DKK3 Regulates Melanogenesis in Melanocytes of Alpaca / 中国生物化学与分子生物学报

Dickkopf-3 (DKK3) , as a critical inhibitor of the Wnt/p-catenin signaling pathway, may he involved in melanogenesis.In the current study, we investigated the effects of DKK3 on melanogenesis in melanocytes of alpaca.Overexpression of DKK3 in alpaca melanocytes, the expression of Wntl, Lefl , Myc and the major target genes termed microphthalmia-associated transcription factor (M1TF) and its downstream genes, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase- related protein 2 (TYRP2) were significantly decreased at both mRNA and protein levels (P<0.05); total alkali melanin, pheomelanin and eumelanin were decreased by 80.30%, 72.17% and 64.60% (P <0.05), respectively.In contrast, in the melanocytes transfected with siRNA-DKK3 (a small interference RNA targeting DKK3) , the expression of Wntl, Lefl, Myc, MITF, TYR, TYRPl and TYRP2 were significantly increased at both mRNA and protein levels (P<0.05) ; total alkali melanin, pheomelanin and eumelanin were significantly increased by 1.65 folds, 1.25 folds and 1.21 folds (P< 0.05) , respectively.These results indicate that DKK3 regulates melanogenesis in alpaca melanocytes via the Wnt/p-catenin signaling pathway and down-regulates MITF.

MicroRNA-100-5p Regulates Melanogenesis in Alpaca Melanocytes by Targeting FGF21 / 中国生物化学与分子生物学报

Although many microRNAs (miRNAs) are known to function as regulators of coat color and melanogenesis, the underlying molecular mechanisms of miR-100-5p governing melanogenesis were not completely known.The goal of this study was to determine the effect of miR-l()()-5p on melanogenesis in alpaca melanocytes.Fibroblast growth factor 21 (FGF21) is a predicted target gene of miR-100-5p and the luciferase reporter assay demonstrated that miR-100-5p regulates FGF21 by binding to its 3' untranslated region (3'UTR).In this study, alpaca melanocytes were transfected with miR-100-5p, inhibitor and negative control plasmid.Results showed that miR-100-5p overexpression significantly decreased mRNA and protein expression of FGF2\.Meanwhile, the ERK signal pathway was inhibited, with subsequent up-regulation of microphthalmia-associated transcription factor (MITF) , tyrosinase (TYR) and tyrosinase-related protein 2 (TYRP2), which increased melanin production.The results suggest that miR-100-5p may regulate melanogenesis by targeting FGF21 via extracellular regulated MAP kinase (ERK) signaling pathway.

Research progress in melanogenesis signaling pathway / 生物工程学报

Melanogenesis is a biosynthetic pathway to produce melanin pigment in melanocyte, involving a series of intricate enzymatic and chemical catalyzed reactions. Melanogenesis involves five signaling pathways that converge on microphthalmia-associated transcription factor. In addition, many cytokines, involved in the regulation of melanogenesis, play an important role in the development, proliferation, differentiation and migration of melanocytes. Polyoxometalate can be used as a potential inhibitor of melanin production. Hence, this paper reviews the signaling pathways of melanogenesis and their regulatory mechanism, to apply polyoxometalates in the melanin production pathway, and briefly introduces the regulatory factors of related pathways.

Effect of latanoprost on cell proliferation of and melanogenesis in human epidermal melanocytes and its mechanism / 中华皮肤科杂志

Objective To evaluate the effect of latanoprost on cell proliferation of and melanogenesis in human epidermal melanocytes,and to explore its mechanism.Methods Latanoprost was added into the 254 medium to prepare latanoprost solutions at different concentrations of 10-5,10-6 and 10-7 mol/L.In vitro cultured human epidermal melanocytes were divided into 4 groups to be cultured with media containing no latanoprost (control group) or 10-5,10-6 and 10-7 mol/L latanoprost for 48 hours.Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative activity of melanocytes,dopa oxidation assay to estimate the activity of tyrosinase.Sodium hydroxide (NaOH)-lysis method was used to determine the content of melanin,and Masson-Fontana staining to observe the number and distribution of melanin granules.Westernblot analysis and real-time fluorescence-based quantitative PCR were performed to determine the protein and mRNA expression of melanogenesis-related genes including microphthalmia-associated transcription factor (MITF),tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1).Comparison among the 4 groups and multiple comparisons were done by using one-way analysis of variance and least significant difference (LSD)-t test.Results Compared with the control group,the 10-6-,10-5-mol/L latanoprost groups showed significantly increased proliferative activity of melanocytes (1.064 ± 0.172 and 1.078 ± 0.080 vs.0.784 ± 0.015;t =3.289,3.454 respectively,both P ＜ 0.05),increased activity of tyrosinase (0.510 ± 0.017 and 0.454 ± 0.009 vs.0.355 ± 0.041;t =6.139,3.939 respectively,P ＜ 0.01 or 0.05),and increased content of melanin (t =7.232,5.967,both P ＜ 0.01).However,there were no significant differences in the proliferative activity of melanocytes,activity of tyrosinase or content of melanin between the 10-7-mol/L latanoprost group and control group (all P ＞ 0.05).Masson-Fontana staining showed more and darker melanin granules on melanocyte dendrites in the 10-5-,10-6-,10-7-mol/L latanoprost groups than in the control group,and the color of melanin granules changed from light brown to black brown along with the increase in the concentration of latanoprost.The mRNA expression of MITF increased along with the increase in the concentration of latanoprost (P ＜ 0.01),and the protein expression of MITF wassignificantly higher in the 10-6,10-5-mol/L latanoprost groups than in the control group and 10-7-mol/L latanoprost group (all P ＜ 0.01).The 10-6-mol/L latanoprost group showed significantly increased mRNA and protein expression of TYR and TYRP1 compared with the control group,10-7-,10-5-mol/L latanoprost groups (all P ＜ 0.01).Conclusion Latanoprost can increase the proliferation of human epidermal melanocytes,and promote tyrosinase activity and melanogenesis likely by enhancing the mRNA and protein expression of MITF,TYR,TYRP1.

Sageretia thea fruit extracts rich in methyl linoleate and methyl linolenate downregulate melanogenesis via the Akt/GSK3β signaling pathway

BACKGROUND/OBJECTIVES: Sageretia thea is traditionally used as a medicinal herb to treat various diseases, including skin disorders, in China and Korea. This study evaluated the inhibitory effect of Sageretia thea fruit on melanogenesis and its underlying mechanisms in B16F10 mouse melanoma cells. The active chemical compounds in anti-melanogenesis were determined in Sageretia thea. MATERIALS/METHODS: Solvent fractions from the crude extract were investigated for anti-melanogenic activities. These activities and the mechanism of anti-melanogenesis in B16F10 cells were examined by determining melanin content and tyrosinase activity, and by performing western blotting. RESULTS: The n-hexane fraction of Sageretia thea fruit (HFSF) exhibited significant anti-melanogenic activity among the various solvent fractions without reducing viability of B16F10 cells. The HFSF suppressed the expression of tyrosinase and tyrosinase-related protein 1 (TRP1). The reduction of microphthalmia-associated transcription factor (MITF) expression by the HFSF was mediated by the Akt/glycogen synthase kinase 3 beta (GSK3β) signaling pathway, which promotes the reduction of β-catenin. Treatment with the GSK3β inhibitor 6-bromoindirubin-3'-oxime (BIO) restored HFSF-induced inhibition of MITF expression. The HFSF bioactive constituents responsible for anti-melanogenic activity were identified by bioassay-guided fractionation and gas chromatography-mass spectrometry analysis as methyl linoleate and methyl linolenate. CONCLUSIONS: These results indicate that HFSF and its constituents, methyl linoleate and methyl linolenate, could be used as whitening agents in cosmetics and have potential for treating hyperpigmentation disorders in the clinic.

Mechanism for synergistic effect of IRF4 and MITF on tyrosinase promoter / 中南大学学报(医学版)

Objective:To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR)promoter.Methods:The synergistic transcriptional effect,subcellular localization,and protein-protein interaction for IRF4 and MITF were observed by luciferase assay,immunofluorescence,GST-pull down,and co-immunoprecipitation,respectively.Results:IRF4 and MITF proteins were co-expressed in the cell nucleus.IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter,but with no effect on other MITF-specific target promoters.IRF4 alone did not affect TYR promoter significantly.No direct interaction between the two proteins was noted.Conclusion:IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF.This synergistic effect is mainly regulated by MITF;DNA might be involved in the interaction between the two proteins.

Role of microphthalmia-associated transcription factor in melanoma metastasis / 国际肿瘤学杂志

In recent years, the function of microphthalmia-associated transcription factor (MITF) is a hot field in melanoma.The abnormal expression of MITF is closely related to the occurrence and metastasis of melanoma.In addition, the down expression of MITF promotes its invasion.Studying the regulation of MITF and its related molecules and signaling pathways will let us further understand the molecule mechanism of malignant melanoma metastasis and provide help to exploit novel molecular targeted drug.

Pigmentation Effect of Electromagnetic Fields at Various Intensities to Melanocytes

Melanogenesis is the biological process that results in the synthesis of skin pigment of melanin and it has various functions in living systems and is synthesized by the melanosome within the melanocytes. A variety of physical treatments are used to promote melanin production in the melanocytes for pigmentation control. The purpose of this study was to evaluate the intensity-dependent effect of extremely low-frequency electromagnetic fields (ELF-EMFs) on melanogenesis by melanocytes in vitro. Melanocytes were exposed to ELF-EMFs at a frequency of 50 Hz and at intensities in the range of 0.5–20 G over 4 days. The results of lactate dehydrogenase assay showed that there were no significant differences between cells exposed to 0.5 G or 2 G groups and the controls. The melanin contents increased 1.2–1.5-fold in cells exposed to ELF-EMFs and tyrosinase activity increased 1.3-fold in cells exposed to ELF-EMFs, relative to the controls. Also, exposure to ELF-EMFs was associated with activation in cyclic-AMP response element binding protein and microphthalmia-associated transcription factor (MITF) was up-regulated. Up-regulation of MITF induces the expression of melanogenesis-related markers, such as tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2. In conclusion, the present study showed that the exposure to ELF-EMFs at low intensities can stimulate melanogenesis in melanocyte, and these results may be used to a therapeutic devices for inducing repigmentation in vitiligo patients.

Promotive effect of puerarin on melanogenesis in melanocytes and its possible mechanisms / 中华皮肤科杂志

Objective To evaluate the effect of puerarin on melanogenesis in melanocytes,and to explore its possible mechanisms.Methods Third-to fifth-passage melanocytes isolated from human foreskin were treated with different concentrations (1,5,10,20,40,80 and 160 μmol/L) of puerarin for 24 hours,with those receiving no treatment as the normal control group.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of melanocytes,a sodium hydroxide solubilization method was used to measure melanin content,and reverse transcription PCR (RT-PCR) and Western blot analysis were performed to quantify the mRNA and protein expressions of microphthalmia-associated transcription factor (MITF),tyrosinase (TYR) and tyrosinase-related protein-1 (TRP-1) respectively.Results There were no significant differences in the proliferative activity of melanocytes between the puerarin (1-40 μmol/L) groups and normal control group (P ＞ 0.05),and 40 μmol/L was chosen as the concentration of puerarin for subsequent experiments.Compared with the normal control group,the 40-μmol/L puerarin group showed increased melanin content as well as mRNA and protein expressions of MITF,TYR and TRP-1 (all P ＜ 0.05).Concretely speaking,the protein expressions of MITF,TYR and TRP-1 in the 40-μmol/L puerarin group were increased by 8.69％,10.28％ and 10.58％ compared with the normal control group respectively (all P ＜ 0.05),and their mRNA expressions were 2.48,1.91 and 1.63 times higher in the 40-μmol/L puerarin group than in the normal control group respectively (all P ＜ 0.05).Conclusion Puerarin can increase the mRNA and protein expressions of MITF,TYR and TRP-1,and promote melanogenesis in melanocytes.

Biological and clinical research progress of MITF in melanoma / 肿瘤

Melanoma arises from the melanocyte cell lineage, it is one of the most notoriously aggressive and treatment-resistant human cancers. Microphthalmia-associated transcription factor (MITF) acts as a master regulator of melanocyte's development, function and survival by modulating various differentiation and cell-cycle progression genes. It also plays an oncogenic role in melanoma. MITF targeting genes important in melanoma progression include promoting survival and blocking apoptosis, stimulating proliferation, and inhibiting cell cycle. This review summarized the molecular function of MITF and its related pathways, and hope it will shed light on strategies for improving therapeutic approaches for melanoma. Copyright © 2013 by TUMOR.

Effects of ginsenoside Rb1 on melanogenesis in human epidermal melanocytes / 中华皮肤科杂志

[Objective] To estimate the effects of ginsenoside Rb1 on melanogenesis in human melanocytes and underlying mechanisms.[Methods] Epidermal melanocytes were obtained from circumcision specimens of children,and subjected to primary culture.After 2 to 5 passages,the melanocytes were treated with different concentrations of ginsenoside Rb1,dimethyl sulfoxide (DMSO,vehicle control),forskolin at 10 μmol/L(positive control) or remained untreated (blank control).After additional culture for 72 hours,methyl thiazolyl tetrazolium (MTT) assay and NaOH lysis method were used to evaluate cell viability and melanin content in melanocytes respectively,spectrophotometer to determine dopa oxidase activity of tyrosinase,Western blot to quantify the protein level of tyrosinase,microphthalmia-associated transcription factor (MITF),phosphorylated and total cAMP response element binding protein (p-CREB and t-CREB) in melanocytes.[Results] After treatment with ginsenoside Rbl of 25,50 and 100 μmol/L for 72 hours,the melanocytes experienced no significant changes in viability (P ＞ 0.05 ),but a significant dose-dependent increase in melanin content (112.4％± 5.7％,155.7％ + 6.3％,217.2％ ± 11.7％ vs.100％,P＜ 0.05 or 0.01) and tyrosinase activity(117.9％ ± 5.7％,158.2％ ± 9.6％,182.6％ ± 10.0％ vs.100％,P＜ 0.05 or 0.01 ) compared with the vehicle control melanocytes.The protein expressions of tyrosinase,MITF and p-CREB were statistically higher in melanocytes treated with ginsenoside Rb1 of 100 μmol/L for 72 hours than in the vehicle control melanocytes (225.4％ ± 12.8％ vs.100％ ± 7.9％,313.5％ ± 16.7％ vs.100％ ± 9.8％,322.5％ ± 21.1％ vs.100％ ± 9.1％,all P＜ 0.01).The increase in MITF protein expression was inapparent in melanocytes at 8 hours after the treatment with ginsenoside Rb1 of 100 μmol/L,but statistically significant at 24 hours compared with the melanocytes at baseline (P＜ 0.01).The pretreatment with H-89 (a 8elective inhibitor of PKA) at 10 μmol/L,significantly suppressed the ginsenoside Rb1 (100 μmol/L for 72 hours) -induced phosphorylation of CREB,increase in MITF,tyrosinase expression,as well as tyrosinase activity and melanin content in melanocytes (all P ＜ 0.01 ).[Conclusion]s Ginsenoside Rb1could enhance the melanogenesis and tyrosinase activity in normal human melanocytes.The PKA/CREB/MITF/ tyrosinase signaling pathway may contribute to the pro-melanogenic effect of ginsenoside Rb1.

Involvement of MITF-A, an alternative isoform of mi transcription factor, on the expression of tryptase gene in human mast cells

Mast cells play a central role in the initiation and development of allergic diseases through release of various mediators. Tryptase has been known to be a key mediator in mast cell-mediated inflammatory reactions. In the present study, we investigated whether the transcription of tryptase gene in human mast cells was induced by microphthalmia (mi)-associated transcription factor (MITF). We observed that the human CD34+ progenitor-derived cultured mast cells and human mast cell line HMC-1 expressed strongly the transcripts of tryptase-beta1 and MITF-A, which is a MITF alterative splicing isoform. The transcriptional activity of tryptase gene was specifically higher in HMC-1 cells compared to the tryptase-negative cells. Using mutant constructs of tryptase promoter, we observed that two E-box (CANNTG) motifs including between -817 to -715 and -421 to -202 are able to involve in the transactivation of tryptase gene by MITF-A. In addition, the binding of these motifs-containing oligonucleotides to MITF proteins was detectable by EMGA using the nuclear extracts of HMC-1 cells and anti-MITF mAb. The overexpression of MITF-A elevated tryptase production by HMC-1 cells, while the introduction of specific siRNA against MITF attenuated the expression and enzymatic activity of tryptase. These data suggest that MITF might play a role in regulating the transcription of tryptase gene in human mast cells.

Ultrastructure and transcriptional activity of MITF in epidermal melanocytes from patients with vitiligo / 中华皮肤科杂志

Objective To analyze the relationship of melanocyte ultrastructure and expression of microphthalmia-associated transcription factor (MITF) as well as tyrosinase-related proteins (TRP) transcriptionally modulated by MITF to clinical types and duration of vitiligo.Methods Epidermal sheets were taken by suction blisters respectively from lesional,perilesional,and normal skin of 12 patients with vitiligo vulgaris (VV) and 8 with segmental vitiligo (SV).The duration of vitiligo varied from 3 to 300 months in these patients. Transmission electron microscopy was performed in 10 patients with vitiligo,including 6 cases of VV and 4 cases of SV.Epidermal melanocytes from normal skin of 20 patients were subjected to culture followed by Western blot to detect the expression level of MITF and some molecules transcriptionally modulated by MITF,including tyrosinase (TYR),TYR-related protein-1(TYRP1),and TYR-related protein-2 (TYRP2) in cultured melanocytes.Results Epidermal melanocytes were absent in lesional skin of 7 out of 10 patients observed for ultrastructural alterations,whereas melanocytes with reduced or absent melanin (melanosome) could accidently be seen in lesional skin of 1 patient with short-standing vitiligo and 2 patients with long-standing vitiligo.In perilesional skin.abnormal ultrastructure of melanocytes was found in 3 with a duration of vitiligo less than 15 months among 6 patients with VV,and in 1 out of 4 patients with SV.The down-regulated expression of MITF was consistent with that of TYR,TYRP1 and TYRP2 in cultured epidermal melanocytes from normal skin of patients with VV;in those from patients with SV,the down-regulated expression was observed only in MITF,while the expressions of TYR,TYRP1 and TYRP2 were nearly normal.Conclusion Differences may exist between VV and SV in the ultrastructure as well as mechanisms of transcriptional modulation by MITF in epidermal melanocytes.

Effect of xanthohumol on melanogenesis in B16 melanoma cells

Xanthohumol (XH), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis in B16 melanoma cells, with little cytotoxicity at the effective concentrations. Decreased melanin content was accompanied by reduced tyrosinase enzyme activity, protein and mRNA expression. The levels of tyrosinase-related protein 1 and 2 mRNAs were decreased by XH. XH also inhibited alpha-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. XH downregulated the protein and mRNA expression of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of key melanogenic enzymes. These results suggest that XH might act as a hypo-pigmenting agent through the downregulation of MITF in the cAMP-dependent melanogenic pathway.