Possible Involvement of Dermal Fibroblasts in Modulating Nrf2 Signaling in Epidermal Keratinocytes.

Epidermal keratinocytes protect themselves by cooperating with neighboring cells against internal and external stresses, which leads not only to the maintenance of cell homeostasis but also to the prevention of skin aging. Although it is known that nuclear factor (NF)-E2-related factor 2 (Nrf2) signaling plays a pivotal role in ameliorating oxidative stress and inflammatory responses under stress situations, it is unclear whether Nrf2 signaling in keratinocytes cooperates with neighboring cells such as dermal fibroblasts. Thus, this study was conducted to examine the influence of dermal fibroblasts on Nrf2 signaling in epidermal keratinocytes using a co-culture system. The results show that expression levels of Nrf2-regulated antioxidant factors, such as glutathione and heme oxygenase-1, in HaCaT keratinocytes (HaCaT KCs) are up-regulated in the presence of normal human dermal fibroblasts (NHDFs). In addition, the secretion of pro-inflammatory molecules, including interleukin-1α (IL-1α) and prostaglandin E2 (PGE2), is suppressed in co-cultures of NHDFs and UVB-irradiated HaCaT KCs. Interestingly, the localization of Nrf2 protein in HaCaT KCs was immediately translocated from the cytoplasm to the nucleus after the co-culture with NHDFs. These results suggest the possibility that Nrf2 signaling in keratinocytes is regulated in cooperation with dermal fibroblasts.

[Case of Spindle Cell Carcinoma of the Breast Diagnosed as Mastitis].

The patient was a 51-year-old woman, who came to our hospital because of pain in her left breast and a tumor. US examination demonstrated a low echoic area with irregular margin and many high echoic spots within the left breast. We diagnosed mastitis. Therefore, we administered an antibiotic. One week later, her pain was improved. However, the low echoic area did not improve. Thus, we performed an aspiration cytology. Pus was aspirated. Cytology revealed a carcinoma. Enhanced CT demonstrated an irregular mass with ring enhancement in the left breast. A mastectomy with sentinel lymph- node biopsy and reconstruction with the latissimus dorsi muscle flap was performed. Post-operative course was uneventful. The pathological finding was of spindle cell carcinoma. After operation, she underwent chemotherapy(AC). Ten years later, she is alive with no recurrence.

Riboflavin Plays a Pivotal Role in the UVA-Induced Cytotoxicity of Fibroblasts as a Key Molecule in the Production of H2O2 by UVA Radiation in Collaboration with Amino Acids and Vitamins.

To investigate environmental factors that contribute to ultraviolet A (UVA)-induced oxidative stress, which accelerates the senescence and toxicity of skin cells, we irradiated human fibroblasts cultured in commonly used essential media with UVA and evaluated their viability and production of reactive oxygen species. The viability of fibroblasts exposed to a single dose of 3.6 J/cm2 UVA was not reduced when cultured in Hanks balanced salt solution, but it was significantly decreased when cultured in Dulbecco's modified Eagle's medium (DMEM), which contains various amino acids and vitamins. Furthermore, cell viability was not reduced when fibroblasts were cultured in DMEM and treated with a hydrogen peroxide (H2O2) scavenger such as glutathione or catalase added after UVA irradiation. In addition, we confirmed that the production of H2O2 was dramatically increased by UVA photosensitization when riboflavin (R) coexisted with amino acids such as tryptophan (T), and found that R with folic acid (F) produced high levels of H2O2 after UVA irradiation. Furthermore, we noticed that R and F or R and T have different photosensitization mechanisms since NaN3, which is a singlet oxygen quencher, suppressed only R and T photosensitization. Lastly, we examined the effects of antioxidants (L-ascorbic acid, trolox, L-cysteine, and L-histidine), which are singlet oxygen or superoxide or H2O2 scavengers, on R and F or on R and T photosensitization, and found that 1 mM ascorbic acid, Trolox, and L-histidine were strongly photosensitized with R, and produced significant levels of H2O2 during UVA exposure. However, 1 mM L-cysteine dramatically suppressed H2O2 production by UVA photosensitization. These data suggest that a low concentration of R-derived photosensitization is elicited by different mechanisms depending on the coexisting vitamins and amino acids, and regulates cellular oxidative stress by producing H2O2 during UVA exposure.

Dermal Fibroblasts Internalize Phosphatidylserine-Exposed Secretory Melanosome Clusters and Apoptotic Melanocytes.

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

Placental extracts regulate melanin synthesis in normal human melanocytes with alterations of mitochondrial respiration.

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese-dependent superoxide dismutase (MnSOD), a mitochondrial-resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract-induced regulation of melanogenesis.

The maximal cumulative solar UVB dose allowed to maintain healthy and young skin and prevent premature photoaging.

The young facial skin of children with a smooth healthy appearance changes over time to photoaged skin having mottled pigmentation, solar lentigines, wrinkles, dry and rough skin, leathery texture, and benign and malignant tumors after exposure to chronic, repeated solar radiation. The first sign of photoaging in Japanese subjects is usually solar lentigines appearing around 20 years of age on the face. Fine wrinkles can then appear after 30 years of age, and benign skin tumors, seborrhoeic keratoses, can occur after 35 years of age in sun-exposed skin. We theoretically calculated the maximal daily exposure time to solar radiation, which could prevent the development of photoaged skin until 60 and 80 years of age, based on published data of personal solar UVB doses in sun-exposed skin. One MED (minimal erythema dose) was determined to be 20 mJ/cm(2) , and 200 MED was used as the average yearly dose of Japanese children. Further, we hypothesized that the annual dose of Japanese adults is the same as that of the children. The cumulative UVB dose at 20 years of age was thus calculated to be 4000 MED, and 22 MED was used as the maximal daily UVB dose based on data measured in Kobe, located in the central area of Japan. We used the solar UVB dose from 10:00 a.m. to 14:00 p.m. which occupies 60% of the total daily UV dose, to obtain the maximal UVB per hour in a day, and calculated the maximal daily UV exposure time that would delay the onset of solar lentigines until 60 or 80 years of age. The mean daily sun exposure time to maintain healthy skin until 80 years of age in the summer was calculated to be 2.54 min (0.14 MED) for unprotected skin and 127 min with the use of a sunscreen of SPF (sun protection factor) of 50. In this study, we did not evaluate the photoaging effect of UVA radiation, but findings of the adverse effects of UVA radiation on the skin have accumulated in the last decade. Therefore, it will be important to estimate the maximal dose of solar UV radiation to retard the onset of photoaging based on an evaluation of both solar UVB and UVA in the future. Finally, we expect that this study may contribute to keeping Japanese and other types of skin young and healthy by limiting the exposure of the skin to solar radiation outdoors during the day.

A Single Intradermal Injection of Autologous Adipose-Tissue-Derived Stem Cells Rejuvenates Aged Skin and Sharpens Double Eyelids.

Facial skin aging is the most visible manifestation of aging in the body. In this study, we aimed to rejuvenate aging skin via a one-time intradermal injection of autologous adipose-derived stem cells (ADSCs). Eight patients were enrolled for study. Photographs of patients taken immediately before and 1, 3, 6, and 12 months after ADSC injections were comparatively evaluated for visible skin manifestations. ADSCs were cultured from the abdominal-skin-derived subcutaneous fat tissue, and 1 × 108 cultured ADSCs were injected intradermally into the facial skin. Cultured myoblasts were incubated with the supernatant derived from ADSCs, and the effect was evaluated via glucose consumption and lactic acid production in the medium. Eight cases showed the shallowing and disappearance of wrinkles, including those of the glabella, lower eyelids, crow`s feet, and forehead and nasolabial grooves, a month to several months after treatment. Double eyelids became prominent, and facial pores significantly reduced in size. These effects lasted for over one year. Myoblasts cultured in the presence of an ADSC-derived exosome were activated compared to that of ADSCs cultured without supernatant. The result supports the role of muscle in ADSC skin rejuvenation. The present study first reports that a single intradermal administration of cultured ADSCs rejuvenates aged facial skin over the course of one year. Further, patients exhibited definite double eyelids and pore shrinkage, strongly indicating the active involvement of muscle, which was supported by an in vitro study. Our study also suggested the important role of biological factors delivered from injected stem cells, although the detailed mechanism of rejuvenation effects of ADSC skin injection remains to be clarified.

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis.

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.

Pigmentation and TYRP1 expression are mediated by zinc through the early secretory pathway-resident ZNT proteins.

Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are essential for pigmentation. They are generally classified as type-3 copper proteins, with binuclear copper active sites. Although there is experimental evidence for a copper cofactor in TYR, delivered via the copper transporter, ATP7A, the presence of copper in TYRP1 and TYRP2 has not been demonstrated. Here, we report that the expression and function of TYRP1 requires zinc, mediated by ZNT5-ZNT6 heterodimers (ZNT5-6) or ZNT7-ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in hypopigmentation in medaka fish and human melanoma cells, and is accompanied by immature melanosomes and reduced melanin content, as observed in TYRP1 dysfunction. The requirement of ZNT5-6 and ZNT7 for TYRP1 expression is conserved in human, mouse, and chicken orthologs. Our results provide novel insights into the pigmentation process and address questions regarding metalation in tyrosinase protein family.