sPmel17 Secreted by Ultraviolet B-Exposed Melanocytes Alters the Intercellular Adhesion of Keratinocytes.

Repigmentation of the skin in patients with vitiligo represents an intricate process in which the depigmented epidermis is replenished by functional melanocytes (MCs) that migrate from undamaged hair follicles and/or surrounding areas. We characterized whether MCs release a secreted form of Pmel17 (sPmel17) protein after exposure to UVB, thereby weakening the cell-cell adhesions of keratinocytes (KCs), which provides MCs the opportunity to migrate to areas devoid of MCs. At first, we examined the interactions of sPmel17 and FHL2 (four-and-a-half LIM domain protein 2) in KCs treated with the conditioned media (CM) from MCs exposed to UVB. The results showed that both the protein and mRNA levels of FHL2 were significantly upregulated in KCs treated with sPmel17-enriched CM from UVB-exposed MCs. We also found that there are physical interactions between sPmel17 and FHL2 as analyzed by reciprocal coimmunoprecipitation assays and double immunofluorescence staining. The CM from UVB-exposed MCs signaled KCs to remodel the actin cytoskeleton and reduce E-cadherin expression. However, the CM from UVB-exposed and Pmel17-silenced or from UVB-unexposed MCs failed to do this. To further determine the in situ distributions of sPmel17, FHL2, and E-cadherin, we examined the expression profiles of those proteins in the skin from healthy subjects and from depigmented or repigmented vitiligo using immunofluorescence and immunohistochemical staining. The results showed that the expression of sPmel17 was positively correlated with FHL2 but not to E-cadherin. The colocalization of FHL2 and sPmel17 was also observed in UVB-exposed mouse tail skin. Together, the upregulation of FHL2 in KCs requires stimulation by sPmel17 secreted from MCs and activation of the sPmel17-FHL2-E-cadherin axis offers a potential therapeutic target to expedite the repigmentation process in patients with vitiligo.

Metformin Promotes the Hair-Inductive Activity of Three-Dimensional Aggregates of Epidermal and Dermal Cells Self-Assembled in vitro.

INTRODUCTION: Although it has been reported that the antidiabetic drug metformin has multiple extra-hypoglycemic activities, such as anti-oxidation, antiaging, and even antitumor, topical metformin also can induce hair regeneration, but the precise mechanism involved in that process is still unclear. OBJECTIVES: The aim of this study was to assess the effect of metformin on hair growth in a mouse hair-follicle reconstitution model generated by in vitro self-assembled three-dimensional aggregates of epidermal and dermal cells (DCs) (3D aggregates). METHODS: Epidermal cells and DCs were isolated and cultured from the mouse skin of 50 C57BL/6 mouse pups (1-day-old). For tracing the distribution of DCs during the self-assembly process of 3D aggregates, the DCs were labeled with Vybrant Dil Cell-Labeling Solution and mixed with epidermal cells at a 1:1 ratio. Formed 3D aggregates were treated with 10 mM metformin and then were grafted into recipient BALB/c nude mice. The biomarkers (hepatocyte growth factor [HGF], prominin-1 [CD133], alkaline phosphatase [ALP], ß-catenin, and SRY-box transcription factor 2 [SOX2]) associated with the hair-inductive activity of DCs were detected in the grafted skin tissues and in cultured 3D aggregates treated with metformin using immunofluorescent staining, quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Furthermore, the expression levels of CD133 were also examined in DCs with different passage numbers using qRT-PCR and Western blotting. RESULTS: Metformin directly stimulates the activity of ALP of cultured 3D aggregates, upregulates both the protein and mRNA expression levels of molecular markers (HGF, CD133, ALP, ß-catenin, and SOX2), and improves the survival rate of reconstituted hair follicles. Moreover, we also found that metformin increases the expression of CD133 in DCs thus maintaining their trichogenic capacity that would normally be lost by serial subculture. CONCLUSIONS: These results suggest that metformin can promote hair follicle regeneration in vitro through upregulation of the hair-inductive capability of DCs, warranting further evaluation in the clinical treatment of male or female pattern hair loss.

Pro-pigmentary action of 5-fluorouracil through the stimulated secretion of CXCL12 by dermal fibroblasts.

BACKGROUND: There is growing evidence that 5-fluorouracil (5-FU) combined with therapeutic trauma can effectively induce skin repigmentation in vitiligo patients who are unresponsive to conventional treatments. Previous studies have mainly focused on identifying the antimitotic activity of 5-FU for the treatment of skin cancer, but few studies have investigated its extra-genotoxic actions favoring melanocyte recruitment. METHODS: We utilized the full thickness excisional skin wound model in Dct-LacZ transgenic mice to dynamically assess the migration of melanocytes in the margins of wounds treated with or without 5-FU. The in-situ expression of CXCL12 was examined in the wound beds using immunofluorescence staining. Quantitative real-time polymerase chain reaction and Western blotting analyses were performed to detect the expression levels of CXCL12 mRNA and protein in primary mouse dermal fibroblasts treated with or without 5-FU. Transwell assays and fluorescein isothiocyanate (FITC)-phalloidin staining were used to observe cell migration and filamentous actin (F-actin) changes of melan-a murine melanocytes. RESULTS: Whole mount and cryosection X-gal staining showed that the cell numbers of LacZ-positive melanocytes were much higher in the margins of dorsal and tail skin wounds treated with 5-FU compared with the controls. Meanwhile, CXCL12 immunostaining was significantly increased in the dermal compartment of wounds treated with 5-FU (control vs. 5-FU, 22.47 ±â€Š8.85 vs. 44.69 ±â€Š5.97, P < 0.05). Moreover, 5-FU significantly upregulated the expression levels of CXCL12 mRNA (control vs. 5-FU, 1.00 ±â€Š0.08 vs. 1.54 ±â€Š0.06, P < 0.05) and protein (control vs. 5-FU, 1.00 ±â€Š0.06 vs. 2.93 ±â€Š0.10, P < 0.05) in cultured fibroblasts. Inhibition of the CXCL12/CXCR4 axis suppressed melanocyte migration in vitro using a CXCL12 small interfering RNA (siRNA) or a CXCR4 antagonist (AMD3100). CONCLUSION: 5-FU possesses a pro-pigmentary activity through activation of the CXCL12/CXCR4 axis to drive the chemotactic migration of melanocytes.

Taurine up-regulated gene 1 and disease development. / ç‰›ç£ºé ¸ä¸Šè°ƒåŸºå› 1ä¸Žç–¾ç— çš„å ³ç³».

Long non-coding RNA (lncRNA) have been attracted attention due to its role in many diseases. Taurine up-regulated gene 1(TUG1)is a non-coding RNA of 7.1 kb in length, which locates on chromosome 22q12. More and more studies have found that TUG1 not only participates in the occurrence and development of tumors, but also plays an important role in the progression of diseases in cardiovascular system, endocrine system, nervous system and so on. It is expected to become the therapeutic targets and indicators for evaluating prognosis of a variety of diseases such as diabetes, myocardial ischemia, osteoarthritis, atherosclerosis and so on.

The Long Noncoding RNA UCA1 Negatively Regulates Melanogenesis in Melanocytes.

The long noncoding RNA UCA1 was first discovered in bladder cancer and is known to regulate the proliferation and migration of melanoma. However, its role in melanogenesis is unclear. In this study, we aimed to explore the role and mechanism of UCA1 in melanogenesis. Our findings showed that the expression of UCA1 was negatively correlated with melanin content in melanocytes and pigmented nevus. Overexpression of UCA1 in melanocytes decreased melanin content and the expression of melanogenesis-related genes, whereas knockdown of UCA1 in melanocytes had the opposite effect. High-throughput sequencing revealed that microphthalmia-associated transcription factor (MITF), an important transcription factor affecting melanogenesis, was also negatively correlated with the expression of UCA1. Furthermore, the transcription factor CRE-binding protein (CREB), which promotes MITF expression, was negatively regulated by UCA1. The cAMP/protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) signaling pathways, which are upstream of the CREB/MITF/melanogenesis axis, were activated or inhibited in response to silencing or enhancing UCA1 expression, respectively. In addition, enhanced UCA1 expression downregulates the expression of melanogenesis-related genes induced by UVB in melanocytes. In conclusion, UCA1 may negatively regulate the CREB/MITF/melanogenesis axis through inhibiting the cAMP/PKA, ERK, and JNK signaling pathways in melanocytes. UCA1 may be a potential therapeutic target for the treatment of pigmented skin diseases.

The role and mechanism of Asian medicinal plants in treating skin pigmentary disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Chloasma, senile plaques, vitiligo and other pigmentary disorders seriously affect patients' appearance and life quality. Medicinal plant is the product of long-term medical practice worldwide, with the advantages of outstanding curative properties and less side effects. Recently, research were made to explore the value of medicinal plants in the treatment of pigmentary disorders, and remarkable results were achieved. AIM OF THE REVIEW: This review outlines the current understanding of the role and potential mechanisms of medicinal plants (including active ingredients, extracts and prescriptions) in pigmentary disorders, especially Chinese medicinal plants, provides the preclinical evidence for the clinical benefits. This study hopes to provide comprehensive information and reliable basis for exploring new therapeutic strategies of plant drugs in the treatment of skin pigmented diseases. METHODS: The literature information was obtained from the scientific databases (up to Oct, 2017), mainly from the PubMed, Web of Science and CNKI databases, and was to identify the experimental studies on the regulating melanogenesis role of the active agents from herbal medicine and the involved mechanisms. The search keywords for such work included: "pigmentary" or "pigmentation", "melanogenesis", and "traditional Chinese medicine" or "Chinese herbal medicine", "herb", "medicinal plant". RESULTS: We summarized the function of medicinal plants involved in melanogenesis, especially Chinese medicine. It was reported that the active ingredients, extracts, or prescriptions of medicinal plants can regulate the expression of genes related to melanogenesis by affecting the signaling pathways such as MAPK and PKA, thereby regulating pigment synthesis. Some of them can promote melanogenesis (such as isoliquiritigenin, geniposide; Cornus officinalis Siebold & Zucc., Eclipta prostrata (L.) L.; the Bairesi complex prescription, etc.). While others have the opposite effect (such as biochanin A, Gomisin N; Panax ginseng C.A. Meyer, Nardostachys chinensis Bat.; Sanbaitang, etc.). CONCLUSION: Asian medicinal plants, especially their active ingredients, have multilevel effects on melanogenesis by regulating melanogenesis-related genes or signaling pathways. They are of great clinical value for the treatment of skin pigmentary disorders. However, the experimental effect, safety, and functional mechanism of the medicinal plants require further determination before studying their clinical efficacy.

Ganoderma lucidum polysaccharide reduces melanogenesis by inhibiting the paracrine effects of keratinocytes and fibroblasts via IL-6/STAT3/FGF2 pathway.

Our previous study found that Ganoderma lucidum polysaccharide (GLP), bioactive ingredients from Ganoderma lucidum, protected fibroblasts from photoaging. However, whether GLP can affect melanogenesis in melanocytes through regulating paracrine mediators that secreted by keratinocytes and fibroblasts is unclear. We aimed to investigate the efficacy and mechanisms of action of GLP in melanogenesis by regulating paracrine effects of keratinocytes and fibroblasts. The effect of GLP on cell viability affected by GLP was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After an immortal keratinocyte line (HaCaT) and primary fibroblasts (FB) were treated with GLP, the supernatants of HaCaT and FB cells were collected and cocultured with an immortalized melanocyte line (PIG1). The expression levels of melanogenesis-associated genes in PIG1 cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Furthermore, FRS-2, ERK, JNK, and p38 phosphorylation levels were measured. Then, major melanogenic paracrine mediators in HaCaT and FB cells treated with GLP were evaluated by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). In addition, the expression of IL-6 and STAT3 was examined in HaCaT and FB cells. GLP was not cytotoxic to HaCaT and FB cells. The supernatants of GLP-treated HaCaT and FB cells downregulated the expression levels of MITF, TYR, TYRP1, TYRP2, RAB27A, and FSCN1 genes and inhibited the phosphorylation of FRS-2, ERK, JNK, and p38 in PIG1 cells. GLP also decreased FGF2 secretion in HaCaT and FB cells. Moreover, GLP reduced IL-6 expression and STAT3 phosphorylation in HaCaT and FB cells. GLP reduced melanogenesis in melanocytes by inhibiting the paracrine effects of keratinocytes and fibroblasts via IL-6/STAT3/FGF2 pathway.

Downregulation of TUG1 promotes melanogenesis and UVB-induced melanogenesis.

Studies have revealed that taurine upregulated gene 1 (TUG1), an important member of the long non-coding RNA family, is involved in the regulation of cell growth, tumorigenesis and invasion, insulin secretion and so on. However, its role in melanogenesis has not been explored. This study attempts to explore the effects of TUG1 on melanogenesis and its regulatory mechanisms. We evaluated the expression changes in melanogenesis-related genes and detected phosphorylation levels of ERK, JNK and P38 in TUG1 downregulated melanocytes. After exposure of melanocytes to UVB irradiation, the expression of TUG1 and melanogenesis-related genes was detected. We found that the expression of tyrosinase (TYR), tyrosine-related protein 1 (TYRP1) and tyrosine-related protein 2 (TYRP2) was upregulated and that the phosphorylation level of ERK was downregulated by downregulating TUG1. Inhibition of TUG1 could further upregulate the expression of UVB-induced melanogenesis-related genes. In conclusion, TUG1 negatively regulates melanocyte melanogenesis via the ERK pathway and plays a negative role in UVB-induced melanogenesis.

Ganoderma lucidum polysaccharide inhibits UVB-induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways.

Ultraviolet (UV)-induced pigmentation is very common in clinical practice, but the current treatments are rarely effective, accompanied by some side effects. Ganoderma lucidum polysaccharide (GLP) is a natural antioxidant with no toxic side effects, which can antagonize UVB-induced fibroblast photo aging. The study aims to explore the role of GLP in inhibiting UVB-induced melanogenesis and its possible mechanism. The expression of melanogenesis genes such as microphthalmia-associated transcription factor (MITF), tyrosine (TYR), tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2), ras-related protein Rab-27A (Rab27A), and Myosin shows an upward trend after exposure of B16F10 and PIG1 cells to UVB irradiation, but GLP can downregulate the expression of genes related to UVB-induced melanogenesis. GLP can inhibit UVB-activated protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signaling pathways. Besides, GLP protects mitochondria from UVB damage and inhibits reactive oxygen species (ROS) production. Also, UVB-induced cyclic adenosine monophosphate (cAMP) can be inhibited. It has been found in the experiments of UVB-induced skin pigmentation in zebrafish that GLP is capable of inhibiting UVB-induced skin pigmentation. Meanwhile, it can greatly relieve erythema reaction in guinea pig skin caused by high-dosage UVB irradiation. In conclusion, this study shows that GLP can inhibit UVB-induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways and is a potential natural safe whitening sunscreen additive.

UVB-inhibited H19 activates melanogenesis by paracrine effects.

The long noncoding RNA H19 was reported to associate with melanogenesis. However, it remains unknown whether H19 expression will be changed by UVB irradiation and whether H19 will regulate melanocytes melanogenesis by paracrine effects. Here, we analysed the expression changes of H19 irradiated by UVB in keratinocytes and explored the mechanism of melanogenesis stimulated by H19 through paracrine effects. First, after keratinocytes were exposed to UVB irradiation, expression of H19 and pro-opiomelanocortin (POMC) was measured by qRT-PCR. Also, α-melanocyte-stimulating hormone (α-MSH) contents in cells supernatant were measured by ELISA. Then, H19 siRNAs were designed and transfected into keratinocytes by liposome. The expression changes of H19, POMC and α-MSH were detected. Besides, expression of p53 was detected by Western blot. After that, supernatant of keratinocytes with H19 siRNAs or negative control siRNA was cocultured with immortalized melanocyte line PIG1. Expression levels of MiTF, TYR, Rab27A, TYRP2, FSCN1 and MYO5A in PIG1 cells were detected by Western blot and qRT-PCR. We found that H19 expression of keratinocytes cells decreased after UVB irradiation. However, the levels of POMC, α-MSH and p53 were upregulated in UVB-irradiated cells. Compared with the negative control, H19 siRNAs could significantly increase the expression of POMC, α-MSH and p53. After supernatant of keratinocytes transfected with H19 siRNAs was cocultured with PIG1 cells, the levels of MiTF, TYR and Rab27A were upregulated in PIG1 cells. In conclusion, UVB-inhibited H19 may promote α-MSH secretion by p53 in keratinocytes and then regulate melanocytes melanogenesis through paracrine effects.