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A kindred with familial progressive hyperpigmentation-like disorder: implication of fibroblast-derived growth factors in pigmentation

European Journal of Dermatology. Volume 19, Number 5, 469-73, September-October 2009, Genes and skin

DOI : 10.1684/ejd.2009.0724

Summary

Author(s) : Giorgia Cardinali , Daniela Kovacs , Micol Del Giglio, Carlo Cota, Nicaela Aspite, Ada Amantea, Giampiero Girolomoni, Mauro Picardo , San Gallicano Dermatological Institute, IRCCS, Via Elio Chianesi 53, 00144 Rome, Italy, Section of Dermatology and Venereology, Department of Biomedical and Surgical Sciences, University of Verona, Italy.

Summary : Two patients with a generalized, progressive dyschromatosis disorder are described and investigated as a model to study the role of fibroblast-derived mediators on skin pigmentation. The patients (father and daughter) had had a widespread hyperpigmentation since early life which then progressively worsened with the appearance of hyperpigmented macules, café-au-lait macules and freckles, also involving the lips, palms and soles, intermixed with small hypopigmented spots. These features resembled those of familial progressive hyperpigmentation (FPH). Histology revealed a normal epidermis with pronounced keratinocyte hyperpigmentation and the presence of dermal melanophages. Ultrastructural analysis showed basal and suprabasal keratinocytes enriched in melanosome complexes. Immunohistochemical staining displayed an increased expression of hepatocyte growth factor (HGF), stem cell factor (SCF) and keratinocyte growth factor (KGF) in fibroblast-like cells of the upper dermis in hyperpigmented lesions of both patients, compared to control healthy skin. Our data suggest that a persistent activation of fibroblasts abnormally stimulating melanocyte functions is involved in hyperpigmentation disorders.

Keywords : fibroblasts, hyperpigmentation, melanogenic growth factors

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ARTICLE

Auteur(s) : Giorgia Cardinali¹, Daniela Kovacs¹, Micol Del Giglio², Carlo Cota¹, Nicaela Aspite¹, Ada Amantea¹, Giampiero Girolomoni², Mauro Picardo¹

¹San Gallicano Dermatological Institute, IRCCS, Via Elio Chianesi 53, 00144 Rome, Italy
²Section of Dermatology and Venereology, Department of Biomedical and Surgical Sciences, University of Verona, Italy

accepté le 7 Avril 2009

Skin pigmentation and photoprotection are related to the type and the amount of melanin synthesized by the melanocytes, and also to the differences in size, number and distribution pattern of melanosomes within neighboring keratinocytes [1-3]. The processes of melanogenesis and melanosome transfer to keratinocytes are induced by ultraviolet (UV) irradiation, and a complex melanogenic paracrine network between different cell types regulates melanocyte survival and functions [4, 5]. Keratinocytes and fibroblasts produce several cytokines and growth factors which support the survival and/or melanization of surrounding melanocytes. In particular endothelin-1 (ET-1), stem cell factor (SCF), melanocyte stimulating hormone (α-MSH) and basic fibroblast growth factor (bFGF) are important mediators for UVB-induced pigmentation [6, 7], whereas granulocyte-monocyte colony-stimulating factor (GM-CSF) plays a crucial role in UVA-induced pigmentation [8]. In different hyperpigmentary disorders such as UVB-melanosis [9, 7], lentigo senilis [10, 11], melasma [4, 12], seborrhoeic keratosis [13] and dermatofibroma [14], an altered expression of growth factors and their receptors has been implicated. An increased production of SCF, KGF and HGF has been demonstrated in lentigo senilis [11, 15]. In type 1 neurofibromatosis (NF-1) a higher expression of HGF, SCF, and bFGF by fibroblasts derived from café-au-lait macules (CALM) compared to fibroblasts from both non-CALM skin of NF-1 patients and healthy control skin has been observed [16]. Moreover, some authors established the role of KGF in combination with IL-1α in increasing melanin deposition both in the basal layer and in the whole epidermis [17].

In the present study, we have evaluated the expression of melanogenic cytokines (KGF, SCF and HGF) in the upper dermis of two cases presenting with a generalized, progressive dyschromatosis disorder resembling familial progressive hyperpigmentation. We observed an increased expression of these cytokines, suggesting that the fibroblast-derived factors are most likely involved in the skin hyperpigmentation.

Materials and methods

Skin biopsies

Tissue samples were obtained from hyperpigmented skin areas of a 59-year-old Caucasian man and a 31-year-old Caucasian woman observed in San Gallicano Dermatological Institute and histologically examined by conventional hematoxylin and eosin. Human skin samples, used as control, were taken from healthy volunteers after informed consent.

Electron microscopy

Skin biopsies of both patients were fixed with 2% glutaraldehyde in PBS (pH 7.4) for 2 h at 25 °C. Samples were post-fixed in 1% osmium tetroxide in veronal acetate buffer (pH 7.4) for 2 h at 25 °C and were stained with uranyl acetate 2% (5 mg/mL) (pH 6.0), dehydrated in acetone and embedded in Epon 812. Thin sections were examined poststained with uranyl acetate and lead hydroxide.

Immunohistochemical analysis

Serial sections (3 μm), derived from formalin-fixed and paraffin-embedded blocks, were dewaxed in xylene and rehydrated through graded ethanols to PBS, pH 7.4. Unfixed cryosections were air-dried and processed for the immunostaining. Endogenous peroxidase activities were blocked by 0.03% hydrogen peroxide for 5 min. Tissue sections were then incubated with the following primary antibodies at room temperature in a humidified chamber: anti-KGF goat or rabbit polyclonal antibodies (C-19; H-73; Santa Cruz Biotechnology Inc.) both diluted 1:100 in PBS for 2 h, anti-HGF goat polyclonal antibody (H 7157; Sigma) diluited 1:200 in PBS for 1h, anti-SCF rabbit polyclonal antibody (H-189; Santa Cruz Biotechnology Inc.) diluited 1:200 in PBS for 1 h. Sections were then treated with peroxidase-labelled polymer conjugated with secondary antibodies for 30 min, incubated with 3-amino-9-ethyl-carbazole substrate chromogen for 10 min (Dako Corp., Carpiteria, CA, USA), counterstained with haematoxylin and mounted under a coverslip. Negative controls were performed by omitting the primary antibodies from the immunohistochemical procedure.

Case reports

A 31-year-old woman and her father, a 59-year-old male, presented with a peculiar type of skin darkening. The same skin changes were referred to be present in other family members through at least four generations, suggesting an autosomal dominant inheritance pattern (figure 1). Both patients showed a diffused dark complexion with skin type III-IV and dark hair and eyes. Skin hyperpigmentation started at the age of few months and progressively worsened. During childhood and adolescence hyperpigmented macules with the appearance of lentigo (diffuse lentiginosis) and 5 CALMs intermixed with small hypopigmented spots appeared and spread out on both sun exposed and unexposed body areas. Both patients showed hyperpigmented palmar creases and lentigo on the palms. A few brown-grey macules were present on the lips and the gingival mucosa. No axillary freckling was present (figures 2 A-F). Both patients stated that new brown macules were still emerging. No other symptoms or signs were present, and both patients were in general good health. The woman first received the diagnosis of neurofibromatosis type 1, but molecular testing for gene mutations of NF-1 (in particular microdeletion of gene 17q.11.2) was negative.

Haematoxylin-eosin stained sections from lesional hyperpigmented areas of both patients showed a normal epidermis with basal keratinocytes strongly hyperpigmented and the presence of a mild perivascular inflammatory infiltrate on the papillary dermis. Moreover, several melanophages were observed on the upper dermis (figures 3A-B). Transmission electron microscopy revealed an increased number of stage IV melanosomes on basal and suprabasal layers of epidermis. Melanosomes are not individually distributed throughout the cytoplasm and appeared densely clustered, forming complexes in perinuclear area, as expected for a Caucasian phototype skin (figures 3C-D, arrows). The skin sample from the father, in comparison to the daughter, showed a higher number of melanosome complexes, confirming a more advanced stage of hyperpigmentation. However, in both patients the increase of epidermal melanosomes did not parallel with an increased number of melanocytes.

Based on the recent evidence on the important role of fibroblast derived growth factors in the regulation of melanocyte functions, we deepened the analysis of the mechanisms underlying the development of this type of hyperpigmentation, focusing on the expression of the melanogenic paracrine factors KGF, SCF and HGF by using immunohistochemistry. In sample skin from normal subjects, only a weak immunoreactivity for KGF, HGF and SCF was detected in the papillary dermis (figures 4G-I). On the contrary, skin samples from both patients showed intense immunostaining for all growth factors, mainly in the subepithelial upper dermis (figures 4A-F). A careful analysis revealed that dermal immunostaining was confined to spindle-like cells most likely represented by fibroblasts, which are known to release melanogenic factors (figure 4 arrows in insert A-C, arrows in D-F). The weak staining for KGF and HGF observed on the epidermal layers may correspond to the growth factor bound to their specific receptor, as previously reported [18-23]. All three factors analysed showed a higher expression in tissue samples from the father compared to the daughter, further indicating their possible involvement in inducing and sustaining skin hyperpigmentation.

Discussion

A complex melanogenic paracrine network between different cell types (keratinocytes, melanocytes and fibroblasts) regulates melanocyte survival and functions [4, 5]. Recently, more attention has been given to mesenchymal-epithelial interactions via fibroblast-derived factors, with the topographical (site-specific) regulation of melanocyte density and differentiation determined by fibroblast populations, which differ in the expression of melanogenic mediators. Palmoplantar fibroblasts, unlike nonpalmoplantar ones, express high levels of the inhibitor of Wnt/β-catenin signaling pathway dickkopf1 (DKK1) which by decreasing the growth and differentiation of melanocytes accounts for the hypopigmentation of palms and soles compared with other areas of the body [24, 25]. Moreover, experimental evidence using conditioned medium from fibroblasts or epidermal reconstructs grafted on nude mice supports the concept of a strong modulatory action of dermal cells on melanocyte functions and skin pigmentation. Fibroblast soluble factors, such as SCF, can influence melanocyte proliferation and melanin distribution or indirectly activate keratinocytes to produce melanogenic factors [5]. We have shown that exposure to UVB is able to trigger keratinocyte growth factor receptor (KGFR) activation and internalization [26-28] and that KGF, similarly to UVB, promotes the phagocytosis of melanosomes by keratinocytes in vitro [29, 30]. A role for KGF in combination with IL-1α in increasing melanin deposition has also been identified [17].

Based on the main role of fibroblast-derived growth factors in regulating pigmentation, we focused our study on the evaluation of melanogenic cytokine expression (KGF, SCF and HGF) in the upper dermis of two cases of generalized, progressive dyschromatosis disorder resembling familial progressive hyperpigmentation. Dyschromatosis is a term used to identify a number of conditions characterized by the simultaneous presence of hyper- and hypopigmentation in variable distribution and patterns. Several disorders have been described with similarities and differences, the recognition of which can also be difficult because of the presence of variants. Genetic studies may help in differentiating these conditions [31, 32]. The patients we present have clinical, histological and ultrastructural similarities to those described by Zanardo et al. [33] and by Betts et al. [34]. In particular, they had diffuse, progressive skin hyperpigmentation since an early age associated with diffuse lentiginosis and CALMs, intermixed with small hypopigmented spots. Both patients showed hyperpigmented palmar creases and lentigo in the palms, lips and the gingival mucosa. However, in neither case did we observe the presence of large hypopigmented ash-leaf-like lesions and macules in the conjunctiva. Histological and ultrastructural analysis of lesional hyperpigmented areas from both patients showed a hyperpigmented epidermis with an increased amount of stage IV melanosomes densely clustered forming complexes in perinuclear area of basal and suprabasal keratinocytes. All together these features allow the differentiation of our patients from dyschromatosis universalis hereditaria, characterized by widespread hyper- and hypopigmented macules, but lacking CALMs and mucosal changes [35-37], dyschromatosis symmetrica hereditaria, which predominantly affects the face, and the back of hands and feet [31, 32], and generalized Dowling-Degos disease, which has typical histological features [38]. The term familial progressive hyperpigmentation may not correctly describe this condition, which indeed belongs to the dyschromatosis disorders.

In this study we evaluated in both patients the involvement of fibroblast derived growth factors analysing the expression of KGF, SCF and HGF by immunohistochemistry. All three factors showed a higher expression in tissue samples compared to normal control skin, indicating their involvement in inducing and sustaining skin hyperpigmentation. The intense immunostaining was detected in the upper dermis and mainly confined to spindle-like cells likely represented by fibroblasts. These results support a central role of mesenchymal cells in influencing melanocyte functionality by the up-regulation of melanogenic factors which in turn can contribute to increase melanin production and melanosome transfer into keratinocytes. Our data are in agreement with other studies on different hyperpigmentary disorders in which an altered expression of growth factors and their receptors has been demonstrated. Solar lentigo results from an up-modulation of ET-1, SCF, HGF and KGF, which stimulates melanogenesis specifically on lesional areas [10, 11, 15]. It has been suggested that solar lentigo could be a consequence of the UV-induced upregulation of genes related to chronic inflammation and fatty-acid metabolism which leads to an increase of melanin production together with an alterated proliferation and differentiation of keratinocytes [39-41]. Moreover, an increased production of melanogenic cytokines HGF, SCF, bFGF by fibroblasts derived from CALMs of NF-1, compared to fibroblasts from both non-CALM skin of NF-1 patients and healthy control skin, has been observed [16].

In conclusion, our results suggest that the activated status of fibroblasts may lead to a sustained release of melanogenic factors responsible for the onset and maintenance of the excessive melanin production.

Acknowledgements

Financial support: this work was partially supported by the grant onc-ord/32/07 from Ministero della Salute, Italy. Conflict of interest: none.

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