate two potassium channels present in the inner mitochondrial membrane of dermal fibroblasts.     

Langerhans cell hyperplasia and IgE expression in canine atopic dermatitis

Langerhans cells appear to be critical for IgE-mediated allergen capture and presentation in human atopic dermatitis. The present study sought to determine whether epidermal (i.e Langerhans cells) and dermal dendritic cells in the skin of dogs with atopic dermatitis are hyperplastic and expressed surface IgE. Frozen sections of lesional or nonlesional atopic and normal control canine skin were immunostained with CD1a-, CD1c-, and IgE-specific monoclonal antibodies. The enumeration of cells was performed by morphometry in both the epidermis and the dermis. Cell counts were compared with each individual’s total serum IgE levels. Higher numbers of epidermal and dermal dendritic cells were present in atopic dogs than in normal control animals. Epidermal Langerhans cell counts were significantly higher in lesional than in nonlesional atopic specimens. IgE⁺ dendritic cells were observed in lesional atopic epidermis and dermis, and nonlesional atopic dermis, but not in normal control skin specimens. The percentages of IgE⁺ dendritic cells were correlated with each patient’s total serum IgE levels. These results demonstrate dendritic cell hyperplasia and IgE expression in canine atopic dermatitis. Increased epidermal Langerhans cell counts in lesional specimens suggest an epidermal allergen contact in canine atopic dermatitis.     

Quantitative analysis of tryptase- and chymase-containing mast cells in atopic dermatitis and nummular eczema

The distribution of mast cells (MCs) containing tryptase (T) and chymase (C) was studied in the non-lesional and lesional skin of 26 patients with atopic dertnatitis (AD) and 23 patients with non-atopic nummular eczema (NE). and in the skin of eight healthy controls. T and C activities were demonstrated enzymehistochemically using 2-Gly-Pro-Arg-MNA and Suc-Val-Pro-Phe-MNA as substrates, respectively. The T- and C-containing MCs were counted separately in the epidermis, in contact with the basement membrane. In the papillary dermis and in different dermal levels (0·2 mm each). Also, the C protein was determined immunohistochemically. T-positive MCs were similarly distributed in non-lesional and lesional skin of both AD and NE. The MC number was relatively high in the upper dermis (papillary dermis and levels I and I!) of non-lesional and lesional skin of AD. In the upper dermis of non-lesional AD and NE skin and in normal skin, about 50% of T-positive MCs displayed C activity, whereas the percentage in lesional AD and NE skin was only about 30%. hi this respect, the non-lesional and lesional samples differed significantly froLu each other in both dennatoses (in AD p = 0%005, in NEP = 0·002. Students' t-test). In all samples the MC number decreased in the deeper dermal levels, although numerous T-containing MCs were still counted in the deeper dermis (dermal levels IV-VII) of lesional AD and NE skiti. differing significantly from the MC number in normal skin (In ADp = 0·005. in NE p=0·041). In the deeper dermis. the percentage of MCs containing active C was about 70% in non-lesional and lesional AD and NE. and about 90% in normal healthy skin. However, in the upper dermis of non-lesional and lesional skin of both AD and NE. about H()% of all MCs contained the C protein, which differed significantly from the value of 100% in normal skin (p<0·5). In conclusion, the increased number of T-positive MCs in the upper dermis of non-lesional and lesional AD contributes to promoting inflammation. C apparently loses its activity in the upper dermis of lesional AD and especially in NE. Thus. Ihe enzyme partially lacks its capability to suppress inflammation, such as degradation of neuropeptides and proteins. The dysregulation of these proteinases exists already in non-lesional skin of AD and NE.     

Functional regulation of tyrosinase and LAMP gene family of melanogenesis and cell death in immortal murine melanocytes after repeated exposure to ultraviolet B

The aim of this study was to investigate the effects of topical α-tocopherol application on epidermal and dermal antioxidants and its ability to prevent ultraviolet (UV)-induced oxidative damage. Hairless mice received topical applications of α-tocopherol 24 h before a single, acute UV irradiation (10 × minimal erythemal dose). The four major antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase), hydrophilic and lipophilic antioxidants, and lipid hydroperoxides, markers of oxidative damage, were assayed in both epidermis and dermis of hairless mice. Topical α-tocopherol treatment increased dermal superoxide dismutase activity by 30% ( P  < 0.01) and protected epidermal glutathione peroxidase and superoxide dismutase from depletion after UV irradiation. Total and reduced glutathione levels in the epidermis increased by 50% after the topical treatment ( P  < 0.05), as did dermal ascorbate levels (by 40%; P  < 0.01). The topical treatment increased α-tocopherol levels both in the epidermis (62-fold) and the dermis (22-fold; P  < 0.001 in each layer). Furthermore, α-tocopherol treatment significantly reduced the formation of epidermal lipid hydroperoxides after UV irradiation ( P  < 0.05). These results demonstrate that topical administration of α-tocopherol protects cutaneous tissues against oxidative damage induced by UV irradiation in vivo , and suggest that the underlying mechanism of this effect involves the up-regulation of a network of enzymatic and non-enzymatic antioxidants.     

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2×10^?10 mol/l) or by pharmacological doses of biotin (10^?5 mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.     

Vitamin A uptake by human skin in vitro

Summary Results are presented establishing that epidermis accumulates vitamin A from serum retinolbinding protein (RBP). Strips of human breast skin (0.2–0.3-mm thick) were incubated in a serum-free medium. From the rate of glucose oxidation, the tissue was viable for at least 48 h at 32°C in 5% CO₂ air. [³H]-Retinol-RBP (10^-6 M) was added to the medium for 1–24 h, after which epidermis and dermis were split and separately extracted with hexane after saponification. [³H]-Retinol was isolated by high performance liquid chromatography (HPLC). Epidermis had 6–7 times higher affinity for [³H]-retinol than dermis. The uptake could be saturated by substrate and was inhibited with unlabelled retinol-RBP but not with serum albumin. Furthermore, although the uptake was temperature-dependent, it seemed independent of cellular energy production. The epidermal accumulation of [³H]-retinol was reduced by the filtering action of dermis. On the basis of these observations, an in vitro model for the delivery of vitamin A to human skin has been proposed.     

A hypothesis on the cause of chronic epidermal hyperproliferation in asebia mice

We compared the skin of the mutant mouse asebia (ab/ab) with that of normal BALB/c mice by examining dermal cell counts, epidermal thickness, epidermal cell labelling index and ultrastructure. In asebia we found epidermal hyperproliferation and dermal inflammation, with much cell debris in the dermis. The very numerous macrophages, both intact and disintegrating, contained electron-lucent crystals, apparently composed of lipid. We hypothesize that these crystals cause macrophage lysis followed by dermal inflammation and finally hyperproliferation in the overlying epidermis.     

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2×10⁻¹⁰ mol/l) or by pharmacological doses of biotin (10⁻⁵ mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.     

On the relationship between the urea-soluble and citric acid-soluble proteins of bovine snout epidermis

This report presents evidence that the urea-extractable proteins and the citric acid-extractable protein (‘prekertin’) of bovine snout epidermis are related. Prekeratin was solubilized by trypsinization, S-carboxymethylation, 6 mol/l urea or 0·05 mol/l NaOH. trypsinization or S-carboxymethylation produced at least two species of soluble proteins. 6 mol/l urea at 37°C solubilized prekeratin, but a homogeneous product was not obtained. Depending upon the duration of exposure, prekeratin was cleaved by 0.05 mol/l NaOH at 25°C to form several soluble derivatives, whereas at comparable time intervals at 4°C both insoluble and soluble derivatives were produced. The various soluble proteins derived from prekeratin showed precipitin bands of identity with the urea-extracted proteins of beef snout epidermis when reacted in agar with antisera raised against the urea-extracted protein which precipitated at pH 6.3 and 5.5. It is suggested 6 mol/l urea and 0.05 mol/l NaOH may cleave prekeratin in situ to release the proteins extracted by urea and alkali respectively.