Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis.

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

Melanin Inhibitory Effect of Tuber himalayense Isolated in Incheon, Korea.

There has been a growing interest in skin beauty and antimelanogenic products. Melanogenesis is the process of melanin synthesis whereby melanocytes are activated by UV light or hormone stimulation to produce melanin. Melanogenesis is mediated by several enzymes, such as tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), and TRP-2. In this study, we investigated the effect of Tuber himalayense extract on melanin synthesis in α-melanocyte-stimulating hormone (α-MSH)-treated B16F10 melanoma cells. We confirmed that T. himalayense extract was not toxic to α-MSH-treated B16F10 melanoma cells and exhibited a significant inhibitory effect on melanin synthesis at concentrations of 25, 50, and 100 µg/ml. Additionally, the T. himalayense extract inhibited melanin, TRP-1, TRP-2, tyrosinase, and MITF, which are enzymes involved in melanin synthesis, in a concentration-dependent manner. Furthermore, T. himalayense extract inhibited the mitogen-activated protein kinase (MAPK) pathways, such as extracellular signal-regulated kinase-1/2 (ERK), c-Jun N-terminal kinase (JNK), and p38. Therefore, we hypothesized that various components of T. himalayense extract affect multiple factors involved in melanogenesis in B16F10 cells. Our results indicate that T. himalayense extract could potentially be used as a new material for preparing whitening cosmetics.

Interruption of p38MAPK-MSK1-CREB-MITF-M pathway to prevent hyperpigmentation in the skin.

Background: Melanocortin 1 receptor (MC1R), a receptor of α-melanocyte-stimulating hormone (α-MSH), is exclusively present in melanocytes where α-MSH/MC1R stimulate melanin pigmentation through microphthalmia-associated transcription factor M (MITF-M). Toll-like receptor 4 (TLR4), a receptor of endotoxin lipopolysaccharide (LPS), is distributed in immune and other cell types including melanocytes where LPS/TLR4 activate transcriptional activity of nuclear factor (NF)-κB to express cytokines in innate immunity. LPS/TLR4 also up-regulate MITF-M-target melanogenic genes in melanocytes. Here, we propose a molecular target of antimelanogenic activity through elucidating inhibitory mechanism on α-MSH-induced melanogenic programs by benzimidazole-2-butanol (BI2B), an inhibitor of LPS/TLR4-activated transcriptional activity of NF-κB. Methods: Ultraviolet B (UV-B)-irradiated skins of HRM-2 hairless mice and α-MSH-activated melanocyte cultures were employed to examine melanogenic programs. Results: Topical treatment with BI2B ameliorated UV-B-irradiated skin hyperpigmentation in mice. BI2B suppressed the protein or mRNA levels of melanogenic markers, such as tyrosinase (TYR), MITF-M and proopiomelanocortin (POMC), in UV-B-exposed and pigmented skin tissues. Moreover, BI2B inhibited melanin pigmentation in UV-B-irradiated co-cultures of keratinocyte and melanocyte cells and that in α-MSH-activated melanocyte cultures. Mechanistically, BI2B inhibited the activation of cAMP response element-binding protein (CREB) in α-MSH-induced melanogenic programs and suppressed the expression of MITF-M at the promoter level. As a molecular target, BI2B primarily inhibited mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)-catalyzed kinase activity on p38MAPK. Subsequently, BI2B interrupted downstream pathway of p38MAPK-mitogen and stress-activated protein kinase-1 (MSK1)-CREB-MITF-M, and suppressed MITF-M-target melanogenic genes, encoding enzymes TYR, TYR-related protein-1 (TRP-1) and dopachrome tautomerase (DCT) in melanin biosynthesis, and encoding proteins PMEL17 and Rab27A in the transfer of pigmented melanosomes to the overlaying keratinocytes in the skin. Conclusion: Targeting the MKK3-p38MAPK-MSK1-CREB-MITF-M pathway was suggested as a rationale to inhibit UV-B- or α-MSH-induced facultative melanogenesis and as a strategy to prevent acquired pigmentary disorders in the skin.

A comparative study on the effects of human serum albumin and α-melanocyte-stimulating hormone fusion proteins on the anti-neuroinflammatory in the central nervous system of adult mice.

The immunomodulatory effects of α-melanocyte stimulating hormone (α-MSH) in the central nervous system (CNS) have been investigated for forty years. The clinical applications of α-MSH are limited due to its short half-life. Our previous study has indicated that the short half-life of α-MSH can be extended by fusion with carrier human serum albumin (HSA) and this fusion protein has also retained the anti-inflammatory effect on the CNS. This improvement is still far from the clinical requirements. Thus, we expected to enhance the half-life and activity of the fusion protein by optimizing the linker peptide to get closer to clinical requirements. In a previous study, we screened out two candidates in vitro experiments with a flexible linker peptide (fusion protein with flexible linker peptide, FPFL) and a rigid linker peptide (fusion protein with rigid linker peptide, FPRL), respectively. However, it was not sure whether the anti-inflammatory effects in vitro could be reproduced in vivo. Our results show that FPRL is the best candidate with a longer half-life compared to the traditional flexible linker peptides. Meanwhile, the ability of FPRL to penetrate the blood-brain barrier (BBB) was enhanced, and the inhibition of TNF-α and IL-6 was improved. We also found that the toxicity of FPRL was decreased. All of the results suggested that trying to choose the rigid linker peptide in some fusion proteins may be a potential choice for improving the unsatisfactory characteristics.

The whitening effect of cuscutin responsible for traditional use of Bergenia purpurascens.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and rhizomes of Bergenia purpurascens (Hook. f. et Thomson) Engl., was used as a sunscreen to protect against ultraviolet rays in Tibet of China historically, but its skin whitening constituents and pharmacological effects of this plant remained unknown. AIM OF THE STUDY: To investigate the anti-melanogenesis effect of B. purpurascens in vitro and in vivo, and then explore the preliminary mechanism. MATERIALS AND METHODS: An ultraviolet B (UVB)-induced skin injury model of mice was used to verify the ameliorative effect of B. purpurascens extract (BPE) on ultraviolet damage. Then, alpha-melanocyte stimulating hormone (α-MSH)-induced murine melanoma cell line (B16F10) melanin generation model was further adopted to approval the effects of BPE and its bioactive compound, cuscutin, in vitro. Moreover, α-MSH stimulated melanogenesis model in zebrafish was employed to confirm the anti-pigmentation effect of cuscutin. Then, proteins expressions associated with melanin production were observed using western blotting assay to explore preliminary mechanism. RESULTS: BPE inhibited UVB-induced mice injury and restored skin barrier function observably in vivo. BPE and cuscutin suppressed the overproduction of melanin in α-MSH induced B16F10 significantly, in which cuscutin exhibited better effect than well-known whitening agent α-arbutin at same 10 µg/mL concentration. Moreover, the pigmentation of zebrafish embryo was decreased by cuscutin. Finally, cuscutin showed significant downregulation of expressions of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1), TRP-2 and microphthalmia-associated transcription factor (MITF) in the melanogenic signaling pathway. CONCLUSION: B. purpurascens extract and its major bioactive constituent, cuscutin, showed potent anti-melanogenesis and skin-whitening effect by targeting TYR and TRP-2 proteins for the first time, which supported its traditional use.

Inhibition of α-melanocyte-stimulating hormone-induced melanogenesis and molecular mechanisms by polyphenol-enriched fraction of Tagetes erecta L. flower.

BACKGROUND: The pursuit for safe and efficacious skin-whitening agents has prompted a dedicated exploration of plant-derived compounds. Notably, Tagetes erecta L. flowers have been used as a medicinal extract and possessed in vitro mushroom tyrosinase activity. However, whether polyphenol-enriched fraction extracted from T. erecta L. flowers (TE) regulates melanogenesis within cellular and animal models has not yet been investigated. PURPOSE: This study aimed to investigate the effect of TE as a prospective inhibitor of melanogenesis. METHODS: Through advanced UPLC-QTof/MS analysis, the components of TE were analyzed. Anti-melanogenic effects of TE were evaluated in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells by measuring cell viability assay, extracellular and intracellular melanin biosynthesis, cyclic adenosine monophosphate (cAMP) production, and melanogenesis-related gene and protein expression. Zebrafish larvae were employed for in vivo studies, assessing both heart rate and melanogenesis. Furthermore, molecular docking analyses were employed to predict the interaction between TE components and the melanocortin 1 receptor (MC1R). Direct binding activity of TE components to MC1R was compared with [Nle4, d-Phe7]-MSH (NDP-MSH). RESULTS: TE was found to contain significant phenolic compounds such as patulitrin, quercetagetin, kaempferol, patuletin, and isorhamnetin. This study revealed that TE effectively inhibits melanin biosynthesis in both in vitro and in vivo models. This inhibition was attributed to interference of TE with the cAMP-cAMP response element-binding protein (CREB)-microphthalmia-associated transcription factor (MITF)-tyrosinase pathway, which plays a pivotal role in regulating melanogenesis. Importantly, TE exhibited the remarkable ability to curtail α-MSH-induced melanogenesis in zebrafish larvae without impacting heart rates. Molecular docking analyses predicted that the components of TE possibly interact with the melanocortin 1 receptor, suggesting their role as potential inhibitors of melanin biosynthesis. However, through the direct binding activity compared with NDP-MSH, any TE components did not directly bind to MC1R, suggesting that TE inhibits α-MSH-induced melanogenesis by inhibiting the cAMP-mediated intracellular signaling pathway. The assessment of anti-melanogenic activity, conducted both in vitro and in vivo, revealed that patulitrin and patuletin exhibited significant inhibitory effects on melanin formation, highlighting their potency as major contributors. DISCUSSION: This investigation demonstrated the considerable potential of TE as a natural remedy endowed with remarkable anti-melanogenic properties. The demonstrated capacity of TE to attenuate melanin production by modulating the cAMP-CREB-MITF-tyrosinase pathway underscores its central role in management of disorders associated with excessive pigmentation. Importantly, the implications of these findings extend to the cosmetics industry, where TE emerges as a prospective and valuable ingredient for the formulation of skin-whitening products. The elucidated interactions between TE components and MC1R not only provide insight into a potential mechanism of action but also elevate the significance of this study. In summary, this study not only contributes to our comprehension of pigmentation-related conditions but also firmly establishes TE as a secure and natural strategy for the regulation of melanin production. The innovative aspects of TE propel it into the forefront of potential interventions, marking a noteworthy advancement in the pursuit of effective and safe solutions for pigmentation disorders.

2,4,6-Triphenyl-1-hexene, an Anti-Melanogenic Compound from Marine-Derived Bacillus sp. APmarine135.

Although melanin protects against ultraviolet radiation, its overproduction causes freckles and senile lentigines. Recently, various biological effects of metabolites derived from marine microorganisms have been highlighted due to their potential for biological and pharmacological applications. In this study, we discovered the anti-melanogenic effect of Bacillus sp. APmarine135 and verified the skin-whitening effect. Fractions of APmarine135 showed the melanin synthesis inhibition effect in B16 melanoma cells, and 2,4,6-triphenyl-1-hexene was identified as an active compound. The melanogenic capacity of 2,4,6-triphenyl-1-hexene (1) was investigated by assessing the intracellular melanin content in B16 cells. Treatment with 5 ppm of 2,4,6-triphenyl-1-hexene (1) for 72 h suppressed the α-melanocyte-stimulating hormone (α-MSH)-induced intracellular melanin increase to the same level as in the untreated control group. Additionally, 2,4,6-triphenyl-1-hexene (1) treatment suppressed the activity of tyrosinase, the rate-limiting enzyme for melanogenesis. Moreover, 2,4,6-triphenyl-1-hexene (1) treatment downregulated tyrosinase, Tyrp-1, and Tyrp-2 expression by inhibiting the microphthalmia-associated transcription factor (MITF). Furthermore, 2,4,6-triphenyl-1-hexene (1) treatment decreased the melanin content in the three-dimensional (3D) human-pigmented epidermis model MelanoDerm and exerted skin-whitening effects. Mechanistically, 2,4,6-triphenyl-1-hexene (1) exerted anti-melanogenic effects by suppressing tyrosinase, Tyrp-1, and Tyrp-2 expression and activities via inhibition of the MITF. Collectively, these findings suggest that 2,4,6-triphenyl-1-hexene (1) is a promising anti-melanogenic agent in the cosmetic industry.

(2-Methylbutyryl)shikonin Naturally Occurring Shikonin Derivative Ameliorates the α-MSH-Induced Melanogenesis via ERK1/2 and p38 MAP Kinase-Mediated Down-Regulation of the MITF Transcription Factor.

Cutaneous pigmentation is an important phenotypic trait whose regulation, despite recent advances, has yet to be completely elucidated. Melanogenesis, a physiological process of melanin production, is imperative for organism survival as it provides protection against the environmental insults that majorly involve sunlight-induced skin photodamage. However, immoderate melanin synthesis can cause pigmentation disorders associated with a psychosocial impact. In this study, the hypopigmentation effect of (2-methylbutyryl)shikonin, a natural product present in the root extract of Lithospermum erythrorhizon, and the underlying mechanisms responsible for the inhibition of melanin synthesis in α-MSH-stimulated B16F10 cells and C57BL/6J mice was studied. Non-cytotoxic concentrations of (2-methylbutyryl)shikonin significantly repressed cellular tyrosinase activity and melanin synthesis in both in vitro and in vivo models (C57BL/6J mice). (2-Methylbutyryl)shikonin remarkably abolished the protein expression of MITF, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2, thereby blocking the production of pigment melanin via modulating the phosphorylation status of MAPK proteins, viz., ERK1/2 and p38. In addition, specific inhibition of ERK1/2 attenuated the inhibitory effects of (2-methylbutyryl)shikonin on melanin synthesis, whereas selective inhibition of p38 augmented the inhibitory effect of BSHK on melanin synthesis. Moreover, topical application of (2-methylbutyryl)shikonin on C57BL/6J mouse tails remarkably induced tail depigmentation. In conclusion, with these findings, we, for the first time, report the hypopigmentation effect of (2-methylbutyryl)shikonin via inhibition of cellular tyrosinase enzyme activity, subsequently ameliorating the melanin production, thereby indicating that (2-methylbutyryl)shikonin is a potential natural therapy for hyperpigmentation disorders.

Targeting the Melanocortin 1 Receptor in Melanoma: Biological Activity of α-MSH-Peptide Conjugates.

Malignant melanoma is one of the most aggressive and resistant tumor types, with high metastatic properties. Because of the lack of suitable chemotherapeutic agents for treatment, the 5-year survival rate of melanoma patients with regional and distant metastases is lower than 10%. Targeted tumor therapy that provides several promising results might be a good option for the treatment of malignant melanomas. Our goal was to develop novel melanoma-specific peptide-drug conjugates for targeted tumor therapy. Melanocortin-1-receptor (MC1R) is a cell surface receptor responsible for melanogenesis and it is overexpressed on the surface of melanoma cells, providing a good target. Its native ligand, α-MSH (α-melanocyte-stimulating hormone) peptide, or its derivatives, might be potential homing devices for this purpose. Therefore, we prepared three α-MSH derivative-daunomycin (Dau) conjugates and their in vitro and in vivo antitumor activities were compared. Dau has an autofluorescence property; therefore, it is suitable for preparing conjugates for in vitro (e.g., cellular uptake) and in vivo experiments. Dau was attached to the peptides via a non-cleavable oxime linkage that was applied efficiently in our previous experiments, resulting in conjugates with high tumor growth inhibition activity. The results indicated that the most promising conjugate was the compound in which Dau was connected to the side chain of Lys (Ac-SYSNleEHFRWGK(Dau=Aoa)PV-NH2). The highest cellular uptake by melanoma cells was demonstrated using the compound, with the highest tumor growth inhibition detected both on mouse (38.6% on B16) and human uveal melanoma (55% on OMC-1) cells. The effect of the compound was more pronounced than that of the free drug.

Dietary flavonoid myricetin 3-O-galactoside suppresses α-melanocyte stimulating hormone-induced melanogenesis in B16F10 melanoma cells by regulating PKA and ERK1/2 activation.

Melanogenesis is the process where skin pigment melanin is produced through tyrosinase activity. Overproduction of melanin causes skin disorders such as freckles, spots, and hyperpigmentation. Myricetin 3-O-galactoside (M3G) is a dietary flavonoid with reported bioactivities. M3G was isolated from Limonium tetragonum and its anti-melanogenic properties were investigated in α-melanocyte stimulating hormone-stimulated B16F10 melanoma cells. The in vitro anti-melanogenic capacity of M3G was confirmed by inhibited tyrosinase and melanin production. M3G-mediated suppression of melanogenic proteins, tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related proteins (TRP)-1 and TRP-2, were confirmed by mRNA and protein levels, analyzed by RT-qPCR and Western blot, respectively. Furthermore, M3G suppressed Wnt signaling through the inhibition of PKA phosphorylation. M3G also suppressed the consequent phosphorylation of CREB and nuclear levels of MITF. Analysis of MAPK activation further revealed that M3G increased the activation of ERK1/2 while p38 and JNK activation remained unaffected. Results showed that M3G suppressed melanogenesis in B16F10 cells by decreasing tyrosinase production and therefore inhibiting melanin formation. A possible action mechanism was the suppression of CREB activation and upregulation of ERK phosphorylation which might cause the decreased nuclear levels of MITF. In conclusion, M3G was suggested to be a potential nutraceutical with anti-melanogenic properties.

A gel-forming α-MSH analog promotes lasting melanogenesis.

The α-MSH peptide plays a significant role in the regulation of pigmentation via the melanocortin 1 receptor (MC1R). It increases the DNA repair capacity of melanocytes and reduces the incidence of skin cancers. As such, α-MSH analogs could have the utility for protecting against UV-induced skin DNA damage in susceptible patients. Recently, α-MSH analogs have been approved for the treatment of erythropoietic protoporphyria, hypoactive sexual desire, or pediatric obesity. However, the delivery of these drugs requires inconvenient implants or frequent injections. We recently found that select palmitoylated melanocortin analogs such as afamelanotide and adrenocorticotropin peptides self-assemble to form liquid gels in situ. To explore the utility of these novel analogs, we studied their pharmacological characteristics in vitro and in vivo. Acylated afamelanotide (DDE 313) and ACTH1-24 (DDE314) analogs form liquid gels at 6-20% and have a significantly increased viscosity at >2.5% compared to original analogs. Using the DDE313 analog as a prototype, we showed gel-formation reduces the passage of DDE313 through Centricon filters, and subcutaneous injection of analog gel in rats leads to the sustained presence of the peptide in circulation for >12 days. In addition, DDE313 darkened the skin of frogs for >4 weeks, whereas those injected with an equivalent dose of afamelanotide lost the tanning response within a few days. Because self-assembled gels allow sustained activation of melanocortin receptors, further studies of these analogs may allow the development of effective and convenient tanning therapies to prophylactically protect against UV-induced malignant transformation of skin cells in susceptible patients.

EGCG, GCG, TFDG, or TSA Inhibiting Melanin Synthesis by Downregulating MC1R Expression.

Without affecting cell viability, epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), theaflavine-3,3'-digallate (TFDG), or theasinensin A (TSA) have been found to effectively reduce intracellular melanin content and tyrosinase (TYR) activity. However, studies on the anti-melanogenic mechanism of the above samples remain weak, and the activities of these samples in regulating melanogenesis at the molecular level lack comparison. Using B16F10 cells with the α-melanocyte-stimulating hormone (α-MSH) stimulation and without the α-MSH stimulation as models, the effects of EGCG, GCG, TFDG, or TSA on cell phenotypes and expression of key targets related to melanogenesis were studied. The results showed that α-MSH always promoted melanogenesis with or without adding the four samples. Meanwhile, the anti-melanogenic activities of the four samples were not affected by whether the α-MSH was added in the medium or not and the added time of the α-MSH. On this basis, the 100 µg/mL EGCG, GCG, TFDG, or TSA did not affect the TYR catalytic activity but inhibited melanin formation partly through downregulating the melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), and the TYR family. The downregulation abilities of catechins on the TYR family and MITF expression were stronger than those of dimers at both the transcription and translation levels, while the ability of dimers to downregulate the MC1R expression was stronger than that of catechins at both the transcription and translation levels to some extent. The results of molecular docking showed that these four samples could stably bind to MC1R protein. Taken together, this study offered molecular mechanisms for the anti-melanogenic activity of the EGCG, GCG, TFDG, and TSA, as potential effective components against the UV-induced tanning reactions, and a key target (MC1R) was identified.

Paeoniflorin found in Paeonia lactiflora root extract inhibits melanogenesis by regulating melanin-related signal transduction in B16F10 cells.

BACKGROUND: Skin pigmentation is modulated by various processes, with melanogenesis playing a key role. Melanin is synthesized by the catalysis of melanogenesis-related enzymes, such as tyrosinase and tyrosine-related proteins TRP-1 and TRP-2. Paeoniflorin is the main bioactive component of Paeonia suffruticosa Andr., Paeonia lactiflora., or Paeonia veitchii Lynch and has been used for centuries for its anti-inflammatory, anti-oxidant, and anti-carcinogenic properties. AIMS & METHODS: In this study, melanin biosynthesis in mouse melanoma (B16F10) cells was induced using α-melanocyte-stimulating hormone (α-MSH), and then cells were co-treated with paeoniflorin to evaluate its potential anti-melanogenic effect. RESULTS: α-MSH stimulation increased melanin content, tyrosinase activity, and melanogenesis-related markers in a dose-dependent manner. However, treatment with paeoniflorin reversed α-MSH-induced upregulation of melanin content and tyrosinase activity. Furthermore, paeoniflorin inhibited cAMP response element-binding protein activation and TRP-1, TRP-2, and microphthalmia-associated transcription factor protein expression in α-MSH-stimulated B16F10 cells. CONCLUSION: Overall, these findings show the potential of paeoniflorin as a depigmenting agent for cosmetic products.

ACTH/cAMP-Mediated Skin Pigmentation Caused by 5-Fluorouracil Administration.

Anticancer drugs exhibit many side effects, including skin pigmentation, which often lowers patient QOL. However, the mechanism of pigmentation caused by anticancer drugs remains unknown. The purpose of this study was to elucidate the mechanism of anticancer drug-induced skin pigmentation using 5-fluorouracil (5-FU), a widely used anticancer drug. Specific pathogen-free, 9-week-old Hos:HRM-2 male mice were intraperitoneally administered 5-FU daily for 8 weeks. Skin pigmentation was observed at the end of the study. Mice treated with 5-FU were also administered inhibitors of cAMP, α-melanocyte-stimulating hormone (α-MSH), and adrenocorticotropic hormone (ACTH) for analysis. Administration of oxidative stress, nuclear factor-kappa B (NF-κB), cAMP, and ACTH inhibitors reduced pigmentation in 5-FU-treated mice. These results indicate that the oxidative stress/NF-κB/ACTH/cAMP/tyrosinase pathway plays an important role in pigmentation in 5-FU-treated mice.

The αMSH-Dependent PI3K Pathway Supports Energy Metabolism, via Glucose Uptake, in Melanoma Cells.

Stimulation of melanocytes and murine melanoma cells with αMSH plus the PI3K inhibitor LY294002 resulted in ROS increase, oxidative DNA damage, and pigment retention. We performed cellular and molecular biology assays (Western blot, FACS, immunofluorescence analysis, scratch assay) on murine and human melanoma cells. Treatment with αMSH plus LY294002 altered cortical actin architecture. Given that cytoskeleton integrity requires energy, we next evaluated ATP levels and we observed a drop in ATP after exposure to αMSH plus LY294002. To evaluate if the αMSH-activated PI3K pathway could modulate energy metabolism, we focused on glucose uptake by analyzing the expression of the Glut-1 glucose translocator. Compared with cells treated with αMSH alone, those exposed to combined treatment showed a reduction of Glut-1 on the plasma membrane. This metabolic alteration was associated with changes in mitochondrial mass. A significant decrease of the cell migratory potential was also observed. We demonstrated that the αMSH-dependent PI3K pathway acts as a regulator of energy metabolism via glucose uptake, influencing the actin cytoskeleton, which is involved in melanosome release and cell motility. Hence, these results could constitute the basis for innovative therapeutical strategies.

Fermented Aronia melanocarpa Inhibits Melanogenesis through Dual Mechanisms of the PI3K/AKT/GSK-3ß and PKA/CREB Pathways.

UV light causes excessive oxidative stress and abnormal melanin synthesis, which results in skin hyperpigmentation disorders such as freckles, sunspots, and age spots. Much research has been carried out to discover natural plants for ameliorating these disorders. Aronia melanocarpa contains various polyphenolic compounds with antioxidative activities, but its effects on melanogenesis have not been fully elucidated. In this study, we investigated the inhibitory effect of fermented Aronia melanocarpa (FA) fermented with Monascus purpureus on melanogenesis and its underlying mechanism in the B16F10 melanoma cell line. Our results indicate that FA inhibited tyrosinase activity and melanogenesis in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. FA significantly downregulated the PKA/CREB pathway, resulting in decreased protein levels of tyrosinase, TRP-1, and MITF. FA also inhibited the transcription of MITF by increasing the phosphorylation levels of both GSK3ß and AKT. Interestingly, we demonstrated that these results were owing to the significant increase in gallic acid, a phenolic compound of Aronia melanocarpa produced after the fermentation of Monascus purpureus. Taken together, our research suggests that Aronia melanocarpa fermented with Monascus purpureus acts as a melanin inhibitor and can be used as a potential cosmetic or therapeutic for improving hyperpigmentation disorders.

Hydrolyzed Conchiolin Protein (HCP) Extracted from Pearls Antagonizes both ET-1 and α-MSH for Skin Whitening.

Pearl powder is a famous traditional Chinese medicine that has a long history in treating palpitations, insomnia, convulsions, epilepsy, ulcers, and skin lightining. Recently, several studies have demonstrated the effects of pearl extracts on protection of ultraviolet A (UVA) induced irritation on human skin fibroblasts and inhibition of melanin genesis on B16F10 mouse melanoma cells. To further explore the effect we focused on the whitening efficacy of pearl hydrolyzed conchiolin protein (HCP) on human melanoma MNT-1 cells under the irritation of alpha-melanocyte-stimulating hormone (α-MSH) or endothelin 1 (ET-1) to evaluate the intracellular tyrosinase and melanin contents, as well as the expression levels of tyrosinase (TYR), tyrosinase related protein 1 (TRP-1), and dopachrome tautomerase (DCT) genes and related proteins. We found that HCP could decrease the intracellular melanin content by reducing the activity of intracellular tyrosinase and inhibiting the expression of TYR, TRP-1, DCT genes and proteins. At the same time, the effect of HCP on melanosome transfer effect was also investigated in the co-culture system of immortalized human keratinocyte HaCaT cells with MNT-1. The result indicated that HCP could promote the transfer of melanosomes in MNT-1 melanocytes to HaCaT cells, which might accelerate the skin whitening process by quickly transferring and metabolizing melanosomes during keratinocyte differentiation. Further study is needed to explore the mechanism of melanosome transfer with depigmentation.

Inhibition of melanogenesis by 3-(1'-methyltetrahydropyridinyl)-2,4-6-trihydroxy acetophenone via suppressing the activity of cAMP response element-binding protein (CREB) and nuclear exclusion of CREB-regulated transcription coactivator 1 (CRTC1).

Exposure to Ultraviolet radiation or α-melanocyte-stimulating hormone (α-MSH) stimulates the Cyclic Adenosine Monophosphate/Protein Kinase A signalling pathway, which leads to the synthesis and deposition of melanin granules in the epidermis. Skin pigmentation is the major physiological defence against inimical effects of sunlight. However, excessive melanin production and accumulation can cause various skin hyperpigmentation disorders. The present study involved the identification of 3-(1'-methyltetrahydropyridinyl)-2,4-6-trihydroxy acetophenone (IIIM-8) as an inhibitor of melanogenesis, IIIM-8 significantly inhibited pigment production both in vitro and in vivo without incurring any cytotoxicity in Human Adult Epidermal Melanocytes (HAEM). IIIM-8 repressed melanin synthesis and secretion both at basal levels and in α-MSH stimulated cultured HAEM cells by decreasing the levels of Cyclic Adenosine Monophosphate (cAMP) and inhibiting the phosphorylation of cAMP response element-binding (CREB) protein, coupled with restoring the phosphorylation of CREB-regulated transcription coactivator 1 (CRTC1) and its nuclear exclusion in HAEM cells. This impeding effect correlates with diminished expression of master melanogenic proteins including microphthalmia-associated transcription factor (MITF), Tyrosinase (TYR), Tyrosinase related protein 1 (TRP1), and Tyrosinase related protein 2 (TRP2). Additionally, topical application of IIIM-8 induced tail depigmentation in C57BL/6J mice. Furthermore, IIIM-8 efficiently mitigated the effect of ultraviolet-B radiation on melanin synthesis in the auricles of C57BL/6J mice. This study demonstrates that IIIM-8 is an active anti-melanogenic agent against ultraviolet radiation-induced melanogenesis and other hyperpigmentation disorders.

Melanocortin/MC5R axis regulates the proliferation of hematopoietic stem cells in mice after ionizing radiation injury.

Hematopoietic stem cells (HSCs) possess great self-renewal and multidirectional differentiation abilities, which contribute to the continuous generation of various blood cells. Although many intrinsic and extrinsic factors have been found to maintain HSC homeostasis, the precise regulation of hematopoiesis under stress conditions is poorly understood. In this study, we show that melanocortin receptor 5 (MC5R) is abundantly expressed in hematopoietic stem progenitor cells (HSPCs). Using an MC5R knockout mouse model, we observed that it is not essential for steady-state hematopoiesis. Interestingly, the levels of α-melanocyte stimulating hormone (α-MSH), an important subtype of melanocortin, were elevated in the serum and bone marrow, and the expression of MC5R was upregulated in HSPCs from mice after irradiation. MC5R deficiency aggravates irradiation-induced myelosuppression because of impaired proliferation and reconstitution of HSCs. Further investigation revealed that the melanocortin/MC5R axis regulates the proliferation of HSCs by activating the PI3K/AKT and MAPK pathways. More importantly, α-MSH treatment can significantly accelerate hematopoietic recovery in irradiated mice. In conclusion, our data demonstrate that the melanocortin/MC5R axis plays a crucial role in regulating HSC proliferation under stress, thus providing a promising strategy to promote hematopoietic regeneration when suffering from injury.

Short-term effects of α-melanocyte-stimulating hormone in three distinct melanin-pigmented cell types of Anura.

Ectothermic animals present melanin-containing cells in their integument and viscera. Besides cutaneous melanophores, amphibians have melanomacrophages in the hepatic parenchyma and melanocytes in the viscera, which are also present in their testicular stroma. The native melanocyte stimulating hormone (α-MSH) is the main hormone that modulates the color change in melanophores. However, we still know too little about how the α-MSH acts in vivo on visceral melanin-containing cells. In this study, we collected 30 adult males of Physalaemus nattereri (Anura, Leptodactylidae) to evaluate the short-term effects of α-MSH on melanophores, melanocytes and melanomacrophages under light microscopy. For this, we injected 0.05 ml of a single intraperitoneal dose containing 2.5x10-7 mmol/10g of α-MSH, diluted in ringer solution, in five experimental groups with five individuals each one. The different groups were analyzed after 1, 3, 6, 12 and 24h. The control group with five other individuals received only 0.05 ml of ringer solution. The skin pigmentation increased quickly after animals received the hormone α-MSH with the consequent darkening of the body (body darkness). Melanophores, melanocytes and melanomacrophages responded similarly to the test, with an increase in the area containing melanin. However, melanophores and melanomacrophages reached their darkest pigmentation in a shorter period of time in comparison to the testicular melanocytes, probably due to specific metabolic characteristics of each organ. Thus, we verified that the three types of cells, although present in different organs, are responsive to the native hormone α-MSH, which enables us to treat them as a pigmentary system.