Atsushi Ohuchi

Functional analysis of keratinocytes in skin color using a human skin substitute model composed of cells derived from different skin pigmentation types

Skin color is one of the most distinct features in the human race. To assess the mechanisms of skin color variation, human skin substitutes (HSS) were constructed by grafting mixtures of cultured keratinocytes and melanocytes from a combination of donor skin types, together with light skin derived fibroblasts, into chambers inserted onto the back skin of severe combined immunodeficient (SCID) mice. The resulting complexion coloration of the HSS was relatively darker and lighter when dark and light skin derived keratinocytes, respectively, were combined with melanocytes derived from either light or dark skin. The melanin content in the epidermis and the maturation stage of melanosomes in basal keratinocytes were significantly increased in the HSS composed of dark compared to light skin derived keratinocytes. In addition, the ratio of individual/clustered melanosomes in recipient keratinocytes was increased in the former as opposed to the latter HSS. The genetic expression of endothelin‐1, proopiomelanocortin, microphthalmia‐associated transcription factor, tyrosinase, GP100, and MART1 were increased in HSS composed of dark vs. light skin derived keratinocytes. These data suggest that our HSS is a promising melanogenic model that demonstrates the role of the keratinocyte in regulating in part both melanogenesis and distribution of transferred melanosomes.—Yoshida, Y., Hachiya, A., Sriwir‐iyanont, P., Ohuchi, A., Kitahara, T., Takema, Y., Visscher, M. O., Boissy, R. E. Functional analysis of keratinocytes in skin color using a human skin substitute model composed of cells derived from different skin pigmentation types. FASEB J. 21, 2829–2839 (2007)

The Essential Role of p53 in Hyperpigmentation of the Skin via Regulation of Paracrine Melanogenic Cytokine Receptor Signaling

Hyperpigmentation of the skin is characterized by increases in melanin synthesis and deposition. Although considered a significant psychosocial distress, little is known about the detailed mechanisms of hyperpigmentation. Recently, the tumor suppressor protein p53 has been demonstrated to promote ultraviolet B-induced skin pigmentation by stimulating the transcription of a melanogenic cytokine, POMC (pro-opiomelanocortin), in keratinocytes. Given that p53 can be activated by various kinds of diverse stresses, including sun exposure, inflammation, and aging, this finding led us to examine the involvement of p53 in cytokine receptor signaling, which might result in skin hyperpigmentation. Immunohistochemical and reverse transcription-PCR analyses revealed the increased expression and phosphorylation of p53 in the epidermis of hyperpigmented spots, accompanied by the higher expression of melanogenic cytokines, including stem cell factor, endothelin-1, and POMC. The involvement of p53 in hyperpigmentation was also indicated by the significantly higher expression of p53 transcriptional targets in the epidermis of hyperpigmented spots. Treatment of human keratinocytes and melanocytes with known p53 activators or inhibitors, including pifithrin-α (PFT), demonstrated significant increases or decreases, respectively, in the expression of melanogenic factors, including cytokines and their receptors. Additionally, PFT administration abolished stem cell factor-induced phosphorylation of mitogen-activated protein kinase in human melanocytes. Furthermore, when organ-cultured hyperpigmented spots, in vitro human skin substitutes, and mouse skin were treated with PFT or p53 small interfering RNA, the expression of melanogenic cytokines and their receptors was significantly decreased, as were levels of tyrosinase and melanogenesis. Taken together, these data reveal the essential role of p53 in hyperpigmentation of the skin via the regulation of paracrine-cytokine signaling, both in keratinocytes and in melanocytes.

Genes for a range of growth factors and cyclin-dependent kinase inhibitors are expressed by isolated human hair follicles

Summary The mRNA expressions of various growth regulatory molecules in single human anagen hair follicles were analysed by reverse transcription and polymerase chain reaction. Approximately 370 hair follicles were isolated from 20 normal individuals, and 0.90 ± 0.34 μg (mean ± SD) total RNA was extracted per whole hair follicle. The mRNAs of fibroblast growth factor (FGF)‐l, FGF‐2, FGl‐5. FGF‐7, transforming growth factor (TGF)‐α. TGF‐β1. hepatocyte growth factor, insulin‐like growth factor (IGF)‐I. tumour suppressor gene p53 and high sulphur protein were detected in most or all of the examined hair follicles per target gene. In contrast, none of the mRNAs of FGF‐3, FGF‐4, FGF‐6, FGF‐9 and IGF‐II was detected, and those of TGF‐β2 and TGF‐β3 were detected in only a limited number of the examined hair follicles. Among cyclin‐dependent kinase inhibitors, the mRNAs of p2lwaf1/cip1 and p27kipl were expressed in almost all the hair follicles, while those of p15INK4B and p161NK4A were not detected. These results suggest that both positive and negative factors for the proliferation and differentiation of follicular epithelial cells coexist in a human anagen hair follicle.

Ovariectomy is sufficient to accelerate spontaneous skin ageing and to stimulate ultraviolet irradiation‐induced photoageing of murine skin

Summary Background  Wrinkling and sagging of the skin during photoageing is physiologically associated with diminished elasticity, which can be attributed to increased fibroblast‐derived elastase activity. This degrades the dermal elastic fibres needed to maintain the three‐dimensional structure of the skin. We previously reported that ovariectomy accelerates ultraviolet (UV)B‐induced wrinkle formation in rat hind limb skin by altering the three‐dimensional structure of elastic fibres.

Mechanistic Effects of Long-Term Ultraviolet B Irradiation Induce Epidermal and Dermal Changes in Human Skin Xenografts

UVB irradiation has been reported to induce photoaging and suppress systemic immune function that could lead to photocarcinogenesis. However, because of the paucity of an UVB-induced photodamaged skin model, precise and temporal mechanism(s) underlying the deleterious effects of long-term UVB exposure on human skin have yet to be delineated. In this study, we established a model using human skin xenografted onto severe combined immunodeficient mice, which were subsequently challenged by repeated UVB irradiation for 6 weeks. Three-dimensional optical image analysis of skin replicas and noninvasive biophysical measurements illustrated a significant increase in skin surface roughness, similar to premature photoaging, and a significant loss of skin elasticity after long-term UVB exposure. Resembling authentically aged skin, UVB-exposed samples exhibited significant increases in epithelial keratins (K6, K16, K17), elastins, and matrix metalloproteinases (MMP-1, MMP-9, MMP-12) as well as degradation of collagens (I, IV, VII). The UVB-induced deterioration of fibrous keratin intermediate filaments was also observed in the stratum corneum. Additionally, similarities in gene expression patterns between our model and chronologically aged skin substantiated the plausible relationship between photodamage and chronological age. Furthermore, severe skin photodamage was observed when neutralizing antibodies against TIMP-1, an endogenous inhibitor of MMPs, were administered during the UVB exposure regimen. Taken together, these findings suggest that our skin xenograft model recapitulates premature photoaged skin and provides a comprehensive tool with which to assess the deleterious effects of UVB irradiation.

Ethnic differences in the structural properties of facial skin

BACKGROUND Conspicuous facial pores are one type of serious aesthetic defects for many women. However, the mechanism(s) that underlie the conspicuousness of facial pores remains unclear. We previously characterized the epidermal architecture around facial pores that correlated with the appearance of those pores. OBJECTIVES A survey was carried out to elucidate ethnic-dependent differences in facial pore size and in epidermal architecture. METHODS The subjects included 80 healthy women (aged 30-39: Caucasians, Asians, Hispanics and African Americans) living in Dallas in the USA. First, surface replicas were collected to compare pore sizes of cheek skin. Second, horizontal cross-sectioned images from cheek skin were obtained non-invasively from the same subjects using in vivo confocal laser scanning microscopy (CLSM) and the severity of impairment of epidermal architecture around facial pores was determined. Finally, to compare racial differences in the architecture of the interfollicular epidermis of facial cheek skin, horizontal cross-sectioned images were obtained and the numbers of dermal papillae were counted. RESULTS Asians had the smallest pore areas compared with other racial groups. Regarding the epidermal architecture around facial pores, all ethnic groups observed in this study had similar morphological features and African Americans showed substantially more severe impairment of architecture around facial pores than any other racial group. In addition, significant differences were observed in the architecture of the interfollicular epidermis between ethnic groups. CONCLUSIONS These results suggest that facial pore size, the epidermal architecture around facial pores and the architecture of the interfollicular epidermis differ between ethnic groups. This might affect the appearance of facial pores.

Gene transfer in human skin with different pseudotyped HIV-based vectors

Pseudotyping lentiviral vector with other viral surface proteins could be applied for treating genetic anomalies in human skin. In this study, the modification of HIV vector tropism by pseudotyping with the envelope glycoprotein from vesicular stomatitis virus (VSV), the Zaire Ebola (EboZ) virus, murine leukemia virus (MuLV), lymphocytic choriomeningitis virus (LCMV), Rabies or the rabies-related Mokola virus encoding LacZ as a reporter gene was evaluated qualitatively and quantitatively in human skin xenografts. High transgene expression was detected in dermal fibroblasts transduced with VSV-G-, EboZ- or MuLV-pseudotyped HIV vector with tissue irregularities in the dermal compartments following repeated injections of EboZ- or LCMV-pseudotyped vectors. Four weeks after transduction, double-labeling immunofluorescence of β-galactosidase and involucrin or integrin β1 demonstrated that VSV-G-, EboZ- or MuLV-pseudotyped HIV vector effectively targeted quiescent epidermal stem cells which underwent terminal differentiation resulting in transgene expression in their progenies. Among the six different pseudotyped HIV-based vectors evaluated, VSV-G-pseudotyped vector was found to be the most efficient viral glycoprotein for cutaneous transduction as demonstrated by the highest level of β-galactosidase expression and genome copy number evaluated by TaqMan PCR.

Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes

The signaling of stem cell factor (SCF) and its receptor KIT (membrane-bound KIT; m-KIT) plays an important role in melanocyte development, survival, proliferation, and melanogenesis. It has been demonstrated in other systems that a soluble form of m-KIT released from the cell surface (s-KIT) regulates SCF signaling, although there have been no reports pertaining to the existence and the biological role of s-KIT in melanocytes. In this study, we therefore examined the involvement of s-KIT in melanogenesis. Western blotting analysis revealed that treatment with phorbol 12-myristate-13-acetate (PMA) or 4-aminophenylmercuric acetate (APMA) induced s-KIT production in cultured human melanocytes. Inhibitors of tumor necrosis factor-alpha-converting enzyme (TACE) and metalloproteinases (MMPs) muted this release of s-KIT into the media. Human recombinant s-KIT added to melanocytes inhibited SCF-induced phosphorylation of m-KIT, resulting in suppression of SCF-induced melanogenesis. Additionally, APMA-induced s-KIT production abolished SCF-induced melanogenesis as effectively as a KIT-neutralizing antibody. Concomitantly, APMA and TACE inhibitors significantly decreased and increased melanin synthesis, respectively, in an in vitro skin model. Taken together, these findings provided an insight into the elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggested that production of s-KIT contributes to the regulation of human skin pigmentation. Journal of Investigative Dermatology (2008) 128, 1763-1772; doi:10.1038/jid.2008.9; published online 31 January 2008.

Ovariectomy Accelerates Photoaging of Rat Skin

We have previously reported the formation of wrinkles, a decrease in skin elasticity and a loss in the linearity of dermal elastic fibers in rat hind limb skin irradiated with ultraviolet radiation in wavelength ranging 290–320 nm (UVB) at a suberythemal dose for 6 weeks. Estrogens are considered effective in preventing photoaging in postmenopausal females, but the role of estrogen in the skin remains unclear. In this study we have evaluated the influence of short‐term chronic UVB irradiation at a suberythemal dose on the skin of ovariectomized rats. An ovariectomy or a sham operation was performed on each 3 week–old female Sprague‐Dawley rat. Starting 1 week after the operation the hind limb skin of each rat was irradiated with UVB at a suberythemal dose (130 mJ/cm2) three times a week for 3 or 6 weeks. Decreases in elasticity and wrinkle formation in the skins of ovariectomized animals were induced more quickly than in the skins of sham‐operated animals following UVB irradiation. The linearity of elastic fibers in the ovariectomy group decreased significantly compared with the sham‐operation group, but erythema in the ovariectomy group was induced more readily than in the sham‐operation group following UVB irradiation. These findings suggest that decreases in the estrogen levels after ovariectomy accelerate photoaging in terms of the morphology and physical properties of the skin surface and the three‐dimensional structure of elastic fibers.

Interaction between stem cell factor and endothelin-1: effects on melanogenesis in human skin xenografts

The two paracrine melanogenic cytokines, stem cell factor (SCF) and endothelin-1 (ET-1), have been demonstrated to play pivotal roles in skin pigmentation including UVB-induced pigmentation and senile lentigo. However, little is known regarding their interactive effect on skin pigmentation. In order to investigate their roles in vivo, facultative pigmentation of human skin xenografts on severe combined immunodeficient (SCID) mice was assessed. After 1 week of acclimation in a pathogen-free barrier, dermatomed fresh cadaveric skin was surgically grafted onto the back of the mice and allowed to heal for 5–6 weeks prior to cytokine administration. Intradermal injections of SCF at 0.7 or 2.0 μg significantly increased skin pigmentation when compared to vehicle control. Despite the lack of a dose-dependent pigmentation response following ET-1 administration, the combination of 0.2 μg SCF and 0.1 μg ET-1 demonstrated a statistically significant increase in tyrosinase gene expression substantiated by the enhancement of melanin content and skin pigmentation compared to treatment with SCF alone or ET-1 alone. These findings establish an in vivo interaction between SCF and ET-1 with regard to their capacity to effect an increase in skin pigmentation.