GPNMB Extracellular Fragment Protects Melanocytes from Oxidative Stress by Inhibiting AKT Phosphorylation Independent of CD44.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that plays an important role in cancer metastasis and osteoblast differentiation. In the skin epidermis, GPNMB is mainly expressed in melanocytes and plays a critical role in melanosome formation. In our previous study, GPNMB was also found to be expressed in skin epidermal keratinocytes. In addition, decreased GPNMB expression was observed in the epidermis of lesional skin of patients with vitiligo. However, the exact role of keratinocyte-derived GPNMB and its effect on vitiligo is still unknown. In this study, we demonstrated that GPNMB expression was also decreased in rhododendrol-induced leukoderma, as seen in vitiligo. The extracellular soluble form of GPNMB (sGPNMB) was found to protect melanocytes from cytotoxicity and the impairment of melanogenesis induced by oxidative stress. Furthermore, the effect of rGPNMB was not altered by the knockdown of CD44, which is a well-known receptor of GPNMB, but accompanied by the suppressed phosphorylation of AKT but not ERK, p38, or JNK. In addition, we found that oxidative stress decreased both transcriptional GPNMB expression and sGPNMB protein expression in human keratinocytes. Our results suggest that GPNMB might provide novel insights into the mechanisms related to the pathogenesis of vitiligo and leukoderma.

Evaluation of subclinical chronic sun damage in the skin via the detection of long-lasting ultraweak photon emission.

BACKGROUND: It is well known that solar radiation accelerates skin photoaging. To evaluate subclinical photodamage in the skin especially from the early phase of ultraviolet (UV)-induced damage, we have focused on ultraweak photon emission (UPE), also called biophotons. Our previous study reported that the amount of long-lasting UPE induced by UV, predominantly from lipid peroxidation, is a valuable indicator to assess cutaneous photodamage even at a suberythemal dose, although it was only applied to evaluate acute UV damage. The aim of this study was to further investigate whether long-lasting UPE could also be a useful marker to assess subclinical chronic sun damage in the course of skin photoaging. MATERIALS AND METHODS: Forty-three Japanese females in their 20s were recruited and were divided into two groups according to their history of sun exposure based on a questionnaire (high- and low-sun-exposure groups). Several skin properties on the cheek and outer forearm were measured in addition to UV-induced UPE. RESULTS: Among the skin properties measured, water content, average skin roughness, and the lateral packing of lipids in the stratum corneum were significantly deteriorated in the high-sun-exposure group as were changes in some skin photoaging scores such as pigmented spots and wrinkles. In addition, those skin properties were correlated with the UPE signals, suggesting the possible impact of oxidative stress on chronic skin damage. CONCLUSION: Subtle oxidative stress detected by long-lasting UPE may contribute to subclinical cutaneous damage at the beginning phase of chronic sun exposure, which potentially enhances skin photoaging over a lifetime.

Exploitation of long-lasting ultraweak photon emission to estimate skin photodamage after ultraviolet exposure.

BACKGROUND: Establishing a noninvasive method to estimate skin damage immediately after ultraviolet (UV) exposure is required to minimize the anticipated severe symptoms triggered by early phase UV-induced reactions in the skin. To develop a suitable method, we focused on ultraweak photon emission (UPE) immediately after UV exposure to characterize the relationship of UPE to skin photodamage caused by the UV exposure. MATERIALS AND METHODS: Analysis of the correlation between UV-induced UPE and erythema formation characterized by skin redness was conducted in a clinical study. To clarify the source of UPE, time-dependent lipid oxidation was analyzed in human epidermal keratinocytes in vitro using a fluorescence indicator as well as the lipid hydroperoxide (LPO) assay. RESULTS: The average amount of UV-induced long-lasting UPE per second, especially from 1 to 3 minutes compared to other time periods after the UV radiation, increased in a dose-dependent manner and was highly correlated with the intensity of cutaneous redness 24 hours after UV exposure. In addition, cellular examinations elucidated that both the long-lasting UPE signals and the increased amounts of LPO 2 minutes after UV radiation were significantly suppressed by Trolox (a vitamin E derivative), which has been shown to inhibit UV-induced erythema formation in human skin. CONCLUSION: Long-lasting UPE generated between 1 and 3 minutes immediately after UV exposure, which is associated with LPO production, is a valuable indicator to estimate and/or avoid severe cutaneous photodamage.

Autophagy Declines with Premature Skin Aging resulting in Dynamic Alterations in Skin Pigmentation and Epidermal Differentiation.

Autophagy is a membrane traffic system that provides sustainable degradation of cellular components for homeostasis, and is thus considered to promote health and longevity, though its activity declines with aging. The present findings show deterioration of autophagy in association with premature skin aging. Autophagy flux was successfully determined in skin tissues, which demonstrated significantly decreased autophagy in hyperpigmented skin such as that seen in senile lentigo. Furthermore, an exacerbated decline in autophagy was confirmed in xerotic hyperpigmentation areas, accompanied by severe dehydration and a barrier defect, which showed correlations with skin physiological conditions. The enhancement of autophagy in skin ex vivo ameliorated skin integrity, including pigmentation and epidermal differentiation. The present results indicate that the restoration of autophagy can contribute to improving premature skin aging by various intrinsic and extrinsic factors via the normalization of protein homeostasis.

The Surprising Effect of Phenformin on Cutaneous Darkening and Characterization of Its Underlying Mechanism by a Forward Chemical Genetics Approach.

Melanin in the epidermis is known to ultimately regulate human skin pigmentation. Recently, we exploited a phenotypic-based screening system composed of ex vivo human skin cultures to search for effective materials to regulate skin pigmentation. Since a previous study reported the potent inhibitory effect of metformin on melanogenesis, we evaluated several biguanide compounds. The unexpected effect of phenformin, once used as an oral anti-diabetic drug, on cutaneous darkening motivated us to investigate its underlying mechanism utilizing a chemical genetics approach, and especially to identify alternatives to phenformin because of its risk of severe lactic acidosis. Chemical pull-down assays with phenformin-immobilized beads were performed on lysates of human epidermal keratinocytes, and subsequent mass spectrometry identified 7-dehydrocholesterol reductase (DHCR7). Consistent with this, AY9944, an inhibitor of DHCR7, was found to decrease autophagic melanosome degradation in keratinocytes and to intensely darken skin in ex vivo cultures, suggesting the involvement of cholesterol biosynthesis in the metabolism of melanosomes. Thus, our results validated the combined utilization of the phenotypic screening system and chemical genetics as a new approach to develop promising materials for brightening/lightening and/or tanning technologies.

Quantitative changes in the secretion of exosomes from keratinocytes homeostatically regulate skin pigmentation in a paracrine manner.

The content and distribution of melanin in the epidermis determines the wide variety of skin colors associated with ethnic/racial diversity. Although it was previously reported that qualitative changes in keratinocyte-derived exosomes regulate melanocyte pigmentation in vitro, their practical involvement, especially in skin color development in vivo, has remained unclear. To address this unexplained scientific concern, the correlation of epidermal exosomes isolated from human skin tissues with melanosomal protein expression levels was demonstrated in this study for the first time. After confirming the quantitative effect of human keratinocyte-derived exosomes on human melanocyte activation, even in the absence of ultraviolet B (UV-B) exposure, the impact of exosomes secreted from UV-B-irradiated keratinocytes on melanogenesis was consistently detected, which suggests their constitutive role in regulating cutaneous pigmentation. Additionally, both a specific exosome secretion inducer and a suppressor were consistently found to significantly control melanin synthesis in a co-culture system composed of keratinocytes and melanocytes as well as in an ex vivo skin culture system. These results suggest that quantitative changes, in addition to already known qualitative changes, in exosomes secreted from human epidermal keratinocytes homeostatically regulate melanogenic activity in a paracrine manner, which leads to skin color determination.

Correction to: Characterization of skin function associated with obesity and specific correlation to local/systemic parameters in American women.

Following publication of the original article [1], the authors identified an error. In the description in Fig. 1b the "solid line" "dashed line" should be exchanged. The original article has been updated.

Characterization of skin function associated with obesity and specific correlation to local/systemic parameters in American women.

BACKGROUND: Obesity is considered problematic not only as a major cause of diabetes, hypertension, and dyslipidemia, but also as a risk of intractable dermatosis; however influence of obesity on skin function has not been clarified. To clarify the mechanism of obesity-associated skin disorders, we aimed to characterize the skin function of subjects with obesity, and identify possible influencing factors. METHODS: Complex analyses including instrumental measurement, biochemical and lipidomics were performed for facial skin and physical evaluation in 93 Caucasian women with obesity (OB) and non-obesity (NOB). RESULTS: In OB, imbalance in metabolism of carbohydrate and lipid, autonomic nerve activity, and secreted factors were confirmed. In the skin properties in OB, surface roughness was higher by 70%, the water content was lower by 12%, and changes in the lipid profile of stratum corneum ceramide were observed; in particular, a 7% reduction of [NP]-type ceramide, compared with NOB. Moreover, significant redness accompanied by a 34% increase in skin blood flow was observed in OB. Correlation analysis elucidated that the water content was strongly correlated with local skin indices, such as the ceramide composition, redness, blood flow, and TNFα in the stratum corneum, whereas roughness was correlated with the systemic indices, such as serum insulin, leptin, and IL-6. CONCLUSIONS: Characteristics of obesity-associated skin were (A) reduction of the barrier and moisturizing function accompanied by intercellular lipid imbalance, (B) increased redness accompanied by hemodynamic changes, and (C) surface roughness. It was suggested that each symptom is due to different causes in local and/or systemic physiological impairment related to the autonomic nerve-vascular system, inflammation and insulin resistance.

Variation in Hsp70-1A Expression Contributes to Skin Color Diversity.

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes, and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either white or African American skin. Two-dimensional gel electrophoresis and mass spectrometry demonstrated that heat shock protein 70-1A (Hsp70-1A) protein levels were significantly higher in African American melanocytes compared with white melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70 family members in tyrosinase post-translational modification. In addition, pharmacologic inhibition and small interfering RNA-mediated downregulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes, and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes.

Cooperation of endothelin-1 signaling with melanosomes plays a role in developing and/or maintaining human skin hyperpigmentation.

Skin hyperpigmentation is characterized by increased melanin synthesis and deposition that can cause significant psychosocial and psychological distress. Although several cytokine-receptor signaling cascades contribute to the formation of ultraviolet B-induced cutaneous hyperpigmentation, their possible involvement in other types of skin hyperpigmentation has never been clearly addressed. Since our continuous studies using skin specimens from more than 30 subjects with ethnic skin diversity emphasized a consistent augmentation in the expression of endothelin-1 (ET-1) and its receptor (Endothelin B receptor, ET-B) in hyperpigmented lesions, including senile lentigos (SLs), the precise function of ET-1 signaling was investigated in the present study. In line with previous studies, ET-1 significantly induced melanogenesis followed by increases in melanosome transport in melanocytes and in its transfer to keratinocytes while inhibition of ET-B function substantially depressed melanogenic ability in tissue-cultured SLs. Additionally, in agreement with a previous report that the formation of autophagosomes rather than melanosomes is stimulated according to starvation or defective melanosome production, ET-1 was found to remarkably augment the expression of components necessary for early melanosome formation, indicating its counteraction against autophagy-targeting melanosome degradation in melanocytes. Despite the lack of substantial impact of ET-1 on keratinocyte melanogenic functions, the expression of ET-1 was enhanced following melanosome uptake by keratinocytes. Taken together, our data suggest that ET-1 plays a substantial role in the development and/or maintenance of skin hyperpigmentation in reciprocal cooperation with increased melanosome incorporation.

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.

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape.

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.

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-alpha (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.

Glucose 1-phosphate increases active transport of calcium in intestine.

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.