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PubMed1993

Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light.

A comprehensive comparison of antioxidant defenses in the dermis and epidermis and their response to exposure to ultraviolet (UV) irradiation has not previously been attempted. In this study, enzymic and non-enzymic antioxidants in epidermis and dermis of hairless mice were compared. Enzyme activities are presented both as units/gram of skin and units/milligram of protein; arguments are presented for the superiority of skin wet weight as a reference base. Catalase, glutathione peroxidase, and glutathione reductase (units/gram of skin) were higher in epidermis than dermis by 49%, 86%, and 74%, respectively. Superoxide dismutase did not follow this pattern. Lipophilic antioxidants (alpha-tocopherol, ubiquinol 9, and ubiquinone 9) and hydrophilic antioxidants (ascorbic acid, dehydroascorbic acid, and glutathione) were 24-95% higher in epidermis than in dermis. In contrast, oxidized glutathione was 60% lower in epidermis than in dermis. Mice were irradiated with solar light to examine the response of these cutaneous layers to UV irradiation. After irradiation with 25 J/cm2 (UVA + UVB, from a solar simulator), 10 times the minimum erythemal dose, epidermal and dermal catalase and superoxide dismutase activities were greatly decreased. alpha-Tocopherol, ubiquinol 9, ubiquinone 9, ascorbic acid, dehydroascorbic acid, and reduced glutathione decreased in both epidermis and dermis by 26-93%. Oxidized glutathione showed a slight, non-significant increase. Because the reduction in total ascorbate and catalase was much more severe in epidermis than dermis, it can be concluded that UV light is more damaging to the antioxidant defenses in the epidermis than in the dermis.

Animals↗

PubMed1986

In vitro synthesis of 12-hydroxy-eicosatetraenoic acid is increased in uninvolved psoriatic epidermis.

Certain arachidonic acid (AA) metabolites have been detected in psoriatic skin lesions. In this study the capacity of normal epidermis and clinically uninvolved psoriatic epidermis to transform AA into lipoxygenase products was determined in vitro. After incubating homogenized epidermis with exogenous AA, the extracted lipids were isolated by reverse-phase high-performance liquid chromatography. Each chromatographic peak was characterized by its UV absorption spectrum and identified by its coelution with the appropriate authentic standard and by radioimmunoassay of its eluate fraction. Identified compounds were quantitated by integrated UV absorbance. Leukotriene B4 (LTB4) was also identified by neutrophil chemokinesis. Normal epidermis generated 15-hydroxy-eicosatetraenoic acid (15-HETE) and 12-HETE, the latter being more abundant. 5-Lipoxygenase products (LTB4, LTC4, and 5-HETE) were not detected. However, an unknown compound exhibiting a triplet UV absorbtion spectrum with maximum at 274 mm was formed. Its formation was inhibited by 5,8,11,14-eicosatetraynoic acid, but not by indomethacin or a specific 5-lipoxygenase inhibitor (REV 5901). These data suggest that a di-HETE with a triene structure is one possible candidate for the unknown compound. Compared with normal epidermis, the formation of 12-HETE and the unknown di-HETE by uninvolved psoriatic epidermis was increased by 54% and 63%, respectively. The formation of 12-HETE and the unknown di-HETE in uninvolved psoriatic epidermis was stimulated to the same degree in the presence of the phospholipase inhibitor quinacrine. These results indicate that uninvolved psoriatic epidermis has an increased capacity to metabolize free AA into 12-lipoxygenase products.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1978

Possible defects in triacylglycerol and phosphatidylcholine metabolism in psoriatic epidermis.

Biopsies of normal skin, psoriatic lesions and the adjacent psoriatic skin were incubated with I-14C-acetate, 32P-orthophosphate and U-14C-glycerol. Total incorporation of I-14C-acetate into psoriatic lesions (17 samples) was 50% higher than in the adjacent uninvolved epidermis (9 samples) and 120% higher than into normal epidermis (10 samples). In the psoriatic lesion a much higher proportion of the total incorporation was into the neutral lipids and was due mainly to a very high incorporation of I-14C-acetate into the triacylglycerols. The I-14C-acetate incorporated into the phospholipids and especially phosphatidylcholine was proportionally much less in the psoriatic lesion and uninvolved psoriatic epidermis than in normal epidermis even though the incorporation of 32P-orthophosphate and U-14C-glycerol, both representing de novo synthesis, into the phospholipids in the psoriatic lesions and uninvolved epidermis were higher (four-fold in lesion) than in normal epidermis. Our findings (1) are evidence for a much increased triacylglycerol synthesis in psoriatic epidermis which would account for the long-known observation of lipid droplet accumulation in psoriatic cells; (2) suggest that in psoriatic epidermis there is a defect in phospholipid metabolism mainly involving phosphatidycholine and the deacylation (phospholipase A)-reacylation (phospholipid acyltransferase) cycle for fatty acid transfer.

Acetates↗

PubMed1983

Altered binding of Ulex europaeus I lectin to psoriatic epidermis.

We have used Ulex europaeus I (UEA I) lectin, specific for alpha-L-fucose-containing glycoconjugates, in fluorescence microscopy to stain cryostat sections of human skin from normal persons and patients with psoriasis and lichen simplex. In normal skin the upper layers of the stratum spinosum and the stratum granulosum were strongly reactive with UEA I, whereas the lower layers of the epidermis did not react. The staining intensity of the upper epidermis was similar to that of the endothelium of dermal blood vessels. Biopsies of the lesional skin of lichen simplex showed an intense UEA I-specific staining throughout the whole epidermis, similar in intensity to that seen in the upper epidermis of normal skin. In psoriatic lesions positive UEA I-specific fluorescence was seen throughout the whole epidermis, but the fluorescence was more faint and often granular. In uninvolved skin of psoriatic patients the whole epidermis showed a diffuse UEA I-specific fluorescence, differing in this respect from normal skin. In normal skin UEA I binds to epidermal cells which are at a certain state of differentiation. The results with psoriatic epidermis confirm that both uninvolved and lesional epidermis have a defect in epidermal maturation, as shown by the altered binding of UEA I lectin.

Epidermis↗

PubMed1990

[The effects of metabolic inhibitors on the excitability of the cells in embryonic epidermis of Cynops orientalis].

It has been reported that the cells in atypical epidermis, which developed from the in vitro cultured ectoderm isolated at early gastrula, showed very low excitability or were even non-excitable at 6 V when examined electro-physiologically. If non-excitable explants were treated with energy supplying substances, such as glucose, the action potential (AP) appeared quickly. It indicates that, the excitability of epidermis cells is related to their energy metabolism. In order to verify the above proposition the effects of metabolic inhibitors on the excitability of the epidermis cells were examined using electrophysiological technique. Two kinds of explants were used: explants which developed from the epidermis underlaid with mesoderm isolated at early neurula (epidermis vesicle) and explant which developed from the ectoderm isolated at early gastrula (atypical epidermis). In all experiments explants were stimulated extracellularly and APs were recorded intracellularly. The specimens were stimulated with electric stimulus at 6 V first, and, if they displayed AP, the strength was lowered to determine the stimulus threshold to evoke AP. The duration of stimulus was fixed at 1 ms. The ratio of the resting potential value during treatment to the original value was taken as index of change level of the resting potential (RP). During treatment of epidermis vesicle with 1 mM NaN3 or 1 mM NaCN or 0.1 mM 2,4-dinitrophenol (DNP), the excitability of the epidermis cells was reduced: the stimulus threshold gradually increased and the cells in most explants lost the excitability. The cells became excitable after the drugs were washed out.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1989

[Ultrastructure of the epidermis in laboratory rats under normal conditions and after percutaneous administration of an organophosphate].

For the study of the normal epidermal layers of the stratum basale, stratum spinosum, stratum granulosum and (male rats at the age of 2 years) were used. Skin samples were obtained from the spinal area and processed by the method of transmission electron microscopy to get semithin and ultrathin sections. An electron microscopical examination preceded by an evaluation of the semithin sections in the light microscope showed that the epidermis of both age groups contained keratinocytes and Langerhans cells. Keratinocytes are typically arranged in epidermis layers of the stratum basale, stratum spinosum, stratum granulosum and stratum corneum. These layers are of different ultrastructure. The Langerhans cells are localized suprabasally in the epithelium. In the course of keratinization, prominent ultrastructural changes in keratinocytes occur. The cell organelles of keratinocytes including the nucleus disappear and the formation of specific cell components--prominent bundles of tonofilaments, keratohyalin granules and keratinosomes--can be observed. Keratinized cells of the stratum corneum exhibit a homogeneous or fibrillar inner structure. Langerhans cells contain characteristic cytoplasmic organelles--Langerhans granules. The epidermis of old rats differs from that of young animals in a special arrangement of the dermo-epidermal junction. The most typical change (in comparison with the epidermis of young rats) is the presence of multiplied lamina densa. The qualitative study of the normal epidermis of the laboratory rat is completed by morphometric analysis. The measurements concern keratinocytes of the stratum basale. Cell dimensions under the light microscope as well as some cell organelles in ultrathin sections are evaluated. Another part of the study is aimed to estimate morphological changes in the epidermis of rats which were percutaneously administered a toxic organophosphate. In this experiment, the tissue processing for electron microscopy could be realized only when the exposed epidermal area had been previously decontaminated. A group of intoxicated animals was therefore compared with a control group, the epidermis of which was affected by the decontaminating agent only. The ultrastructural changes noted in the epidermis of intoxicated animals show no specificity. Damaged membrane organelles and vacuoles in keratinocytes, dense cells, dilated epidermal intercellular spaces containing the material of medium density, etc. are present. Furthermore, the above-mentioned changes proved to be manifestation of a combined effect of the proper toxic substance and the decontaminating agent.

Aging↗

PubMed1980

[Prostaglandin biosynthesis in psoriatic epidermis (author's transl)].

The biosynthesis of prostaglandins E2 (PGE2) and F2 alpha (PGF2) from exogenous arachidonic acid (AA) was investigated in 12 involved and uninvolved psoriatic epidermis and in 6 normal epidermis. PGE2 and PGF2 were determined by radio-immuno-assay after addition of 25 microgram of AA and 1 hour incubation. The biosynthesis of PGE2 and PGF2 alpha was higher in psoriatic epidermis than in control epidermis but lower in involved than in uninvolved epidermis. The ratio PGE2/PGF2 alpha is decreased in involved epidermis when compared with uninvolved epidermis and with controls. These quantitative and qualitative disturbances of the PG metabolism in psoriatic epidermis might play an important role in the pathogenesis of the disease. They could be responsible for abnormal keratinocyte differentiation and proliferation, lipid membrane abnormalities and abnormal immune response of psoriatic patients.

Epidermis↗

PubMed1994

Recovery of antioxidants and reduction in lipid hydroperoxides in murine epidermis and dermis after acute ultraviolet radiation exposure.

In previous studies we have found that a single acute dose of ultraviolet radiation to murine skin causes a large degree of destruction of enzymic and non-enzymic antioxidants immediately after irradiation. In the present study, we wished to elucidate the recovery of antioxidants after a single dose of ultraviolet (UV) radiation. We measured antioxidants and lipid hydroperoxides (as a marker of membrane damage) in murine epidermis and the dermis at 0, 3, 12, 24, 72 and 120 h after exposure to UV radiation (25 J/cm2, UVA+UVB). Lipid hydroperoxides showed the highest values immediately after UV exposure and returned to control values within 24 h in both epidermis and dermis. The activities of catalase, glutathione peroxidase and glutathione reductase showed the lowest activities immediately after UV exposure; superoxide dismutase activities reached a minimum at 3 h postexposure. The pattern of recovery was different for each enzyme and for epidermis and dermis. The activities of superoxide dismutase and catalase decreased remarkably and recovered slowly. Superoxide dismutase in the dermis recovered full activity by 120 h and in the epidermis by 12 h. Catalase activity in both epidermis and dermis had returned to only 50% of control activity at 120 h, although the epidermis showed a temporary increase (to 93%) at 24 h. Glutathione peroxidase and glutathione reductase were slightly decreased immediately after irradiation, recovered to 100% at 3 h and then increased to 200-250% in both the epidermis and the dermis at various times; values had returned to 100% in epidermis by 120 h but remained elevated in dermis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Detection of interleukin-1 receptors in human epidermis. Induction of the type II receptor after organ culture and in psoriasis.

Normal human epidermis is a rich source of biologically active interleukin-1 alpha (IL-1 alpha). Keratinocytes both synthesize this cytokine and respond to it via cell surface receptors (IL-1R), suggesting that the IL-1 system may play an important role in normal epidermal physiology and inflammation. In this study, we have examined the expression of IL-1R in normal and psoriatic epidermis, as judged at a functional level by the capacity to bind 125I-labeled IL-1 alpha (the principal IL-1 species present in epidermis) and by immunostaining with antibodies specific for each species of IL-1R. IL-1R was not readily detectable by either technique in normal, freshly isolated human epidermis. However, in lesional psoriasis or normal epidermis after 24 hours of organ culture, expression of IL-1R was dramatically induced, especially in basal keratinocytes. Immunostaining and antibody blocking studies demonstrated the induced IL-1R to be the type II species, a nonsignal transducing molecule previously demonstrated only on leukocytes. The Ka of this receptor was comparable to that previously demonstrated in vitro. mRNA for both species of IL-1R could be demonstrated by reverse transcriptase-polymerase chain reaction in fresh and cultured epidermis. These in vivo findings were confirmed in culture, where normal human keratinocytes expressed few IL-1R at rest but large numbers of type II IL-1R after activation by phorbol ester or interferon-gamma. We conclude that under resting conditions, epidermal expression of IL-1R is low. However, the potential for keratinocytes in vivo to express large numbers of the nonsignal transducing type II IL-1R is evident from both organ cultured and psoriatic epidermis. The in vitro induction of keratinocyte IL-1R by interferon-gamma suggests that this cytokine may be involved in the induction of type II IL-1R in inflammatory skin disease. The presence of bioactive IL-1 in epidermis, coupled with the inducible expression of the decoy type II IL-1R, indicates the existence of a highly regulated system of autocrine stimulation of keratinocytes by IL-1.

Autoradiography↗

PubMed2002

Alterations in cytokine regulation in aged epidermis: implications for permeability barrier homeostasis and inflammation. I. IL-1 gene family.

Acute disruption of the cutaneous permeability barrier with either solvents or tape-stripping stimulates a homeostatic metabolic response in the subjacent nucleated layers of the epidermis that results in a rapid restoration of normal permeability barrier function. When the aged epidermal permeability barrier is stressed, it reveals a diminished capacity for recovery, in comparison to young epidermis, analogous to other organs in the aged when stressed. Although the signals that regulate this homeostatic response by the epidermis have not yet been resolved, acute permeability barrier disruption stimulates release of prestored IL-1alpha, and increased production of potentially regulatory cytokines, including IL-1alpha and TNFalpha in the epidermis. In these studies, we addressed the hypothesis that cytokine dysregulation explains the permeability barrier abnormality in aged epidermis, assessing the regulation of IL-1 and TNF signaling in aged vs young mice. To determine whether the IL-1 family of cytokines plays a key role in the permeability barrier abnormality of the aged, permeability barrier recovery rates were compared in transgenic mice lacking the functional IL-1 type 1 receptor vs wild-type mice at various ages. Knockout of the IL-1 type 1 receptor exacerbates the defect in permeability barrier homeostasis that is seen in age-matched, wild-type counterparts. Furthermore, the sluggish permeability barrier recovery in aged epidermis is associated with, and at least in part attributable to, altered expression of the IL-1 family of cytokines and receptors both under basal conditions and after acute barrier perturbations. Whereas modulations in cytokine expression with epidermal permeability barrier perturbation are qualitatively similar in aged epidermis, they greatly differ quantitatively. In contrast, examination of TNFalpha mRNA and protein basally, and following barrier perturbation revealed no alterations in aged epidermis. Together, these results show that selective alterations in the IL-1 family of cytokines occur with aging and that defects in IL-1 signaling may contribute to the epidermal permeability barrier abnormality of aged skin.

Animals↗

PubMed1994

Cells of the Upper and Lower Epidermis of Barley (Hordeum vulgare L.) Leaves Exhibit Distinct Patterns of Vacuolar Solutes.

Vacuolar saps were extracted from individual, anatomically uniform cells of the upper (adaxial) and lower (abaxial) epidermis of the third leaf of barley (Hordeum vulgare L.) using a modified pressure probe. Saps (volume 80-200 pL) were sampled at various times between 3 d before and 7 d after full-leaf expansion and were analyzed for their osmolality and their concentrations of NO3-, malate, CI-, K+, and Ca2+. The osmolalities of upper and lower epidermis both increased with time but were similar to each other. In young leaves, K+ and Ca2+ were evenly distributed between the two epidermal layers, but as the leaf aged, the upper epidermis accumulated high (40-100 mM) Ca2+, whereas cells of the lower epidermis accumulated K+ instead. Nitrate concentration was 100 to 150 mM higher in the upper than in the lower epidermis, whereas CI- was 50 to 120 mM higher in the lower epidermis. These differences did not depend on the leaf developmental stage. The uneven distribution of epidermal NO3- and CI- was maintainedover a wide range of epidermal sap concentrations of these ions and was not affected by NO3- or CI- starvation or by an increase in the light intensity from 120 to 400 [mu]mol m-2 s-1. However, the latter did cause a decrease in epidermal NO3- and the appearance and accumulation of epidermal malate, particularly in the upper epidermis. The physiological implications of the results for solute storage in leaves and for the pathways of ion distribution to the epidermis are discussed.

Journal Article↗

PubMed1993

Adherens junctions: demonstration in human epidermis.

Adherens junctions are intercellular and cell-matrix junctions that, like desmosomes and hemidesmosomes, mediate adhesion of cells to each other or to matrix structures. These junctions have been detected recently in cultured human keratinocytes, indicating that they may be of importance in epidermis. To investigate the localization of adherens junctions in normal epidermis, we examined human epidermis, human oral mucosa, and monkey esophagus for the presence of vinculin, a major protein of the intracellular plaques of adherens junctions that is thought to be present in all adherens junctions. Western blot analysis demonstrated vinculin in extracts of epidermis. Immunohistochemistry of vinculin in these tissues displayed two distinct locations for adherens junctions: i) at the dermal-epidermal junction, and ii) in the region of cell-cell contacts in all layers of the epidermis. The location of vinculin in the region of the epidermal-dermal junction is reminiscent of the distribution of vinculin-containing focal contacts in cultured keratinocytes, and the intercellular staining of vinculin in epidermis is consistent with the presence of vinculin in adherens junctions in cultured keratinocytes at sites of cell-cell contact. These results demonstrate that adherens junctions are present in human epidermis, oral mucosa, and monkey esophagus. Vinculin-containing junctions in epidermis may be important in the pathogenesis of skin diseases involving alterations in intercellular integrity.

Cell Adhesion↗

PubMed1996

Plasminogen activator inhibitor type-2 in the lesional epidermis of lupus erythematosus.

Under certain pathophysiological conditions epidermal keratinocytes produce urokinase-type plasminogen activator (uPA) or tissue-type PA (tPA). These PAs are subject to regulation by PA inhibitors (PAI), including PAI type-2 (PAI-2). In the normal epidermis, PAI-2 is present in the differentiating suprabasal layers, albeit in the apparent absence of PAs. It has, therefore, been suggested that PAI-2 plays a role in epidermal differentiation not linked to its ability to inhibit PAs. In line with this hypothesis, we have studied, by immunohistochemistry, the distribution of PAI-2, uPA and tPA in the normal and in the lesional epidermis of patients with lupus erythematosus (LE), a disease in which epidermal differentiation is disturbed. The PAI-2 antigen was detectable in the normal epidermis and in the lesional epidermis of LE. In the normal epidermis, the PAI-2 antigen was most pronounced in the granular layer. In the hyperkeratotic epidermal lesions of LE, the PAI-2 antigen was increased. In normal and lesional skin, PAI-2 was distributed along the cell periphery, indicating its association with the cornified envelope. Neither uPA nor tPA was detectable in normal or lesional epidermis. Our findings show that PAI-2 is a major type of PAI in normal epidermis and in the lesional epidermis of LE, and that increased epidermal PAI-2 is observed in a disease which is not associated with an increase in epidermal PAs. The data support the hypothesis that epidermal PAI-2 may have other functions than the regulation of PA activity.

Humans↗

PubMed2000

Immunocytochemical and electrophoretic distribution of cytokeratins in the regenerating epidermis of the lizard Podarcis muralis.

Using immunocytochemistry at light- and electron-microscope levels, we studied the distribution of three monoclonal antibodies (AE1, AE2, AE3) specific for mammalian alpha-keratins in regenerating lizard epidermis. We also characterized the keratins expressed during this process by immunoblotting after electrophoretic separation. The AE1 antibody is localized in the basal and suprabasal layers of prescaling and scaling epidermis. During the first stages of scale neogenesis, the AE1 antibody also marks the differentiating oberhautchen and beta-layer, but it disappears from these layers as they mature. This antibody does not stain the prekeratinized and keratinized outermost layers in the hinge region. The AE2 antibody labels the superficial wound epidermis, prekeratinizing and keratinized beta- and alpha-layers, but not basal and suprabasal cells. The AE3 antibody labels all living and keratinized epidermal layers, although AE3 immunoreactivity decreases and disappears as the beta-layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron-dense areas within the beta-layer, suggesting retention of alpha-keratins. In the stages of tail regeneration examined, immunoblotting with the three antibodies used for the immunolocalization gives a pattern similar to that of the normal epidermis, except distally, where the process of scale differentiation begins. In this region, in addition to the keratin forms discovered in the normal and in proximal regenerating epidermis, an intense low molecular weight band at 40-41 kDa, positive to all three antibodies, is clearly detectable. Furthermore, in the distal region AE1 and AE3 antibodies, but not the AE2, recognize a weak band at 77-78 kDa not present in the normal and proximal epidermis. The localization and the possible role of the different keratins in the regenerating epidermis is discussed.

Animals↗

PubMed1992

Avian scale development. XVII: The epidermis of the scaleless (sc/sc) anterior metatarsal skin is determined, but the dermis lacks permissive cues for the patterned expression of the determined state.

Embryos homozygous for the gene scaleless (sc/sc) completely lack scutate scales and the beta strata which characterize terminal differentiation of the scale ridges located on the anterior metatarsal region of the foot. Although the sc/sc epidermis cannot undergo scale morphogenesis, it can respond to the inductive dermal ridges of normal scutate scales by generating beta strata. Recently, we discovered that the anterior metatarsal epidermis of normal embryos becomes committed to the formation of beta strata prior to morphogenesis of definitive scale ridges. Here, we examined the possibility that the sc/sc anterior metatarsal epidermis also becomes determined, i.e., committed to scutate scale-specific terminal differentiation. Experimental tissue recombinants were used to assess the ability of the sc/sc epidermis to generate beta strata. The results show that the germinative cells of the 15-day sc/sc epidermis are committed to generating beta strata, even though they have not undergone scutate scale morphogenesis. Thus, the mechanisms involved in establishing epidermal determination must differ form those regulating scale morphogenesis. In addition, we examined the formation of patterned, permissive cues in the anterior metatarsal and footpad dermises of sc/sc embryos. Analysis of recombinants showed that both the 15- and 20-day dermises from the sc/sc anterior metatarsal region fail to provide cues for beta stratum formation, when associated with the determined 15-day scutate scale epidermis. Likewise, the 15-day sc/sc footpad dermis cannot support beta stratum formation. However, 20-day sc/sc footpad dermis is able to support the generation of a few abnormally patterned beta strata, demonstrating that sc/sc dermis which has experienced even limited morphogenesis is able to provide permissive cues for the terminal differentiation of the scutate scale epidermis.

Animals↗

PubMed1992

Sheets of embryonic epidermis can be isolated and cultured with or without the basal lamina.

In the current study, we determine the concentration of Dispase II, a neutral protease, and the incubation time necessary for isolation of embryonic avian epidermis. The epidermis can be isolated from the dermis consistently with or without the basal lamina. Because the action of Dispase II is selective and easily controlled, this study suggests that Dispase can serve as a powerful tool for studying epidermal-extracellular matrix interactions. In further experiments we study the response of sheets of epidermis isolated with and without its basal lamina to extracellular matrix. When the epidermis is isolated without the basal lamina, the basal surface blebs and the actin in the basal cytoplasm are disrupted. Epidermis isolated without the basal lamina and cultured on extracellular matrix reorganizes the basal actin mat and retracts the blebs. However, epidermis isolated without the basal lamina and cultured in the absence of extracellular matrix continues to have blebs and a disorganized actin cortical mat. In contrast, the control epidermis isolated with the basal lamina retains a flat basal surface and organized actin cortical mat after culturing either with or without extracellular matrix. The extracellular matrix has been shown by many investigators to play an important role in cytoskeletal organization, metabolism, and differentiation. How the extracellular matrix interacts and influences these functions has not been completely elucidated. The ability to isolate tissue consistently with and without its basal lamina using Dispase II will facilitate addressing these and other epithelial cell biology questions.

Animals↗

PubMed1997

A set of novel tadpole specific genes expressed only in the epidermis are down-regulated by thyroid hormone during Xenopus laevis metamorphosis.

Four genes were identified in a screen for thyroid hormone-induced down-regulation of gene expression in Xenopus laevis tadpole tails. All four encode extracellular glycoproteins that are expressed exclusively in the apical cell layer of the entire tadpole epidermis, which is the equivalent of the mammalian fetal periderm. The onset of the four novel genes' expression late in embryogenesis, their activity throughout the life of the tadpole, their repression by exogenously added thyroid hormone, and the spontaneous cessation of their expression at the end of tadpole life are closely coordinated. These facts suggest that the protein products of these genes form a novel albeit temporary barrier or other structure in the tadpole epidermis that functions in lieu of the cornified, stratified epithelium of the adult epidermis. We have exploited the cloning of these genes for use as cell-specific markers to follow the appearance and loss of apical cells during development. We were able to demonstrate directly that the apical cells are derived from a stratification of the embryonic ectoderm at the onset of the formation of a true epidermis. The apical cells uniformly cover the surface of the tadpole until metamorphosis, when the expression of the four larval epidermis-specific genes is lost coordinately over the entire tadpole. In contrast, the adult epidermis develops with a distinct regional specificity: adult keratin is first expressed up to a line separating the body and tail epidermis and finally appears in the tail only at metamorphic climax. Finally, our analysis reveals that the TH-induced down-regulated gene expression program during metamorphosis is very different from the previously described up-regulated program which involves multiple cell types and several waves of gene expression changes. The down-regulated program only consists of the repression of a small number of genes which are expressed in larval cells preprogrammed to die during the larval to adult transition at metamorphosis.

Amino Acid Sequence↗

PubMed1997

Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta.

To determine which ecdysteroids may be biologically active in the larval epidermis of the tobacco hornworm, Manduca sexta, we studied the action of several known ecdysteroids and metabolites on the expression of the genes encoding the ecdysone receptor (EcR), Manduca hormone receptor 3 (MHR3), dopa decarboxylase (DDC), and a larval cuticle protein (LCP-14). Both Day 2 fourth- and Day 2 fifth-instar larval epidermis contained significant 3 beta-reductase activity which metabolized 3-dehydroecdysone (3DE) and 3-dehydro-20-hydroxyecdysone (3D20E) to ecdysone (E) and 20-hydroxyecdysone (20E), respectively, but had only very low amounts of ecdysone oxidase activity (E to 3DE) and no detectable ecdysone 20-monooxygenase activity (E to 20E). When the expression of the various genes was studied in the epidermis in vitro, 20E and 3D20E had similar effects, whereas E, 3DE, 26-hydroxyecdysone and 20,26-dihydroxyecdysone were ineffective. Exposure of Day 2 fifth-instar epidermis to 500 ng/ml of either 20E or 3D20E for 24 hr caused a rapid, biphasic increase in EcR-B1 mRNA. By contrast, EcR-A mRNA showed a less rapid initial increase followed by a slow steady rise and was less responsive to 3D20E. Ecdysone in a 1:1 mixture with 20E effectively halved the concentration of 20E needed to induce EcR-B1 mRNA but showed no synergism in the induction of EcR-A mRNA. The induction of MHR3 mRNA and of DDC mRNA in Day 2 fourth-instar epidermis as well as the suppression of DDC and LCP-14 gene expression by 3D20E was indistinguishable from that of 20E. Therefore, for Manduca larval epidermis, only 20E and 3D20E are biologically active ecdysteroids. Since the 3D20E can be converted to 20E by the epidermis, its effects are likely mediated by 20E.

Animals↗