Kiyoshi Sato

Effects of fibroblast-derived factors on the proliferation and differentiation of human melanocytes in culture

BACKGROUNDnAlthough keratinocyte-derived factors are known to promote the proliferation and differentiation of human epidermal melanocytes, it is not fully understood whether fibroblast-derived factors work in a similar way.nnnOBJECTIVEnThe aim of this study is to clarify whether fibroblast-derived factors are involved in regulating the proliferation and differentiation of human melanocytes with or without keratinocytes using serum-free culture system.nnnMETHODSnHuman epidermal melanoblasts and melanocytes were cultured in a serum-free growth medium supplemented with fibroblast-derived factors such as keratinocyte growth factor (KGF) with or without keratinocytes, and the effects of KGF on the proliferation and differentiation of melanocytes were studied.nnnRESULTSnKGF stimulated the proliferation of melanoblasts in the presence of dibutyryl cAMP (DBcAMP), basic fibroblast growth factor (bFGF), transferrin (Tf), and endothelin-1 (ET-1). Although KGF stimulated the differentiation, melanogenesis, and dendritogenesis in the presence of DBcAMP, Tf, and ET-1 without keratinocytes, KGF required the presence of keratinocytes for the stimulation of melanocyte proliferation.nnnCONCLUSIONnThese results suggest that fibroblast-derived KGF stimulates the proliferation of human melanoblasts in synergy with cAMP, bFGF, Tf, and ET-1, the differentiation of melanocytes in synergy with cAMP, Tf, and ET-1, and the proliferation of melanocytes in synergy with cAMP, Tf, ET-1, and undefined keratinocyte-derived factors.

Inhibitory Effect of Elephantopus mollis H.B. and K. Extract on Melanogenesis in B16 Murine Melanoma Cells by Downregulating Microphthalmia-Associated Transcription Factor Expression

In this study, the inhibitory effect of Elephantopus mollis H.B. and K. extract on melanogenesis in B16 murine melanoma cells was examined and possible mechanisms were elucidated. The melanin content in B16 cells decreased when they were treated with E. mollis extract. Inhibition was accompanied by reduced expression of tyrosinase (TYR) and tyrosinase-related protein 1 (TRP1). Furthermore, the expression level of microphthalmia-associated transcription factor (MITF), a major transcriptional regulator of genes encoding melanogenic enzymes such as Tyr and Trp1, decreased as assessed by western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). These results suggest that E. mollis extract reduces melanogenesis by downregulating Mitf expression, leading to reduced expression of Tyr and Trp1. In addition, melanocortin-1 receptor (MC1R) expression was downregulated by E. mollis extract, suggesting desensitization to α-melanocyte-stimulating hormone (α-MSH) of cells treated with the extract.

Increased blood flow and vasculature in solar lentigo

Solar lentigo (SL) is a hallmark of ultraviolet (UV)‐induced photoaged skin and growing evidence implicates blood vessels in UV‐associated pigmentation. In this study, we investigated whether the vasculatures are modified in SL. Twenty‐five women with facial SL were enrolled and colorimetric and blood flow studies were performed. There was a significant increase in erythema which was associated with increased blood flow in the lesional skin compared with perilesional normal skin. Immunohistochemical studies with 24 facial SL biopsies consistently revealed a significant increase in vessel density accompanied by increased levels of vascular endothelial growth factor expression. CD68 immunoreactivity was significantly higher in lesional skin suggesting increased macrophage infiltration in SL. In conclusion, SL is characterized by increased blood flow and vasculature. These findings suggest the possible influence of the characteristics of vasculature on development of SL.

Possible Involvement of Keratinocyte Growth Factor in the Persistence of Hyperpigmentation in both Human Facial Solar Lentigines and Melasma

Dear Editor: n nAcute pigmentation due to tanning is commonly understood as increased melanization of the epidermis observed in the skin after ultraviolet (UV) exposure, and the mechanisms underlying this condition are well understood now. Keratinocyte-derived gene products are upregulated by UV irradiation and act as paracrine factors in the skin to stimulate melanogenesis and melanin transfer by melanocytes1. Although acute pigmentation disappears over time, some types of hyperpigmentary disorders such as freckles, solar lentigines, and melasma, tend to persist if patients do not receive any treatments such as topical cosmetic products, medication, or laser. n nSolar lentigines are dark brown spots that occur on sun-exposed areas2, typically on the face, upper back, and shoulders. Multiple solar lentigines are considered a hallmark of aged skin. It is thought that cumulative UV exposure causes these spots. Therefore, pigmented spots of solar lentigines can be considered as indications of photoaging. Melasma is a common acquired symmetrical hypermelanosis on sun-exposed areas of the skin and is very common among Oriental women3. The major etiological factors include genetic influences, exposure to UV radiation, and sex hormones. However, the mechanisms underlying the persistence of hyperpigmentation in solar lentigines and melasma are not yet fully understood. n nKeratinocyte growth factor (KGF) or fibroblast growth factor-7 (FGF-7) is a member of the FGF family4. KGF is secreted from cultured stromal fibroblasts derived from the skin and gastrointestinal tract, and is expressed in vivo in dermal cells, but not in epidermal cells5. In addition, this paracrine growth factor also plays a role in the stimulation of melanogenesis5,6, proliferation of human melanoblasts, and differentiation of melanocytes7. A previous study reported higher levels of KGF in five patients with solar lentigines, suggesting the permeation of KGF from the dermis to the epidermis, which may result in the persistence of solar lentigines6. In this study, we quantitatively investigated the accumulation of KGF in the epidermis of patients with two major types of hyperpigmentary disorders, facial solar lentigines, and melasma to identify novel effective topical measures for their treatment. n nWe examined 24 Korean women with newly diagnosed facial solar lentigines and 13 others with newly diagnosed melasma, which were determined on physical examination and histological examination. This study was approved by the ethics committee of Ajou University Hospital (No. MED-KSP-12-171). Punch biopsies from lesions and perilesional normal skin were obtained from each patient. The perilesional normal skin was taken from the area within 1 cm away from the lesional border. Twenty-four pairs of facial solar lentigines and 13 pairs of melasma tissue were prepared for immunohistopathological examination. n nWe examined KGF protein accumulation in the epidermis of both facial solar lentigines and melasma. Paraffin-embedded sections of both lesional and perilesional normal skin were processed with monoclonal antibodies against KGF (dilution, 1:100; Abcam, Boston, MA, USA) for 20 min at 48℃. The stained area per epidermal area (SA/EA) and SA per single rate ridge length (SA/1R) of lesional and perilesional skin were measured. In both facial solar lentigines and melasma lesions, distinctly positive immunoreactivity against KGF was noticed in the epidermis, whereas perilesional normal skin only showed weak immunoreactivity (Fig. 1). The SA/EA of perilesional normal skin samples was 0.184±0.139 for facial solar lentigines and 0.134±0.071 for melasma, and that of lesional skin samples was 0.237±0.107 for facial solar lentigines and 0.210±0.084 for melasma (Fig. 2A). The differences were statistically significant (p=0.014 for facial solar lentigines and p=0.016 for melasma). The SA/1R of perilesional normal skin samples was 10.216±7.194 for facial solar lentigines and 8.699±4.923 for melasma, and that of lesional skin samples was 19.350±8.744 for facial solar lentigines and 13.172±4.709 for melasma (Fig. 2B). The differences were statistically significant (p=0.000035 for facial solar lentigines and p=0.014 for melasma). n n n nFig. 1 n nImmunostaining with an antibody against keratinocyte growth factor (KGF). Significant amount of KGF protein was accumulated in the epidermis of lesional skin compared to that in perilesional normal skin of (A) solar lentigines and (B) melasma (A, B: ×400). ... n n n n n nFig. 2 n nQuantitative analysis of immunostaining. Immunohistochemical analysis for keratinocyte growth factor (KGF) was quantified in two ways: the stained area per epidermal area (SA/EA) (A) and the stained area per single rate ridge length (SA/1R) (B). Each ... n n n nIn the present study, we demonstrated that KGF protein accumulation in the epidermis of facial solar lentigines and melasma was significantly increased. We performed the following two analytical techniques: first, the SA for KGF was normalized to the EA; second, the SA for KGF was normalized with 1R, which is independent from epidermal thickness. In both analyses, we found a statistical difference between perilesional normal and lesional skin of both facial solar lentigines and melasma. In this study, we did not take the duration and stage of solar lentigines and melasma into consideration because of the small number of patients, although they could have affected the results. We expect that a study with a larger number of patients in varied stages and duration of solar lentigines and melasma will demonstrate the correlation between KGF protein accumulation level and the duration or stage of solar lentigines and melasma. n nIriyama et al.8 revealed that the degradation of heparan sulfate at the dermal-epidermal junction in photoaged skin impaired the function of the basement membrane, which regulates the transfer of several growth factors between the epidermis and dermis. Moreover, they demonstrated that heparan sulfate at the dermal-epidermal junction was specifically reduced in solar lentigines on the human back, and that the degradation of heparan sulfate enhanced melanogenesis in a skin equivalent model. In addition, recent histological studies on melasma described changes in the basement membrane in the lesional skin of melasma9,10. The basement membrane structure in lesional skin was not intact and appeared disrupted. Thus, the loss of heparan sulfate at the dermal-epidermal junction of facial solar lentigines and the loosening of the basement membrane in melasma may enhance the transfer of dermis-derived factors such as KGF. Our present study strongly supports the hypothesis that in hyperpigmentary disorders, such as facial solar lentigines and melasma, the fibroblast-derived KGF is transferred into the epidermis in which it accumulates, thereby leading to the persistence of hyperpigmentation. Thus, targeting the dermal-derived KGF itself and the degradation of heparan sulfate at the dermal-epidermal junction, which allow an excess amount of KGF to transfer into the epidermis, could be effective topical measures like cosmetic or medication use for treating hyperpigmentary disorders.

Platelet-derived growth factor regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner

BACKGROUNDnAlthough many kinds of keratinocyte-derived factors are known to regulate the proliferation and differentiation of human melanocytes, it is not well defined whether dermis-derived factors work in a similar way.nnnOBJECTIVEnThe aim of this study is to clarify whether dermal factors are involved in regulating the proliferation and differentiation of human melanocytes.nnnMETHODSnHuman epidermal melanoblasts were cultured serially in a serum-free growth medium. Platelet-derived growth factor-BB (PDGF-BB) was supplemented to the medium, and the effects on the proliferation of melanoblasts/melanocytes and the differentiation of melanocytes were studied.nnnRESULTSnPDGF-BB stimulated the proliferation of melanoblasts cultured in melanoblast-proliferation medium, but inhibited the proliferation of melanocytes cultured in melanocyte-proliferation medium. By contrast, PDGF-BB stimulated the differentiation, dendritogenesis, and melanogenesis of melanocytes through the stimulation of tyrosinase activity and the expressions of tyrosinase and tyrosinase-related protein-1.nnnCONCLUSIONnThese results suggest that PDGF-BB regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner. PDGF-BB seems to be one of the dermal factors that regulate the proliferation and differentiation of human melanocytes.

Human fibroblasts treated with hydrogen peroxide stimulate human melanoblast proliferation and melanocyte differentiation, but inhibit melanocyte proliferation in serum-free co-culture system

BACKGROUNDnOxidative stress caused by hydrogen peroxide (H2O2) elicits harmful effects on human melanocytes such as DNA damage and cell death. On the contrary, H2O2 is known to possess beneficial effects on melanocytes. However, mechanisms of the beneficial effects of H2O2 on melanocytes have not been fully understood, especially the indirect effects on melanocyte proliferation and differentiation from cells constituting surrounding tissue environment such as fibroblasts.nnnOBJECTIVEnThe aim of this study was to clarify whether H2O2-treated human fibroblasts affect the proliferation and differentiation of human melanocytes using serum-free co-culture system.nnnMETHODSnEpidermal melanoblasts and melanocytes were co-cultured with H2O2-treated or control fibroblasts in serum-free culture media. The effects of H2O2-treated fibroblasts were detected by changes in the proliferation and differentiation of melanoblasts/melanocytes.nnnRESULTSnH2O2-treated fibroblasts stimulated the proliferation of melanoblasts and the differentiation, melanogenesis, and dendritogenesis of melanocytes, but inhibited the proliferation of melanocytes. In the melanocytes co-cultured with H2O2-treated fibroblasts, the expression of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and KIT was increased, whereas TYRP2 and microphthalmia-associated transcription factor showed no change.nnnCONCLUSIONnThese results suggest that H2O2-treated fibroblasts can produce and release some mitogenic and melanogenic factors toward melanoblasts in addition to some proliferation-inhibiting factors toward melanocytes. The stimulation of melanocyte differentiation seems to be performed through the upregulation of TYR, TYRP1, and KIT.