Normal Adult Human Skin Research Articles

Connective tissue cells of the skin: heterogeneity of fibroblasts and general properties of their subpopulations

Directing of experimental and clinical research requires integration of information on morphological and functional features of organs and tissue structures. One of the examples of such integration is the analysis of the histophysiology of the skin, in particular dermal fibroblasts – a mandatory cellular component of the dermis. They not only synthesize and organize the components of the extracellular matrix of the dermis, but also interact with each other and with other types of cells, playing a decisive role in the regulation of skin histophysiology. Other resident cells include epidermal, vascular, and nerve cells. In addition, the skin contains various cells of hematogenous origin. They contain a constitutive population of dendritic cells and a more dynamic population of leukocytes, which includes monocytes (macrophages), neutrophils, and lymphocytes. Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast histophysiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous structural transformations.

Dermal fibroblasts: the terminology, heterogeneity of subpopulations and common properties

Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast histophysiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous structural transformations.

Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) “premalignant” keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

Tissue-resident macrophages can be generated de novo in adult human skin from resident progenitor cells during substance P-mediated neurogenic inflammation ex vivo.

Besides monocyte (MO)-derived macrophages (MACs), self-renewing tissue-resident macrophages (trMACs) maintain the intracutaneous MAC pool in murine skin. Here, we have asked whether the same phenomenon occurs in human skin using organ-cultured, full-thickness skin detached from blood circulation and bone marrow. Skin stimulation ex vivo with the neuropeptide substance P (SP), mimicking neurogenic skin inflammation, significantly increased the number of CD68+MACs in the papillary dermis without altering intracutaneous MAC proliferation or apoptosis. Since intraluminal CD14+MOs were undetectable in the non-perfused dermal vasculature, new MACs must have differentiated from resident intracutaneous progenitor cells in human skin. Interestingly, CD68+MACs were often seen in direct cell-cell-contact with cells expressing both, the hematopoietic stem cell marker CD34 and SP receptor (neurokinin-1 receptor [NK1R]). These cell-cell contacts and CD34+cell proliferation were up-regulated in SP-treated skin samples. Collectively, our study provides the first evidence that resident MAC progenitors, from which mature MACs can rapidly differentiate within the tissue, do exist in normal adult human skin. That these NK1R+trMAC-progenitor cells quickly respond to a key stress-associated neuroinflammatory stimulus suggests that this may satisfy increased local MAC demand under conditions of wounding/stress.

Presence of galectin-1 in the epidermal and dermal thickening of keloid tissues

Background: Keloids are recognized as benign dermal fibroproliferative tumors that result from aberrant wound healing in susceptible individuals. Nevertheless, the etiology and pathophysiology of keloids is not fully elucidated yet. A protein that has been implicated in the wound healing process, conversion of fibroblasts into myofibroblasts and production of ECM components, inflammation, and generation of galectin-glycan complexes, is galectin-1 (Gal-1). However, its presence as well as its implications in benign dermal fibroproliferative tumors such as keloids, has not been considered. Therefore, the aim of this study is to ascertain whether Gal-1 is present in keloid tissues. Methods: Seven biopsies from patients diagnosed with keloid were selected. Four normal skin specimens were obtained from patients undergoing aesthetic surgery procedures. To identification of collagen bundles, some histological sections were stained following the Mason´s trichrome protocol. The presence of Gal-1 was assessed by immunofluorescence staining using confocal laser scanning microscopy. Results: Immunostaining revealed that Gal-1 was present in the uppermost cell layers of the thickened epidermis with a gradient of expression that seems to increase from basal layer to suprabasal layers. Surprisingly, Gal-1 was particularly found outlining the cellular margin and intercellular bridges of the upper-spinous and granular keratinocytes and was absent in the epidermal keratinocytes of normal adult human skin. Interestingly, in the reticular dermis the presence of Gal-1 was noticeable in the cytoplasm of elongated fibroblasts that were forming cellular bundles of collagen and along the thick and compact collagen bundles, whereas in the dermis of normal skin, Gal-1 immunoreactivity was weak in some fibroblasts. Conclusions: Our findings suggest that in keloid tissues Gal-1 would not only be regulating the dermal fibroblast proliferation, ECM production, particularly collagen, and abnormal vascularization, but would also be contributing to the altered stratification and terminal keratinocyte differentiation and importantly, to epidermal thickening.

Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

Differential translocation of heat shock factor-1 after mild and severe stress to human skin fibroblasts undergoing aging in vitro.

Repeated exposure to mild heat shock (HS) has been shown to induce a wide range of health promoting hormetic effects in various biological systems, including human cells undergoing aging in vitro. In order to understand how cells distinguish between mild and severe stress, we have investigated the extent of early and immediate HS response by analyzing the nuclear translocation of the transcription factor heat shock factor-1 (HSF1), in serially passaged normal adult human facial skin fibroblasts exposed to mild (41°C) or severe (43°C) HS. Cells respond differently when exposed to mild and severe HS at different passage levels in terms of the extent of HSF1 translocation. In early passage young cells there was a 5-fold difference between mild and severe HS in the extent of HSF1 translocation. However, in near senescent late passage cells, the difference between mild and severe stress in terms of the extent of HSF1 translocation was reduced to less than 2-fold. One of the reasons for this age-related attenuation of heat shock response is due to the fact there was a higher basal level of HSF1 in the nuclei of late passage cells, which is indicative of increased intrinsic stress during cellular aging. These observations are consistent with previously reported data that whereas repeated mild stress given at younger ages can slow down aging and increase the lifespan, the same level of stress given at older ages may not provide the same benefits. Therefore, elucidating the early and immediate steps in the induction of stress response can be useful in deciding whether a particular level of stress is potentially hormetically beneficial or not.

P38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

Human Wharton’s jelly mesenchymal stem cells promote skin wound healing through paracrine signaling

IntroductionThe prevalence of nonhealing wounds is predicted to increase due to the growing aging population. Despite the use of novel skin substitutes and wound dressings, poorly vascularized wound niches impair wound repair. Mesenchymal stem cells (MSCs) have been reported to provide paracrine signals to promote wound healing, but the effect of human Wharton’s jelly-derived MSCs (WJ-MSCs) has not yet been described in human normal skin.The aim of this study is to examine the effects of human WJ-MSC paracrine signaling on normal skin fibroblasts in vitro, and in an in vivo preclinical model.MethodsHuman WJ-MSCs and normal skin fibroblasts were isolated from donated umbilical cords and normal adult human skin. Fibroblasts were treated with WJ-MSC-conditioned medium (WJ-MSC-CM) or nonconditioned medium.ResultsExpression of genes involved in re-epithelialization (transforming growth factor-β2), neovascularization (hypoxia-inducible factor-1α) and fibroproliferation (plasminogen activator inhibitor-1) was upregulated in WJ-MSC-CM-treated fibroblasts (P ≤ 0.05). WJ-MSC-CM enhanced normal skin fibroblast proliferation (P ≤ 0.001) and migration (P ≤ 0.05), and promoted wound healing in an excisional full-thickness skin murine model.ConclusionsUnder our experimental conditions, WJ-MSCs enhanced skin wound healing in an in vivo mouse model.

Defining the lymphoid stress surveillance response in human skin

BackgroundThe skin contains many tissue-resident immune cells increasingly acknowledged as having key roles in maintaining tissue integrity and excluding infection. In lymphoid stress surveillance, lymphocytes are activated rapidly and in synchrony by stress ligands expressed by tissue cells after cellular stress and engage activating receptors such as NKG2D. Although much of this process has been worked out in model systems, whether this capability exists in human skin is not entirely clear. Therefore, we have characterised the human skin-resident lymphocyte compartment, and measured functional responsiveness of cells to see whether certain T cells have the capacity to respond rapidly to tissue stress. MethodsWe used normal adult human skin discarded after cutaneous surgery. Protocols were established to isolate and characterise lymphocytes by tissue disaggregation or three-dimensional explant culture. Skin-resident lymphocytes were characterised by multicolour flow cytometry, including cell-surface receptor expression and cytokine production after in-vitro stimulation. FindingsAfter ex-vivo isolation, distinct skin-resident lymphocyte subsets were identified, including the unconventional lymphocytes, γδ T cells and natural killer (NK) cells. Subsets were consistent across individuals; expressed as a mean (SD) percentage of leucocytes isolated, there were 46·1% CD4 T cells (15·2), 20·2% CD8 T cells (14·8), 1·6% γδ T cells (1·0), and 6·2% NK cells (5·0) (n=7–10). Explant culture greatly increased lymphocyte yield, enabling detailed functional characterisation. In addition, culture conditions greatly amplified the γδ T-cell compartment (1·6–4·0%, p=0·01, Student's t test). Skin-resident lymphocytes displayed skin-homing surface markers and a memory phenotype. Lymphocytes differed in expression of co-stimulatory receptors, with CD8 T cells, γδ T cells, and NK cells expressing the activating receptor NKG2D. Upon in-vitro activation, skin-resident lymphocytes readily produced cytokines and displayed distinct cytokine-producing profiles, with the response dependent on the strength of T-cell receptor-mediated activation. Skin-derived NKG2D+ lymphocytes had clear potential to produce both tumour necrosis factor α and interferon γ. InterpretationSkin-resident lymphocytes could be isolated from healthy human skin. Consistent with published data, cultured skin-resident αβ T cells maintained a tissue-resident memory (TRM) phenotype. In addition, that skin γδ T cell and NK cells also had a TRM-type phenotype was a novel finding. Distinct NKG2D+ lymphocytes were identified, which showed variable expression of additional activatory or inhibitory co-receptors, and were functionally competent with distinct cytokine-producing subsets. This system provides a platform to further characterise human skin-resident lymphocytes and their potential to be directly activated by surrounding tissue dysregulation. FundingUK Medical Research Council.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Spatial-temporal modulation of CCN proteins during wound healing in human skin in vivo

CCN proteins are important modulators of development and function of adult organs. In this study, we examined the localization and expression of the six CCN family members in normal adult human skin and during wound healing in vivo. Transcript and protein expression were studied by laser-capture microdissection-coupled real-time PCR and immunohistochemistry, respectively. Our results demonstrate that CCN1, CCN4, and CCN6 are expressed at relatively low levels in normal human skin. CCN2, CCN3, and CCN5 are the most highly expressed transcripts in the epidermis. CCN3 and CCN5 proteins are prominent in epidermal keratinocytes, whereas CCN2 is primarily expressed in melanocytes. Differential expression within epidermal layers suggests that CCN3 and CCN5 are linked with keratinocyte differentiation. CCN2, CCN3 and CCN5, are the three most highly expressed transcripts in the dermis. Their respective proteins are produced to various extents by dermal fibroblasts, blood vessels, eccrine sweat glands and hair follicles. We find that most CCN family members are temporally and specifically regulated during different phases (inflammation, proliferation, and remodeling) of partial thickness wound repair. By highlighting spatial-temporal regulations of CCN family member expression in relation to cell proliferation and differentiation, our results suggest a diverse range of functions for CCN proteins in both epidermal and dermal cells, and provides a solid reference for interpretation of future studies aimed at understanding the role of CCN proteins in human skin physiology and diseases.

Densities, distribution and phenotypic expression of T cells in human fetal skin

T cells are present in normal adult human skin, but their occurrence in fetal skin is unknown. T cell and Langerhans cell (LC) populations were studied using single or double immunohistochemical staining on cryostat-section. Skin samples taken from different body regions of 17 fetuses ranging from 18 to 30 weeks estimated gestational-age (w-EGA), were examined. In all specimens but one, we did not find any epidermal T cell. In contrast, dermal CD3(+) T cells occurred at all w-EGA. The density of these cells increased with increasing age. Double staining showed that CD3(+) T cells were predominantly CD4(+)/CD45RA(+). On the other hand, LC, as assessed by CD1a expression, was evenly distributed within the interfollicular epidermis and papillary dermis at all gestational ages. Analysis of T cell and LC density in different body regions did not show significant topographic differences. We suggest that lack of epidermal T cells, although the LC network was fully represented, might reflect the scarce opportunity of fetal LC to contact foreign antigens in utero.

Culture of normal adult human melanocytes.

Melanocytes isolated from normal adult human skin were cultured in vitro. Separation of the epidermis from the dermis by trypsin flotation proved better than collagenase treatment for providing viable cultures of melanocytes with a minimum of fibroblast contamination. Centrifugation on a discontinuous, rather than a continuous Percoll gradient, was more efficient in separating the epidermal cell types. Most of the melanocytes were usually found in one particular layer, and most of the viable keratinocytes were in the sediment. None of the layers produced a uniformly high percentage of melanocytes on routine culture, but enriched melanocyte cultures could be obtained by seeding the epidermal cells in magnesium- and calcium-free medium for 24 to 48 hours, and then transferring them to fibroblast-conditioned medium containing horse serum and polyamines. Melanocytes were identified by their dendritic morphology, ultrastructure, reaction to cholera toxin and pigment production after treatment with melanocyte stimulating hormone. Pure cultures of melanocytes have been cultivated by this method for more than 43 weeks (ten passages).

Epithelial regeneration from bioengineered skin explants in culture

Artificial skin substitutes are beneficial in the treatment of chronic wounds although their performance relative to authentic human skin is unclear. We compared the rate of outgrowth and morphology of neoepidermis from a bioengineered skin construct (Apligraf) with normal adult human skin explants on de-epidermized human dermal growth substrate with or without intact epidermal basement membrane zone. Epithelial outgrowth of air-exposed cultures in serum-supplemented keratinocyte medium was quantified by fluorescence imaging, morphology by light microscopy, biomarkers of keratinocyte activation, proliferation and migration by immunohistochemical analysis, and gelatinases by zymography. Resurfacing from bioengineereed skin explants started earlier than from normal skin but subsequently, from day 3 to day 9, the rate of epidermalization from bioengineered skin was only 40% (206 +/- 23 microm day(-1), mean +/- SEM) of that of authentic skin (521 +/- 17 microm day(-1), P < 0.001). At culture termination at day 11, normal human skin had formed a multilayered and well-structured neoepidermis covering 41.0 +/- 1.2 mm2 of the dermal substrate while bioengineered skin produced a thinner, less organized epithelium covering 20.4 +/- 3.0 mm2. At this later stage, a higher expression of beta-defensin-2, keratin 16, Ki67 and matrix metalloproteinase (MMP)-9 was found in neoepidermis formed from authentic skin than from bioengineered skin. Activated MMP-2 was elevated in bioengineered skin-derived neoepidermis. Minor epithelial outgrowth was noted with either skin type on the dermal substrate devoid of basement membrane zone. Cultured normal skin explants produced a more uniform and expansive in vivo-like neoepidermis than bioengineered skin explants.

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

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. To identify genes that plays a role in AD. 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. 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. 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.

β‐endorphin modulates lipogenesis in human sebocytes

Previous research in our laboratories has demonstrated that the human sebaceous gland is a target organ for several stress hormones including α‐melanocyte‐stimulating hormone (α‐MSH) and corticotropin‐releasing hormone (CRH). To further investigate the role of stress hormones in acne, we examined the expression of opioid receptors (ORs) and the biological actions of β‐endorphin (β‐ED), a natural high‐affinity ligand for the μ‐OR (MOR) and low‐affinity ligand for the Δ‐OR (DOR), in human sebocytes. RT‐PCR, Western immunoblotting and immunofluorescence studies identified the MOR but not the DOR in the human sebocyte cell line SZ95. Expression of the MOR was also confirmed in the sebaceous gland in normal human adult skin and was detectable primarily in peripherally located sebocytes. SZ95 sebocytes did not express the β‐ED precursor pro‐opiomelanocortin (POMC) as shown by RT‐PCR analysis and Western immunoblotting and stimulation of cells with prototypical POMC inducers such as tumour necrosis factor‐α, α‐MSH, dbcAMP, phorbol ester and CRH failed to induce POMC. On the functional level, β‐ED significantly suppressed the growth of SZ95 sebocytes induced by epidermal growth factor in chemically defined calcium‐rich medium. On the other hand, β‐ED enhanced lipogenesis as shown by Nile red staining and gas chromatography. Accordingly, β‐ED dramatically increased the amount of C16 : 0, C16 : 1, C18 : 0, C18 : 1 and C18 : 2 fatty acids in an extent similar to linoleic acid used as a positive stimulus. Our data demonstrate that human sebocytes express the MOR and respond to β‐ED with reduced in vitro proliferation and increased lipogenesis. These data establish a further link between stress and acne, the latter in which excessive sebum production is an invariable feature.

Fibroblast heterogeneity: more than skin deep.

Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast physiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous wound repair and an ever-increasing role in bioengineering of skin. Bioengineered skin currently performs important roles in providing (1) a basic understanding of skin biology, (2) a vehicle for testing topically applied products and (3) a resource for skin replacement.

Collagen Metabolism Is a Novel Target of the Neuropeptide α-Melanocyte-stimulating Hormone

Suppression of collagen synthesis is a major therapeutic goal in the treatment of fibrotic disorders. We show here that alpha-melanocyte-stimulating hormone (alpha-MSH), a neuropeptide well known for its pigment-inducing capacity, modulates collagen synthesis and deposition. Alpha-MSH in vitro suppresses the synthesis of collagen types I, III, and V and down-regulates the secretion of procollagen type I C-terminal peptide (PICP) in human dermal fibroblasts treated with the fibrogenic cytokine transforming growth factor-beta1 (TGF-beta1). Alpha-MSH did not interfere with TGF-beta1 signaling, because TGF-beta1-induced expression of collagen mRNA was not affected, implying a posttranscriptional mechanism. Human dermal fibroblasts in vitro express a high affinity binding site for MSH, which was identified by reverse transcription PCR and immunofluorescence analysis as the melanocortin-1 receptor (MC-1R). Immunohistochemical studies on normal adult human skin confirmed MC-1R expression in distinct dermal fibroblastic cells. The MC-1R on fibroblasts appears to be functionally relevant because alpha-MSH increased the amount of intracellular cAMP, and coincubation with a synthetic peptide corresponding to the human Agouti signaling protein abrogated the inhibition of TGF-beta1-induced PICP secretion by alpha-MSH. To assess the in vivo relevance of these findings, a mouse model was used in which dermal fibrosis was induced by repetitive intracutaneous injections with TGF-beta1. The inductive activity of TGF-beta1 on collagen deposition and the number of dermal cells immunoreactive for vimentin and alpha-smooth muscle actin was significantly suppressed by injection of alpha-MSH. Melanocortins such as alpha-MSH may therefore represent a novel class of modulators with potential usefulness for the treatment of fibrotic disorders.

Human Dermal Fibroblasts Express Prohormone Convertases 1 and 2 and Produce Proopiomelanocortin-Derived Peptides

In the last few years it has become apparent that the skin is a locoregional source for several proopiomelanocortin-derived peptides including alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. The enzymes that regulate expression of these neuropeptides are the prohormone convertases 1 and 2. In this study we demonstrate, by reverse transcriptase polymerase chain reaction and Western immunoblotting, that cultured human dermal fibroblasts express prohormone convertases 1 and 2 as well as 7B2, which is an essential cofactor for enzymatic activity of prohormone convertase 2. Immunofluorescence studies revealed prohormone convertase 1 to be mainly expressed in the perinuclear region in vesicular structures resembling the trans-Golgi network, whereas prohormone convertase 2 was found in the trans-Golgi network as well as in vesicular structures diffusely distributed in the peripheral cytoplasm. Expression of both enzymes was also confirmed in fibroblasts of normal adult human skin by immunohistochemistry using antibodies against prohormone convertases 1 and 2 and vimentin. To assess the relevance of prohormone convertase 1 and 2 expression in human dermal fibroblasts, we studied the expression of proopiomelanocortin and proopiomelanocortin-derived peptides. Proopiomelanocortin expression was detected by reverse transcriptase polymerase chain reaction and Western immunoblotting. Alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin were mainly located in vesicular structures as demonstrated by immunofluorescence. Production of these peptides was confirmed by radioimmunoassay, immunoradiometric assay, or enzyme immunoassay. Among several stimuli tested, interleukin-1 was found to upregulate production of alpha-melanocyte-stimulating hormone in human dermal fibroblasts. In summary, we have shown that human dermal fibroblasts express the enzymatic machinery for proopiomelanocortin processing and make proopiomelanocortin, alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. Production of proopiomelanocortin peptides by human dermal fibroblasts may be relevant for fibroblast functions such as collagen degradation and/or regulation of dermal immune responses.