Cloning and characterization of retinol dehydrogenase transcripts expressed in human epidermal keratinocytes.

The normal growth and differentiation of the epidermis require an adequate supply of vitamin A. The active form of vitamin A for normal epidermal homeostasis is retinoic acid (RA). Retinoic acid controls the expression of retinoid-responsive genes via interactions of the retinoic acid/nuclear receptor complexes at specific DNA sequences in their control regions. The message conveyed by RA is likely modulated by the concentration of the ligand available for binding to the receptors. Following the uptake of plasma retinol, epidermal keratinocytes synthesize retinoic acid via two sequential reactions with retinaldehyde as an intermediate. Several retinol dehydrogenase (RDH) enzymes, members of the short-chain dehydrogenase/reductase (SDR) gene superfamily, catalyze the first and rate-limiting step that generates retinaldehyde from retinol bound to cellular retinol-binding protein (holo-CRBP). However, little is known about these enzymes and their genes in the epidermal cells. Our work describes the first member of the RDH family found in epidermis. We show that this gene is expressed predominantly in the differentiating spinous layers and that it is under positive, feed-forward regulation by retinoic acid. It encodes a protein that, using NAD+ as a preferred cofactor, utilizes free and CRBP-bound all-trans-retinol and steroids as substrates.     

Vitamin D3 induces caspase-14 expression in psoriatic lesions and enhances caspase-14 processing in organotypic skin cultures

Caspase-14 is a nonapoptotic caspase family member whose expression in the epidermis is confined to the suprabasal layers, which consist of differentiating keratinocytes. Proteolytic activation of this caspase is observed in the later stages of epidermal differentiation. In psoriatic skin, a dramatic decrease in caspase-14 expression in the parakeratotic plugs was observed. Topical treatment of psoriatic lesions with a vitamin D3 analogue resulted in a decrease of the psoriatic phenotype and an increase in caspase-14 expression in the parakeratotic plugs. To investigate whether vitamin D3 directly affects caspase-14 expression levels, we used keratinocyte cell cultures. 1alpha,25-Dihydroxycholecalciferol, the biologically active form of vitamin D3, increased caspase-14 expression, whereas retinoic acid inhibited it. Moreover, retinoic acid repressed the vitamin D3-induced caspase-14 expression level. In addition, the use of organotypic skin cultures demonstrated that 1alpha,25-dihydroxycholecalciferol enhanced epidermal differentiation and caspase-14 activation, whereas retinoic acid completely blocked caspase-14 processing. Our data indicate that caspase-14 plays an important role in terminal epidermal differentiation, and its absence may contribute to the psoriatic phenotype.     

Thymic hormones in human fetal skin epidermis

Thymic hormone thymalin is detected in young epidermal cells of human fetuses. Its content varies with gestation age. Maturation of keratinocytes in the epidermis is paralleled by a decrease in the population of young thymalin-positive cells. By birth they are located on the basal membrane and in some adjacent layers. This regularity was seen in different parts of the body.     

Expression of human epithelial characteristics in late passages of interspecific somatic hybrids derived from normal and hand wart keratinocytes

The expression of epithelial features has been investigated from the 9th to the 30th passage in cells derived from the fusion of mouse 3T3.4E cells with normal or hand wart human keratinocytes. These cells stratified and grew either on plastic or collagen substrate without modifications. Such growth characteristics are similar to those observed in the epidermis. The doubling time of hybrid cells is nearer to that of 3T3.4E than to that of keratinocytes. Some epithelial markers were detected by immunofluorescence staining. Bullous pemphigoid antigen characteristic of the basal keratinocyte was detected, and cytoplasmic antigen of basal cells was positive with BL7 monoclonal antibody. Among the antigens of the suprabasal cell layers of the epidermis, the pemphigus antigen was present in all hybrids; epidermal keratins recognized by the monoclonal antibodies KL1 and KL2 were detected in wart hybrids up to the 9th passage. When hybrids were cultured in delipidized serum or in methylcellulose, few cells (less than 10%) reacted with KL2. Karyological analysis revealed both murine acrocentric and human submetacentric chromosomes. Thus, hybrid cells obtained from normal and wart keratinocytes were new cell types with a phenotype between keratinocytes and 3T3.4E cells.     

Alterations in the expression of two epidermal differentiation antigens in human epidermal disorders

The expression of an epidermal keratin subunit and a specific antigen of the keratinocyte membrane, two differentiation antigens in normal human epidermis, was studied in benign and malignant epidermal lesions by use of monoclonal antibodies KL1 (anti 55-57 Kd keratins) and KL3 (anti keratinocyte membrane antigen). In normal human epidermis, KL1 labelled all keratinocytes from the suprabasal layers, KL3 stained the intercellular spaces in all epidermal layers with a fluorescence intensity increasing from the basal to the more upper layers and recognized a keratinocyte membrane antigen as demonstrated in electron microscopy. Frozen or deparaffinized sections of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) malignant melanomas, warts, and skin biopsies from benign lesions (psoriasis, lichen planus, bullous pemphigoid, lupus erythematodes, pemphigus, vasculitis) were tested with either KL1 or KL3 by indirect immunofluorescence and/or immunoperoxidase. Benign and malignant lesions in which modifications of the keratinization process and cell differentiation are known to occur (BCC, SCC, warts, psoriasis) showed the most severe alterations as compared to normal epidermis. With KL1 we observed an irregular staining of basal cells; a reorganization of keratin filaments and variable staining intensities within tumoral cells which did not express high MW keratins. With KL3 drastic alterations in the epidermal intercellular patterns and loss of reactivity of tumoral cells were noted. Conversely, the positivity of epidermal basal cells with KL1, in some cases, was the only modification noted in other skin lesions.     

Localization of epidermal hyaluronan in the foot pads of the North American raccoon (Procyon lotor)

The localization of the epidermal hyaluronan in the digital foot pads of the North American raccoon (Procyon lotor) was examined by light and electron microscopic histochemical methods. The thick epidermis contained five epidermal layers composed of typical keratinocytes. In the stratum basale and stratum spinosum, hyaluronic acid was clearly detectable at cytoplasmic processes, in close vicinity to the surface coat of the plasma membrane, and/or in the intercellular space between the basal and spinous cells. The results suggest that epidermal hyaluronan that fills the intercellular space may particularly control water maintenance in the epidermis of the raccoon digital pads to guarantee the optimal mechanoreceptive function of the very specific and sensitive sensoric subepidermal equipment in this mammalian species.     

The significance of epidermal changes in the early diagnosis and development of mycosis fungoides

In 47 patients with stage I mycosis fungoides, biopsy of skin has revealed substantial changes in epidermis acanthosis with confluent epidermal processes, focal dystrophy of basal cells, mitoses in various epidermal layers, parakeratotic foci without a granular layer. Histoautoradiographic and immunomorphological studies demonstrated that enhanced proliferative activity of keratinocytes that was more pronounced in stages I and II mycosis fungoides and impaired epidermal differentiation that was predominant in neoplastic stage III underlay the above changes. There was ultrastructural evidence for abnormal epidermal keratinization. It was proposed that epidermal abnormalities made an important contribution to the development of mycosis fungoides.     

Notch Signaling May Be Involved in the Abnormal Differentiation of Epidermal Keratinocytes in Psoriasis

Localization of each keratin isoform differs among epidermal layers. Proliferating basal cells synthesize keratin 14 (K14) and suprabasal cells express keratin 10 (K10) in normal skin. Notch signaling is essential for keratinocyte differentiation. Notch1 is expressed in all epidermal layers, Notch2 in the basal cell layer and Notch3 in basal cell and spinous cell layers in normal epidermis. It has been poorly elucidated how localization and expression levels of Notch molecules are related to epidermal molecular markers K10 and K14 in psoriatic skin with abnormal differentiation of epidermal tissue. This study aimed to investigate the relationship between abnormal differentiation of epidermal cells in psoriatic skin and expression of Notch molecules. We investigated keratins (K14 and K10) and Notches (1, 2, 3 and 4) using immunohistochemistry in psoriatic skin (n=30) and normal skin (n=10). In normal skin, K14 and K10 were discretely observed in the basal cell layer and suprabasal layer, respectively. In psoriatic skin, K14 was expressed in the pan epidermal layer while it and K10 were co-expressed in some middle suprabasal layer cells. Notch1, 2, 3, and 4 localized in all epidermal layers in normal skin. In psoriatic skin, Notch1, 2, and 4 mainly localized in suprabasilar layers and Notch3 is lacalized in pan epidermal, suprabasilar, and basilar layers. Protein and mRNA of Notch1, 2, and 3 isoforms decreased in psoriatic epidermis compared with normal epidermis. These data suggest that decrements in these Notch molecules might cause aberrant expression of K10 and K14 leading to anomalous differentiation of the epidermis in psoriatic lesions.     

Effects of all-trans retinoic acid and Ca++ on human skin in organ culture.

In this study, we have established an organ culture model of human skin and examined the effects of both all-trans retinoic acid (RA) and extracellular Ca++ on the epidermal and dermal components of the organ-cultured skin. Our data show that while organ cultures maintained in serum-free, growth factor-free culture medium containing 0.15 mM Ca++ degenerated rapidly, those treated with concentrations of RA that have been shown previously to stimulate fibroblast and keratinocyte proliferation in monolayer culture (J Invest Dermatol 1989, 93:449; 1990, 94:717; Am J Pathol 1990, 136:1275) demonstrated a healthy appearance for up to 12 days. Degeneration of the control cultures was characterized by separation of the epidermis from the underlying dermis, progressive cell necrosis leading to a complete absence of viable cells from both the dermal and epidermal compartments, disintegration and fibrillation of the dermal connective tissue, and a cessation of protein synthesis. RA-treated organ cultures contained large numbers of healthy-appearing cells in both the epidermal and dermal compartments. One or several layers of viable basal cells in the epidermis could be seen at least through day 12. However, the upper layers of the epidermis frequently separated from the cells in the basal layer. The dermal connective tissue was histologically well-preserved. Furthermore, the level of protein synthesis was higher in the RA-treated cultures than in the control cultures. In addition to treating organ cultures with RA, other cultures were exposed to serum-free, growth factor-free culture medium containing 1.4 mM Ca++. The presence of the elevated Ca++ concentration also preserved cellular and connective tissue structures in the dermal and epidermal compartments. In comparison to RA there was better preservation of the overall epidermal structure. The upper layers of epidermal cells did not separate from the basal cells, and the various stages of epithelial differentiation could be seen. Histologically, the dermis was well-preserved in the presence of elevated extracellular Ca++. Specimens treated with a combination of Ca++ and RA demonstrated features consistent with the features induced by each treatment separately. This included an expanded basal layer of epithelial cells and a prominent keratotic layer with a fairly orderly pattern of differentiation. The tendency of the upper epidermis to separate from the basal cells was partially mitigated. Taken together, these data indicate that both RA and extracellular Ca++ act to prevent the degeneration of human skin in organ culture but probably do so through different mechanisms.     

Effects of retinoic acid and Gbx1 on feather‐bud formation and epidermal transdifferentiation in chick embryonic cultured dorsal skin

Background: Retinoic acid, an active metabolite of retinol, is known to regulate cell proliferation, differentiation, and morphogenesis during normal development of many tissues. Using chick embryonic tarsometatarsal skin, we showed previously that the expression of Gbx1, a divergent homeobox gene, is increased in the epidermis through interaction with retinol‐pretreated dermal fibroblasts followed by epidermal transdifferentiation to mucous epithelium. This present study was performed to elucidate the effects of retinoic acid and Gbx1 on feather‐bud formation and epidermal transdifferentiation. Results: We showed that Gbx1 was expressed in the chick embryonic dorsal epidermis as early as at placode stage (Hamburger and Hamilton stage 31) and increased in amount during feather‐bud formation. Treatment with 1 μM retinoic acid for 24 hr inhibited feather‐bud formation and induced the transdifferentiation of the epidermis to a mucosal epithelium with a concomitant increase in Gbx1 mRNA expression in the epithelium. Furthermore, transient transfection of the epidermis with Gbx1 cDNA by electroporation induced elongation of the feather bud, but did not result in transdifferentiation. Conclusions: These results indicate that Gbx1 was involved in the feather‐bud formation and was one of target genes of retinoic acid and that other signals in addition to Gbx1 were required for epidermal mucous transdifferentiation. Developmental Dynamics 241:1405–1412, 2012. © 2012 Wiley Periodicals, Inc.     

Patterns of mitosis in hamster epidermis

Alignment of the flattened keratinizing cells of the upper strata of mammalian epidermis leads to the formation of columnar units of structure. In mouse epidermis, mitoses have been found to occur relatively infrequently in the region beneath the center of each cell column where a non-keratinocyte dendritic cell, usually with features typical of an epidermal Langerhans cell, is situated. The observed pattern of mitosis could therefore be due either to displacement of central keratinocytes by Langerhans cells or indicate some control of keratinocyte proliferation related either to the Langerhans cells or to the overlying cell columns. No relationship exists between the position of Langerhans cells and epidermal cell columns in hamster epidermis but measurement of the position of mitosis has shown a reduced frequency of occurrence of mitosis beneath the central region. This pattern of mitosis is therefore unrelated to Langerhans cells and appears to reflect differences in the mitotic potential of basal keratinocytes which could be associated with feedback from the overlying cell columns or with an intrinsic pattern of basal cell activity.     

Efficient production of bioactive insulin from human epidermal keratinocytes and tissue-engineered skin substitutes: implications for treatment of diabetes

Despite many years of research, daily insulin injections remain the gold standard for diabetes treatment. Gene therapy may provide an alternative strategy by imparting the ability to secrete insulin from an ectopic site. The epidermis is a self-renewing tissue that is easily accessible and can provide large numbers of autologous cells to generate insulin-secreting skin substitutes. Here we used a recombinant retrovirus to modify human epidermal keratinocytes with a gene encoding for human proinsulin containing the furin recognition sequences at the A-C and B-C junctions. Keratinocytes were able to process proinsulin and secrete active insulin that promoted glucose uptake. Primary epidermal cells produced higher amounts of insulin than cell lines, suggesting that insulin secretion may depend on the physiological state of the producer cells. Modified cells maintained the ability to stratify into 3-dimensional skin equivalents that expressed insulin at the basal and suprabasal layers. Modifications at the furin recognition sites did not improve proinsulin processing, but a single amino acid substitution in the proinsulin B chain enhanced C-peptide secretion from cultured cells and bioengineered skin substitutes 10- and 28-fold, respectively. These results suggest that gene-modified bioengineered skin may provide an alternative means of insulin delivery for treatment of diabetes.     

Production and characterization of monoclonal antibodies reactive with subsets of epidermal cells

Monoclonal antibodies were raised against an extract of human callus to provide markers of cell differentiation. Three antibodies, LMM1, LMM2 and LMM3, have been isolated and react with different histological structures in skin. LMM1 reacts with two distinct proteins of 62-65 kD molecular weight and stains cells in the stratum spinosum and stratum granulosum of normal epidermis. This antibody stains a high proportion of cultured epidermal cells showing a diffuse cytoplasmic reactivity with nucleolar staining also being present in some cells. LMM2 reacts with several lower molecular weight proteins, the two major bands being 38 kD and 44 kD. It stains basal epidermal cells strongly and stratum spinosum cells less strongly. A population of cultured keratinocytes reacts with an intense fibre stain suggesting that this antibody is directed against an intermediate filament or intermediate filament-associated protein. LMM3 reacts with a 48 kD protein. Although it does not stain cells in normal epidermis its staining of a subpopulation of hair follicle cells is consistent with this protein being a cytokeratin. It also stains a population of cultured keratinocytes, the main reactivity being against mitotic cells. The anti-cytokeratin or anti-cytokeratin-associated reactivity of these three antibodies is supported by their altered reactivities against psoriasis epidermis, where modifications of cytokeratin biosynthesis are known to occur.     

Mast cells and vasculature in atopic dermatitis--potential stimulus of neoangiogenesis

BACKGROUND: Atopic dermatitis skin lesions are characterized by inflammatory changes and epithelial hyperplasia requiring angiogenesis. As mast cells may participate in this process via bidirectional secretion of tissue-damaging enzymes and pro-angiogenic factors, the present study aimed to assess the occurrence and possible function of mast cells in the papillary dermis and in epidermal layers of atopic dermatitis lesions. METHODS: Semi-thin and serial sections in combination with immunohistochemistry, histochemistry and proliferating cell nuclear antigen (PCNA)-activity assays were used and related to epidermal thickness and targeted gene expression studies. RESULTS: Mast cells were located in the papillary dermis and migrated through the basal lamina into the epidermis of atopic dermatitis lesions. An increased PCNA-activity in cells of superficial epidermal layers indicated an activation of keratinocytes and stimulation of endothelial growth. Only approximately 30% of the papillary mast cells stained with the tryptase were toluidin-blue-positive, and approximately 80% were chymase positive. A high number of mast cells expressed c-kit. Most papillary and epidermal mast cells were localized close to endothelial cells. Vascular expression of endoglin (CD105) demonstrated neoangiogenic processes. Mast cells stimulation led to the expression of proangiogenic factors. Also, gene expression of tissue-damaging factors such as matrix metalloproteinases was increased. CONCLUSIONS: These data suggest that in atopic dermatitis, mast cells are abundantly localized close to and within the epidermis where they may stimulate neoangiogenesis. Via the new vessels, inflammatory cells, together with complement components and antibodies, can be transported to the epidermis to aid in the defense against environmental antigens and to maintain chronic inflammation.     

Generation of human epidermal constructs on a collagen layer alone

Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.     

The skin as an immunologic organ

The epidermis is a heterogeneous tissue comprised of cells that may play a role in various types of immunologic responses. This review focuses on the importance of epidermal Langerhans' cells in antigen presenting and accessory cell functions, on the role of la-positive keratinocytes in generating certain types of immune responses, on the role of normal keratinocytes in the liberation of cytokines, on the identification of murine Thy-1-bearing dendritic epidermal cells, and on the modulation of epidermal immune functions by various physical and chemical agents.     

Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion

In adults, bone marrow-derived cells (BMDC) can contribute to the structure of various non-haematopoietic tissues, including skin. However, the physiological importance of these cells is unclear. This study establishes that bone marrow-derived epidermal cells are proliferative and, moreover, demonstrates for the first time that BMDC can localize to a known stem cell niche: the CD34-positive bulge region of mouse hair follicles. In addition, engraftment of bone marrow cells into the epidermis is significantly increased in wounded skin, bone marrow-derived keratinocytes can form colonies in the regenerating epidermis in vivo, and the colony-forming capacity of these cells can be recapitulated in vitro. In some tissues this apparent plasticity is attributed to differentiation, and in others to cell fusion. Evidence is also provided that bone marrow cells form epidermal keratinocytes without undergoing cell fusion. These data suggest a functional role for bone marrow cells in epidermal regeneration, entering known epidermal stem cell niches without heterokaryon formation.     

Retinoic acid imprints gut-homing specificity on T cells

For a preferential homing of T cells to the gut, expression of the integrin alpha4beta7 and the chemokine receptor CCR9 is essential and is induced by antigenic stimulation with dendritic cells from the gut-associated lymphoid organs. Here, we show that the vitamin A (retinol) metabolite, retinoic acid, enhances the expression of alpha4beta7 and CCR9 on T cells upon activation and imprints them with the gut tropism. Dendritic cells from the gut-associated lymphoid organs produced retinoic acid from retinol. The enhanced alpha4beta7 expression on T cells by antigenic stimulation with these dendritic cells was suppressed by the retinal dehydrogenase inhibitor citral and the retinoic acid receptor antagonist LE135. Accordingly, vitamin A deficiency caused a reduction in alpha4beta7(+) memory/activated T cells in lymphoid organs and a depletion of T cells from the intestinal lamina propria. These findings revealed a novel role for retinoic acid in the imprinting of gut-homing specificity on T cells.