Interactions between fibroblasts and keratinocytes in morphogenesis of dermal epidermal junction in a model of reconstructed skin

De novo dermal epidermal junction morphogenesis was studied in a skin model including dermal fibroblasts and epidermal keratinocytes. Sequential gene expression, protein deposition, and localization of basement membrane zone components were studied during 15 days. The morphogenesis of dermal epidermal junction is characterized by an implementation of the different components and then a subsequent plateau phase occurring at day 11. Three groups of genes were identified depending on cellular origin and expression profile: 1/genes of fibroblastic origin (col I alpha1, col III alpha1, nidogen, and fibrillin 1); 2/genes expressed in fibroblasts and keratinocytes with symmetrical expression pattern between both cell types (col IV alpha1, col VII alpha1, and tenascin C); 3/laminin beta3 only expressed in keratinocytes. Use of modified organotypic models excluding one cell type revealed a tight interplay between fibroblasts and keratinocytes for synthesis and localization of the components of dermal epidermal junction. Keratinocytes downregulated mRNA and proteins of fibroblastic origin, upregulated col VII in fibroblasts and were absolutely required for dermal-epidermal junction localization of fibroblastic proteins. Fibroblasts downregulated mRNA of keratinocytes and were needed for extracellular secretion and correct localization of type VII collagen and laminin 5.     

P-glycoprotein (ABCB1) expression in human skin is mainly restricted to dermal components

Several transport proteins are constitutively expressed in skin cells, but the putative role of the ABC transporter P-glycoprotein (P-gp) in human skin is yet unknown. Therefore, we analysed mRNA and protein expression and localization of P-gp in human skin. Using qRT-PCR, we demonstrated a strong MDR1 mRNA expression in whole skin specimens and dermis, whereas the expression of MDR1 in epidermis, epidermal keratinocytes or dermal fibroblasts was only weak. Immunohistochemistry confirmed mRNA data and revealed a marked expression of P-gp within sweat ducts, vessels, nerve sheaths and muscles of human skin and a moderate expression in basal epidermis. Our findings closely correlate with previous studies in murine skin supporting the role of P-gp in the uptake of compounds from the epidermal compartment and their secretion into the bloodstream and sweat ducts. It may also prevent the uptake of xenobiotics into the skin by functioning as a barrier located in the dermal vasculature.     

Tenascin-C is upregulated in the skin lesions of patients with atopic dermatitis

BACKGROUND: Tenascin-C is a large, hexameric extracellular matrix glycoprotein that is expressed during embryogenesis, carcinogenesis and wound healing. In normal adult human skin the expression level of tenascin-C is low, but levels are elevated in skin tumors and rise significantly in the dermal compartment during wound healing. Although the expression of tenascin-C could be upregulated by inflammatory cytokines, the role of tenascin-C in atopic dermatitis (AD) is still unclear. OBJECTIVE: To identify genes that plays a role in AD. METHODS: We screened for differentially expressed genes in lesional and non-lesional skin of AD patients using DNA microarray. Then we monitored with quantitative PCR the expression of the novel disease related genes in human keratinocytes or pinnae from NC/Nga mice. RESULTS: We found that tenascin-C gene expression was expressed at higher levels in lesional skin compared to non-lesional skin of the patients, whereas it was not upregulated in the skin of psoriatic patients or healthy controls. In human cultured keratinocytes, tenascin-C was markedly upregulated by IL-4 and IL-13, and moderately upregulated by IFN-gamma. Tenascin-C expression was also upregulated in the AD-like skin lesions induced in NC/Nga mice ears by intradermal injection of mite antigen, and this upregulation was inhibited by prednisolone. CONCLUSION: These results suggest that upregulation of the tenascin-C expression is specific to AD lesions, and that tenascin-C may therefore play a critical role in regulating the underlining inflammatory processes, which are involved in the pathology of AD.     

Spatial expressions of fibronectin and integrins by human and rodent dermal fibroblasts

BACKGROUND: Human skin shows various morphological characteristics, depending on the body site. As these distinct phenotypes have been explained on the basis of the variance in epidermal keratinocytes and the presence of skin appendages, the spatial distinction of the dermal components has not been fully elucidated. OBJECTIVES: To identify and characterize the profiles of mRNAs that are abundantly or specifically expressed by fibroblasts derived from trunk skin, but not from palmoplantar skin or oral mucosa. METHODS: In order to identify the distinct mRNA expression by trunk skin fibroblasts, a subtraction cDNA screening was performed first, followed by Northern blotting, Western blotting and immunohistochemistry for cultured human and rat dermal fibroblasts and those skin tissues. Finally, whole mount in situ hybridization (WISH) was performed to examine the differences in the expression of the corresponding gene during the developmental stage of mouse embryos. RESULTS: We identified three cDNA clones encoding fibronectin (FN), pregnancy-specific beta1-glycoprotein 5 and beta-actin, respectively, whose mRNAs were abundantly or specifically expressed by trunk skin fibroblasts. FN and some integrins were further confirmed to be expressed more selectively in human and rat trunk skin fibroblasts, both in terms of the RNA and the protein levels, compared with the fibroblasts derived from plamoplantar skin and oral mucosa. WISH demonstrated that FN was localized around the hair follicles of mouse embryos. CONCLUSIONS: FN, one of most potent extracellular matrix molecules, was demonstrated to be spatially transcribed depending on the body sites. The distinct expression of FN was suggestive of the essential commitment in the process of cutaneous development and morphogenesis of appendages originated from hair germ. The paucity of FN in palmoplantar skin and oral mucosa might explain the characteristics of these skin phenotypes.     

UV-induced DNA damage initiates release of MMP-1 in human skin

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.     

Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes

Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.     

UV radiation induces CXCL5 expression in human skin

CXCL5 has recently been identified as a mediator of UVB‐induced pain in rodents. To compare and to extend previous knowledge of cutaneous CXCL5 regulation, we performed a comprehensive study on the effects of UV radiation on CXCL5 regulation in human skin. Our results show a dose‐dependent increase in CXCL5 protein in human skin after UV radiation. CXCL5 can be released by different cell types in the skin. We presumed that, in addition to immune cells, non‐immune skin cells also contribute to UV‐induced increase in CXCL5 protein. Analysis of monocultured dermal fibroblasts and keratinocytes revealed that only fibroblasts but not keratinocytes displayed up regulated CXCL5 levels after UV stimulation. Whereas UV treatment of human skin equivalents, induced epidermal CXCL5 mRNA and protein expression. Up regulation of epidermal CXCL5 was independent of keratinocyte differentiation and keratinocyte‐keratinocyte interactions in epidermal layers. Our findings provide first evidence on the release of CXCL5 in UV‐radiated human skin and the essential role of fibroblast‐keratinocyte interaction in the regulation of epidermal CXCL5.     

Regulation of keratin 9 in nonpalmoplantar keratinocytes by palmoplantar fibroblasts through epithelial-mesenchymal interactions

Palms and soles differ from other body sites in terms of clinical and histologic appearance, response to mechanical stress, and the distribution of keratin 9. Because keratin 9 is exclusively expressed in the palmoplantar suprabasal keratinocyte layers, it is considered a differentiation marker of palms and soles. We studied palmoplantar mesenchymal influences on keratin 9 induction in nonpalmoplantar epidermis. Although palmoplantar keratinocytes when cultured alone continued to express keratin 9 mRNA in 12 (100%) of 12 cultures, nonpalmoplantar keratinocytes did not express it in 16 of 17 cultures. Although nonpalmoplantar keratinocytes did not express keratin 9 mRNA when cultured with nonpalmoplantar fibroblasts, they did express it within 2 h in cocultures with palmoplantar fibroblasts derived from papillary dermis. Grafting of these coculture sheets on severe combined immunodeficient mice resulted in an epidermis, which histologically showed hyperkeratosis and acanthosis and immunohistochemically expressed keratin 9. Furthermore, pure epidermal sheets from nonpalmoplantar skin grafted on the human sole wounds due to burn, injury, and the resection of acral lentiginous melanoma, demonstrated adoption of palmoplantar phenotype and expressed keratin 9. Our report indicates extrinsic keratin 9 regulation by signals from dermal fibroblasts. This is also the first to suggest the possibility of treating palmoplantar wounds with nonpalmoplantar epidermis, which is much easier to obtain and harvest.     

Emerging role of dermal compartment in skin pigmentation: comprehensive review

The variations in human skin colour mainly occur due to differences in the distribution of melanin pigment throughout the body, synthesized by epidermal melanocytes which are further taken up by keratinocytes present in epidermis. Recently, it has been discovered that besides these cells, dermis derived fibroblast factors also play a prominent role in regulating skin pigmentation. There exists a signal crosstalk between epidermal melanocytes, keratinocytes and dermal fibroblasts and any impairment in these signalling pathways may give rise to pigmentary disorders. Vitiligo is a hypopigmentary disorder and alteration in the expression level of several fibroblast-specific factors has been reported in the lesional skin of vitiligo patients. In such patients, there is decrease in the expression levels of factors such as basic fibroblast growth factor, stem cell factor (SCF) and keratinocyte growth factor (KGF) along with a steep increase in the expression levels of Dickkopf 1. Patients affected with hyperpigmentary disorder like melasma exhibit a marked increase in SCF and KGF expression levels leading to increase in melanin production and those affected with solar lentigo experience upregulation in the expression levels of SCF, KGF and HGF (hepatocyte growth factor). Hence, we conclude that new therapeutic strategies can be adopted to cure these pigmentary disorders by targeting factors involved in crosstalk signalling between epidermal melanocytes, keratinocytes and dermal fibroblasts.     

Production of fibronectin by epithelium in a skin equivalent

Although human keratinocytes in vitro have been shown to produce fibronectin, whether keratinocytes can contribute fibronectin to the dermal-epidermal junction or wound matrix is unknown. In order to approach this problem experimentally, we used the "skin equivalent" model composed of a native collagen gel populated with cultured fibroblasts and covered by cultured keratinocytes. By using bovine fibroblasts to populate the gel, fetal bovine serum in the culture medium, and human keratinocytes to form the epithelium, we were able to be certain that any human fibronectin produced in the culture was synthesized by the keratinocytes. A monoclonal antibody to fibronectin was found to recognize human but not bovine fibronectin. When the skin equivalent was stained by indirect immunofluorescence with antifibronectin, fibronectin was visible as an intensely staining band at the dermal-epidermal junction. In sections in which the dermis and epidermis had separated, the staining was usually limited to the dermal aspect of the skin equivalent. The results indicate that epithelium can contribute fibronectin to the dermal-epidermal junction and suggest that dermal staining in skin sections may originate from the epidermis. Since the developing skin equivalent has a rapidly growing epithelium and simulates a healing wound, contribution of fibronectin by the epithelium, in addition to that possibly contributed by serum and fibroblasts, may be of importance in wound healing.     

Dermcidin is constitutively produced by eccrine sweat glands and is not induced in epidermal cells under inflammatory skin conditions

BACKGROUND: Antimicrobial peptides (AMPs) are important effector molecules of innate immunity, protecting epithelial surfaces of multicellular organisms. In human skin two classes of AMPs-the beta-defensins and the cathelicidins-are produced by keratinocytes primarily under inflammatory conditions. In contrast, dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is expressed in eccrine sweat glands and transported via sweat to the epidermal surface. OBJECTIVES: To investigate whether DCD expression is induced under inflammatory conditions in epidermal keratinocytes. METHODS: Lesional skin of the inflammatory skin diseases atopic dermatitis, psoriasis and lichen planus was analysed by immunohistochemistry using a polyclonal anti-DCD antiserum. We also examined whether DCD RNA expression is induced in cultured human keratinocytes, fibroblasts, melanocytes and melanoma cells. RESULTS: Whereas DCD was constitutively expressed in eccrine sweat glands of all skin biopsies, we found that, independent of the type of the inflammatory skin lesion, DCD protein expression was not induced in human epidermal keratinocytes. In contrast, beta-defensin 2 was expressed in epidermal keratinocytes of inflammatory human skin, but not in keratinocytes of healthy human skin. Upon stimulation of the cultured cells with 12-O-tetradecanoyl-phorbol-13-acetate, tumour necrosis factor-alpha, lipopolysaccharide or H2O2, DCD mRNA expression was not detected in primary keratinocytes, fibroblasts and melanocytes, but was detected in MeWo and SKMEL28 melanoma cells. CONCLUSIONS: These results indicate that, unlike human cathelicidins and beta-defensins which are inducible peptides that primarily function in response to injury and inflammation, DCD is exclusively part of the constitutive innate defence of human skin. By modulating surface colonization, DCD may help to prevent local and systemic invasion of pathogens.     

Microarray analysis of gene expression in cultured skin substitutes compared with native human skin

Cultured skin substitutes (CSS), prepared using keratinocytes, fibroblasts, and biopolymers, can facilitate closure of massive burn wounds by increasing the availability of autologous tissue for grafting. But because they contain only two cell types, skin substitutes cannot replace all of the functions of native human skin. To better understand the physiological and molecular differences between CSS and native skin, we undertook a comprehensive analysis of gene expression in native skin, cultured keratinocytes, cultured fibroblasts, and skin substitutes using Affymetrix gene chip microarrays. Hierarchical tree clustering identified six major clusters of coordinately regulated genes, using a list of 1030 genes that were the most differentially expressed between groups. These clusters correspond to biomarker pools representing expression signatures for native skin, fibroblasts, keratinocytes, and cultured skin. The expression analysis revealed that entire clusters of genes were either up- or downregulated upon combination of fibroblasts and keratinocytes in cultured skin grafts. Further, several categories of genes were overexpressed in CSS compared with native skin, including genes associated with hyperproliferative skin or activated keratinocytes. The observed pattern of expression indicates that CSS in vitro, which display a well-differentiated epidermal layer, exhibit a hyperproliferative phenotype similar to wounded native skin.