Yoshinori Takema

Age-related changes in the elastic properties and thickness of human facial skin

Using recently designed, commercially available, non‐invasive instruments, we measured the thickness and elasticity of the skin of the face and ventral forearm in 170 women, and evaluated the effects of age and exposure to sunlight. Skin thickness decreased with age in ventral forearm skin, which has limited exposure to sunlight, but increased significantly in the skin of the forehead, corners of the eyes, and cheeks, which are markedly exposed to sunlight. Skin elasticity (Ur/Uf) decreased with age on both the face and forearm. The ratio of viscosity element to elasticity element (Uv/Ue) increased with age at all sites. However, delayed distension (Uv), immediate retraction (Ur), final distension (Uf), and immediate distention (Ue), as individual elements, decreased on the face and increased on the forearm with age. This tendency was more marked after correction for skin thickness. These results suggested the specificity of skin thickness and elasticity in the facial skin. Analysis using a four‐element model showed no changes in the elasticity coefficient of Maxwell element on the forearm, but an increase on the face. This indicates quantitative or qualitative changes in elastic fibres in facial skin. Thus, sunlight appears to have a considerable effect on the thickness and physical properties of facial skin.

Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of α-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C18)-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.

The Role of Elastases Secreted by Fibroblasts in Wrinkle Formation: Implication Through Selective Inhibition of Elastase Activity ¶

Abstract We have previously demonstrated that decreases in skin elasticity, accompanied by increases in the tortuosity of elastic fibers, are important early events in wrinkle formation. In order to study the role of elastases in the degeneration of elastic fibers during wrinkle formation we examined the effects of an inhibitor of skin fibroblast elastase, N-phenethylphosphonyl-l-leucyl-l-tryptophane (NPLT), on wrinkle formation in hairless mice skin following UV irradiation. Dorsal skins of hairless mice were exposed daily to UV light for 18 weeks at doses of 65–95 mJ/cm2 and treated topically with 100 μL of 1 mM NPLT immediately after each UV irradiation. Wrinkles on dorsal skins were evaluated from week 6 through week 18. The daily exposure of mouse skin to UV light with less than 1 minimal erythemal dose significantly enhanced the activity of elastase in the exposed skin by week 4, and the elevated levels of elastase activity were significantly reduced by the in vitro incubation with NPLT in a dose-dependent manner to a level similar to that in unexposed mice skin, indicating that NPLT can efficiently inhibit the UV-inducible elastase activity. Topical application of NPLT significantly suppressed wrinkle formation when compared with vehicle controls by week 15 of treatment (P < 0.05). Histochemistry of elastic fibers with Orcein staining demonstrated that there were no obvious decreases of the fine elastic fibers in UV-exposed NPLT-treated skin in contrast to their marked decreases in the UV-exposed vehicle-treated skin. These findings suggest that skin fibroblast elastase plays a decisive role in wrinkle formation through the degeneration of elastic fiber.

Age-related changes in skin wrinkles assessed by a novel three-dimensional morphometric analysis.

Summary Background A system has been developed whereby the morphology of the skin surface can be evaluated directly in three dimensions. This system employs a non‐invasive device that utilizes white light of halogen origin, and which allows the computation of wrinkle depth and width, and other parameters of skin surface morphology. Using innovative engineering, an optical system has been devised so that light is transmitted via a slit and can be used to measure not only replicas of the skin but also the skin surface directly. The measurement area is 6·4 × 6·4 mm, and the theoretical resolution with a × 50 magnification lens is within 12·5 µm.

Biphasic Expression of Two Paracrine Melanogenic Cytokines, Stem Cell Factor and Endothelin-1, in Ultraviolet B-Induced Human Melanogenesis

Stem cell factor (SCF) and endothelin-1 (ET-1) have been reported to be up-regulated at the protein and gene levels in human epidermis after ultraviolet B (UVB) irradiation and to play central roles in UVB-induced pigmentation. However, little is known about the time sequence of SCF and ET-1 expression in UVB-exposed human epidermis and the coordination of their roles during epidermal pigmentation. To clarify such parameters in UVB-exposed human skin, we measured the expression patterns of SCF and ET-1 (as well as of their corresponding receptors) at the gene level at various times during UVB-induced human pigmentation. When human forearm skin was exposed to UVB radiation at two minimal erythemal doses, the expression of SCF mRNA transcripts was significantly enhanced at 3 days after irradiation with an early decrease and subsequently constant expression of SCF receptor (c-KIT) mRNA transcripts. In contrast, up-regulation of ET-1 and endothelin B receptor (ET(B)R) mRNA expression was synchronized at 5 to 10 days after irradiation in concert with an increased expression of tyrosinase mRNA transcripts and the increase in pigmentation. In parallel the expression of tyrosinase and ET(B)R proteins as well as ET-1 was up-regulated at 7 to 10 days after irradiation, whereas KIT protein decreased at 3 days after irradiation and returned to the nonirradiated control level at 5 days after irradiation. When cultured human melanocytes were treated with human recombinant SCF, ET(B)R protein expression and the binding of (125)I-labeled ET-1 to the ET(B)R were significantly increased, further suggesting the preferential and coordinated role of early expression of SCF in UVB-induced melanogenesis. These findings suggest that SCF/KIT signaling is predominantly involved in the early phase of UVB-induced human pigmentation during which it stimulates the ET-1/ET(B)R linkage that is associated with the later phase of UVB-induced melanogenesis.

Autophagy Has a Significant Role in Determining Skin Color by Regulating Melanosome Degradation in Keratinocytes

Melanin in the epidermis determines the wide variation in skin color associated with ethnic skin diversity. Ethnic differences exist regarding melanosome loss in keratinocytes, but the mechanisms underlying these differences, and their contribution to the regulation of skin color, remain unclear. Here, we explored the involvement of autophagy in determining skin color by regulating melanosome degradation in keratinocytes. Keratinocytes derived from Caucasian skin exhibit higher autophagic activity than those derived from African American (AA) skin. Furthermore, along with the higher autophagy activity in Caucasian skin-derived keratinocytes compared with AA skin-derived keratinocytes, Caucasian skin-derived keratinocytes were more sensitive to melanosome treatment as shown by their enhanced autophagic activity, which may reflect the substantial mechanisms in the human epidermis owing to the limitations of the models. Melanosome accumulation in keratinocytes was accelerated by treatment with lysosomal inhibitors or with small interfering RNAs specific for autophagy-related proteins, which are essential for autophagy. Furthermore, consistent with the alterations in skin appearance, the melanin levels in human skin cultured ex vivo and in human skin substitutes in vitro were substantially diminished by activators of autophagy and enhanced by the inhibitors. Taken together, our data reveal that autophagy has a pivotal role in skin color determination by regulating melanosome degradation in keratinocytes, and thereby contributes to the ethnic diversity of skin color.

Inhibition of ultraviolet‐B‐induced wrinkle formation by an elastase‐inhibiting herbal extract: implication for the mechanism underlying elastase‐associated wrinkles

Background  Previously, we have demonstrated that fibroblast‐derived elastase plays an essential role in the increased three‐dimensional tortuosity of elastic fibers, contributing to the loss of skin elasticity in UV‐B‐exposed skin. This decrease in skin elasticity is closely associated with the formation of wrinkles induced by UV exposure.

Dermal fluid translocation is an important determinant of the diurnal variation in human skin thickness.

Background Ultrasonography has been used as a non‐invasive approach to measure skin thickness. To date there have been no studies on diurnal variations in skin thickness.

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.