Characteristics of cultivated adult human nevocellular nevus cells

Nevus cells are of biologic interest because of their uncertain relationship to epidermal melanocytes and of clinical interest because of their statistical association with melanoma. We report a technique that allows reliable cultivation of nevus cells from small acquired and congenital nevi and permits in vitro characterization of this cell type. Morphologically, cultured nevus cells were found to closely resemble epidermal melanocytes from the same or comparably aged donors, manifesting marked dendricity and specific ultrastructural features characteristic of melanocytes; but could be distinguished by the presence of occasional large binucleate or trinucleate cells and by the frequent finding of grouped melanosomes in nevus cell cytoplasm. Growth kinetics were also similar for nevus cells and epidermal melanocytes, with population doubling times of 1-2 weeks in hormone-supplemented serum-free medium, and substantial growth enhancement by fetal bovine serum. As previously noted for epidermal melanocytes, nevus cells in serum-free culture demonstrated striking substrate responsiveness, with far greater attachment rates and degree of cytoplasmic spreading on fibronectin or type I/III collagen than on laminin, type IV collagen, or uncoated plastic. These strong similarities in vitro suggest that morphologic and behavioral differences observed between epidermal melanocytes and nevus cells in the skin may result from local environmental influences rather than from intrinsic cellular differences. The availability of a satisfactory culture system for nevus cells may facilitate future investigations into their malignant potential and other biologic features.     

Ligation of the beta4 integrin triggers adhesion behavior of human keratinocytes by an "inside-out" mechanism

Carcinogenesis is considered as a multistep process involving functional changes in the hemidesmosomal organization. In normal skin keratinocytes, expression of the alpha(6)beta(4) integrin is restricted to the proliferative basal layer and mediates stable adhesion to the underlying basement membrane. Observations in carcinoma cells show a functional and spatial dissociation of the alpha(6)beta(4) integrin from the hemidesmosomal complex, which stimulates cell migration and, therefore, may contribute to carcinoma invasion. We now have evaluated the adhesion behavior of epithelial cells at different stages of transformation in response to activation of the beta(4) integrin. It is demonstrated that ligation of the beta(4) integrin augmented adhesion of carcinoma and pre-carcinoma cells to non-modified plastic. In contrast, adhesion behavior of normal human keratinocytes was not influenced by ligation of the beta(4) integrin. In order to explain the mechanism of beta(4)-mediated adhesion, the hypothesis of an "inside-out" activation of integrins was tested. Evidence is given that for cells expressing the alpha(6)beta(4) integrin, ligation of the beta(4) integrin increased beta(1) integrin-mediated adhesion. Furthermore, ligation of the beta(4) integrin led to phosphorylation of PKB/Akt at both phosphorylation sites. Functional blocking of PKB/Akt by dominant-negative overexpression decreased cell adhesion in response to beta(4) integrin ligation. Taken together, the present data establish a link between the ligation of the beta(4) integrin and beta(1) integrin-mediated cell adhesion in carcinoma and pre-carcinoma cells. Hence, these findings provide further insight into the conversion processes during carcinogenesis and show the beta(4) integrin to be a key regulator of cellular adhesion.     

Proteinase-activated receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity

Prostaglandins (PG) are key mediators of diverse functions in the skin and several reports suggest that PG mediate post-inflammatory pigmentary changes through modulation of melanocyte dendricity and melanin synthesis. The proteinase-activated receptor 2 (PAR-2) is important for skin pigmentation because activation of keratinocyte PAR-2 stimulates uptake of melanosomes through phagocytosis in a Rho-dependent manner. In this report, we show that activation of keratinocyte PAR-2 stimulates release of PGE(2) and PGF(2alpha) and that PGE(2) and PGF(2alpha) act as paracrine factors that stimulate melanocyte dendricity. We characterized the expression of the EP and FP receptors in human melanocytes and show that human melanocytes express EP1 and EP3, and the FP receptor, but not EP2 and EP4. Treatment of melanocytes with EP1 and EP3 receptor agonists resulted in increased melanocyte dendricity, indicating that both EP1 and EP3 receptor signaling contribute to PGE(2)-mediated melanocyte dendricity. Certain EP3 receptor subtypes have been shown to increase adenosine 3',5'-cyclic monophosphate (cAMP) through coupling to Gs, whereas EP1 is known to couple to Gq to activate phospholipase C with elevation in Ca(2+). The cAMP/protein kinase A system is known to modulate melanocyte dendrite formation through modulation of Rac and Rho activity. Neither PGF(2alpha) or PGE(2) elevated cAMP in human melanocytes showing that dendricity observed in response to PGE(2) and PGF(2alpha) is cAMP-independent. Our data suggest that PAR-2 mediates cutaneous pigmentation both through increased uptake of melanosomes by keratinocytes, as well as by release of PGE(2) and PGF(2alpha) that stimulate melanocyte dendricity through EP1, EP3, and FP receptors.     

Adhesion molecule mapping in normal human skin

Summary Adhesion molecules are a rapidly growing group of cell surface receptors providing cell-cell and cell-matrix interactions. Their physiological role in tissue homeostasis as well as cellular migration and differentiation is increasingly appreciated. In the present study we have analyzed the expression pattern of most adhesion molecules of the integrin family as well as of adhesion molecules belonging to the immunoglobulin superfamily in normal human skin. We provide evidence that expression of adhesion molecules in the various cutaneous cell systems follows a constant distribution. Moreover, the physiological mononuclear infiltrate of the skin also expresses a variety of adhesion molecules enabeling these cells to migrate or to reside within the skin. Furthermore, our results indicate that intercellular adhesion molecule-1 is not a prerequisite for lymphocyte epidermotropism as frequently stated. Our data provide a rational basis to analyze changing adhesion molecule expression in inflammatory and neoplastic skin diseases.     

Defective beta1-integrins expression in arsenical keratosis and arsenic-treated cultured human keratinocytes

BACKGROUND: beta1-integrins, which localize to the basolateral surface of basal keratinocytes, are important in the differentiation control and proliferation of the epidermis. Many cutaneous diseases with perturbed differentiation, including arsenical keratosis, show altered patterns of integrin distribution and expression. Arsenic may induce arsenical keratosis through the differentiation and apoptosis aberration by integrins. The purpose of this study is to investigate the role of integrin and arsenic in the pathogenesis of arsenical keratosis. METHODS: Twenty-five specimens obtained from 25 patients with arsenical keratosis disease were studied. Immunohistochemistry staining to beta1, alpha2beta1, or alpha3beta1 integrins was performed in arsenical keratosis and clinically normal perilesional skin. Western blotting was used to assess the expression of integrin beta1 and focal adhesion kinase (FAK) in arsenic-treated cultured keratinocytes. RESULTS: A decreased expression of beta1, alpha2beta1, or alpha3beta1 integrins was demonstrated in arsenical keratosis and clinical normal perilesional skin in a large proportion of arsenical keratosis cases studied. The expressions of integrin beta1 and FAK were both decreased in arsenic-treated keratinocytes. CONCLUSIONS: Our results suggest that arsenic induces abnormal differentiation in arsenical keratosis via the effects of integrin expression in keratinocytes.     

Increased adhesion of fibroblasts from patients with scleroderma to extracellular matrix components: in vitro modulation by IFN-gamma but not by TGF-beta.

A characteristic feature of systemic scleroderma is fibrosis of the skin and eventually of internal organs resulting from an overproduction of collagen and other connective tissue components by the resident fibroblasts. The balance between the cells and the amount of the surrounding extracellular matrix is then altered. Because cellular metabolism depends to a large extent on cellular contacts and communications with connective tissue molecules, we have therefore investigated the interactions with extracellular matrix components of fibroblasts obtained from skin of patients affected with scleroderma. In comparison to fibroblasts from healthy skin, all fibroblasts from scleroderma patients had an increased adhesion capacity to collagens I, IV, VI, fibronectin, and laminin. In addition, whereas adhesion of control fibroblasts was stimulated by a pre-treatment with transforming growth factor-beta, adhesion patterns of scleroderma fibroblasts remained unchanged. However, pre-incubation of the cells with interferon-gamma decreased the adhesion of both scleroderma and control fibroblasts.     

Human epidermal Langerhans cells express beta 1 integrins that mediate their adhesion to laminin and fibronectin.

Members of the beta 1 or very late antigen (VLA) integrin family represent the predominant class of integrin extracellular matrix receptors. Adhesion assays were developed for the identification of the beta 1 integrins involved in the adhesive interactions between Langerhans cells (which mainly express alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1) and extracellular matrix proteins. For this purpose, binding assays were performed on fibronectin-, laminin-, collagen type IV-, and collagen type I-coated plates. 59% +/- 21% of Langerhans cells (LC) specifically attached to fibronectin. Using as inhibitory probes monoclonal antibodies against the beta 1, alpha 5, and alpha 3 chains and the synthetic peptide GRGDSP resulted in a decrease of 43%, 41%, 15%, and 42% respectively of LC binding to fibronectin. 76% +/- 20% of LC specifically adhered to laminin. Anti-alpha 6 monoclonal antibody potently inhibited this adhesion, which dropped to 36%, whereas the synthetic peptide GRGDSP was ineffective. A low number of LC adhered to type I and type IV collagen (13-15%). These results indicate that alpha 5 beta 1 and alpha 6 beta 1 were the major beta 1 integrins involved in LC adhesion to fibronectin and laminin. Ultrastructural cell morphology of adherent cells was examined and showed that LC were largely spread on laminin and became tightly bound to the substrate on a large portion of membrane. On fibronectin surface, the contact between LC and substrate was smaller, thus cells could conserve their general round aspect. Moreover, LC binding to fibronectin and laminin induced a significative decrease of the Birbeck granule number. The finding that LC attach to LM and FN in vitro suggests they exist similarly in vivo. By mediating a passage through basement membrane and migration throughout the fibronectin network of the dermis, alpha 5 beta 1 and alpha 6 beta 1 could contribute to the ability of LC to migrate into and out of the epidermis.     

Localization of the laminin alpha4 chain in the skin and identification of a heparin-dependent cell adhesion site within the laminin alpha4 chain C-terminal LG4 module

The laminin alpha4 chain, a component of laminin-8/9, is expressed in basement membranes of endothelial cells, the peripheral nerves, and muscle fibers. The localization and functions of laminin alpha4 chain in the skin have not been elucidated. By immunostaining with specific antibodies, we demonstrate here that the alpha4 chain is located in the basement membrane zones of blood vessels and is also associated with fibroblast-like cells in the dermis. Western blot showed that cultured fibroblasts secreted a laminin trimer containing the alpha4 chain. We have also focused on the cell adhesion activities of the human laminin alpha4 LG4 module since the corresponding LG4 module of laminin alpha3 was previously identified as active for cell adhesion. Recombinant human alpha4 LG4 was active for heparin-dependent fibroblast adhesion. Screening assays with 19 synthetic peptides covering the entire alpha4 LG4 module identified three peptides (HA4G82: TLFLAHGRLVYM; HA4G83: LVYMFNVGHKKL; and HA4G90: TEATWKIKGPIYL) as active sites for heparin- and heparan sulfate-dependent cell adhesion. Serine-substituted peptides demonstrated that two basic residues, His and Arg, within HA4G82 were essential for cell adhesion activity. The cell surface heparan sulfate proteoglycans (HSPGs), syndecan-2, -4, and glypican-1, were stably expressed in 293T cells to estimate whether they function as cell adhesion receptors. 293T cells overexpressing syndecan-2 or -4 bound to recombinant alpha4 LG4 and to HA4G82, but parental or glypican-1-overexpressing 293T cells did not. Therefore, syndecan-2 and -4 could mediate cell adhesion to the laminin alpha4 LG4 module. Our study suggests that the laminin alpha4 LG4 module may play an important role in cell adhesion and/or vessel wall formation in the skin by interacting with syndecan-2 and/or -4.     

Interleukin-6 enhances whereas tumor necrosis factor alpha and interferons inhibit integrin expression and adhesion of human mast cells to extracellular matrix proteins

Integrins are expressed on mast cells and constitute an essential prerequisite for the accumulation of the cells at sites of inflammation. In order to clarify a potential contribution of inflammatory cytokines to this process, we have studied the modulation of integrin expression and adhesion of immature human mast cells (HMC-1) to extracellular matrix proteins by interleukin-6, tumor necrosis factor alpha, interferon-alpha and interferon-gamma. Corticosteroids were used for comparison. On fluorescence-activated cell sorter analysis, preincubation of cells for 48 h with different concentrations of interleukin-6 induced a significant, up to 40%, increase of alpha v alpha 5, CD49b (alpha 2), CD49e (alpha 5), CD49f (alpha 6), and CD51 (alpha v). In contrast, different concentrations of tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone (10-8-10-10 M) inhibited expression of adhesion receptors by up to 60%, reaching significance for some but not all integrins. On semiquantitative polymerase chain reaction analysis, interleukin-6, the other cytokines, and corticosteroids significantly modulated expression of alpha1, alpha v and alpha 5 integrin chains at mRNA level. Functional significance of these findings was proven in adhesion assays using fibronectin, laminin, and vitronectin, with interleukin-6 causing significant enhancement of adhesion in all cases, tumor necrosis factor alpha and dexamethasone inducing significant reduction of adhesion to fibronectin and laminin, and interferon-gamma significantly inhibiting adhesion to fibronectin only. Specificity of interleukin-6-induced changes was demonstrated using antibodies against alpha1 and alpha 5 integrins in unstimulated and interleukin-6-prestimulated cells. These data show that interleukin-6 stimulates mast cell adhesion to extracellular matrix and thus allows for the accumulation of the cells at tissue sites by enhancing integrin expression, whereas tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone downmodulate this process.     

The distribution of α6β4 integrins in lesional and non-lesional skin in bullous pemphigoid

The alpha 6 beta 4 integrin is associated ultrastructurally with the hemidesmosomes of the basal keratinocytes and with the bullous pemphigoid antigen (BPA), suggesting an important role in adhesion of epidermal cells to the basement membrane. Using an immunofluorescence technique with chain-specific monoclonal antibodies to the alpha and beta subunits we have investigated the distribution of the alpha 6 beta 4 integrin in normal skin (n = 3) and in BP skin (uninvolved, perilesional and lesional) [n = 11]). The findings have been compared with other types of subepidermal blisters and with normal skin split by chemical means (n = 2) and by suction (n = 2). The distribution of alpha 6 beta 4 integrin was compared with that of bullous pemphigoid antigen (BPA) and with other basement membrane zone (BMZ) macromolecules, laminin, collagen type IV, collagen type VII and the BM600 antigen. In uninvolved, perilesional and early pre-blistered lesional BP skin the distribution of both the alpha 6 and beta 4 integrin subunits, BPA laminin, collagen types IV and VII and the BM600 antigen was identical to normal skin, i.e. a linear band in the BMZ. Within BP blisters, both alpha 6 and beta 4 integrin subunits and BPA were absent, except in two blisters in which the integrin expression was retained in the blister roof, despite loss of BPA. The other BMZ components were expressed on the blister floor.(ABSTRACT TRUNCATED AT 250 WORDS)     

A ''hot'' new twist to hair biology – involvement of vanilloid receptor-1 signaling in human hair growth control

The melanocortin hormones act on epidermal melanocytes to increase eumelanogenesis, melanocyte dendricity and likely melanosome transfer to keratinocytes. These actions are mediated by the melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function Mc1r alleles are associated with dark, eumelanic skin. Conversely, loss-of-function variants or overexpression of agouti, the natural antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. An induction of constitutive pigmentation in amelanotic mouse melanoma cells following the expression of MC1R has been reported, suggesting that this receptor might display agonist-independent activity, although this aspect has not yet been comparatively studied for MC1R and Mc1r. We show that the expression of MC1R in heterologous systems leads to high agonist-independent increases in intracellular cAMP. This basal signalling is a function of the quantity of receptor expressed, is considerably higher for MC1R than Mc1r and is also observed in human melanoma cells overexpressing MC1R. Moreover, MC1R basal signalling is abolished or reduced by point mutations impairing the response to agonists. Lastly, the expression of wild-type MC1R, but not of loss-of-function mutants potently stimulates forskolin activation of adenylyl cyclase, a feature characteristic of constitutively active G-coupled receptors. Therefore, we conclude that MC1R displays a strong agonist-independent constitutive activity.     

Regulation of human melanocyte growth, dendricity, and melanization by keratinocyte derived factors

Epidermal pigmentation involves the synthesis of melanin in melanocytes and its transfer to surrounding keratinocytes, where it functions in photoprotection. To investigate the possible role of the keratinocyte in regulating pigmentation, human keratinocytes were incubated for 24 h in a defined culture medium, which was then transferred to pure human melanocyte cultures. After 1 week, the conditioned medium produced a fourfold increase in melanocyte yield and a seven-fold increase in total melanin. Increased melanocyte dendricity was clearly visible within 24 h as well. Ultrafiltration of the keratinocyte-conditioned medium suggested approximately one-half of the growth promoting activity as well as most of the dendricity and melanization stimulating activities were of low molecular weight (less than 500 Da). High molecular weight fractions stimulated only melanocyte growth. Of the several known keratinocyte-derived factors tested, none could be implicated as a mediator of the observed effects. Basic fibroblast growth factor, known to stimulate melanocyte growth in some culture systems, failed to stimulate growth, dendricity, or melanin content when added to the complete non-conditioned medium. Interleukin-1 alpha, interleukin-1 beta, 12-hydroxyeicosatetraenoic acid, prostaglandin E2, leukotriene B4, and adenosine 3',5'-cyclic monophosphate analogues also had no effect. These studies demonstrate that keratinocytes in vitro release factors that modulate melanocyte behavior and expand our understanding of controls for human epidermal pigmentation.     

Ultraviolet B radiation suppresses Langerhans cell migration in the dermis by down-regulation of alpha4 integrin

Background/Purpose: Ultraviolet B (UVB) radiation affects the migration and function of epidermal Langerhans cells (LC) and causes immunosuppression of contact hypersensitivity. It is known that LC leaves the epidermis after exposure to UVB. To know the behavior of LC in the dermis after UVB radiation, we studied the effect of UVB radiation on the expression of integrin families on freshly isolated or cultured murine LC. We also examined whether UVB radiation affects the migration of LC to secondary lymphoid tissue chemokine (SLC/6Ckine).
Methods: Integrin expressions of murine LC cultured in epidermal cell suspension were analyzed using flowcytometry. We used murine LC sorted flowcytometrically for binding assay to extracellular matrix and for migration assay to chemokine. Skin explant assay and immnohistochemical staining for 'cords formation' were performed as previously described.
Results: Twenty and 40 mJ/cm² of UVB radiation down-regulated the expression of α4 integrin on 24 h-cultured LC, but not that of α6, β1, or β4 integrin. The number of cultured LC adhered to fibronectin, a ligand for α4 integrin, was decreased after UVB irradiation, while that to laminin, a ligand for α6 integrin, was not influenced. UVB radiation reduced the number of migrating LC to SLC. Furthermore, skin sheet explant experiments showed that UVB radiation inhibited the 'cords' formation in dermal vessels of the 48 h-cultured skin.
Conclusions: These data suggest that UVB radiation may suppress the migration of LC from the dermis to lymphatic vessels. UVB radiation may downregulate the adherence of LC to dermal fibronectin and migration to SLC, and consequently suppress the migration of LC from the UVB-irradiated dermis to lymphatics.     

Adhesion molecules in keratinocytes

Adhesion molecules are proteins on the cell surface that are involved in the interactions between lymphocytes and antigen-presenting cells, especially in inflammatory skin diseases and autoimmune bullous disorders. Adhesion molecules include cadherins (subgroups E, N, P, M), integrins, selectins, and the immunoglobulin gene family. Cadherins E in the epidermis including desmocollin 1 and 2 and desmoglein 1 and 3 are essential transmembrane components of desmosome glycoproteins, which play a major role in bullous diseases, pemphigus in particular. Also important are integrin beta 1 alpha 1 and other integrins, which connect ligands of the collagen, laminin, and fibronectin. Selectins (E, P) are important for leukocyte migration on endothelial cells. Adhesion molecules from the immunoglobulin superfamily (intercellular adhesion molecule-1, 3 [ICAM-1,3]; vascular adhesion molecule-1 [VCAM-1]) have significant roles in the immune and inflammatory mechanisms.     

整合素α3β1在相关皮肤病发病机制的研究进展

整合素α3β1是一类重要的细胞黏附分子,在维持皮肤结构稳态中发挥重要作用.整合素α3β1通过与细胞外基质中的不同配体相互作用,时空特异性的激活下游信号分子,参与调控构成皮肤稳态的复杂网络,直接或间接的调节细胞外微环境,介导细胞增殖、分化、黏附和迁移等,参与基底膜形成、创伤修复、肿瘤侵袭等过程.本文就整合素α3β1在相关...     

Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation

Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.