Microphthalmia-associated Transcription Factor Expression Research Articles

Eribulin Suppresses Clear Cell Sarcoma Growth by Inhibiting Cell Proliferation and Inducing Melanocytic Differentiation Both Directly and Via Vascular Remodeling.

Clear cell sarcoma (CCS) is a rare but chemotherapy-resistant and often fatal high-grade soft-tissue sarcoma (STS) characterized by melanocytic differentiation under control of microphthalmia-associated transcription factor (MITF). Eribulin mesilate (eribulin) is a mechanistically unique microtubule inhibitor commonly used for STS treatment, particularly liposarcoma and leiomyosarcoma. In this study, we examined the antitumor efficacy of eribulin on four human CCS cell lines and two mouse xenograft models. Eribulin inhibited CCS cell proliferation by inducing cell-cycle arrest and apoptosis, shrunk CCS xenograft tumors, and increased tumor vessel density. Eribulin induced MITF protein upregulation and stimulated tumor cell melanocytic differentiation through ERK1/2 inactivation (a MITF negative regulator) in vitro and in vivo Moreover, tumor reoxygenation, probably caused by eribulin-induced vascular remodeling, attenuated cell growth and inhibited ERK1/2 activity, thereby upregulating MITF expression and promoting melanocytic differentiation. Finally, downregulation of MITF protein levels modestly debilitated the antiproliferative effect of eribulin on CCS cells. Taken together, eribulin suppresses CCS through inhibition of cell proliferation and promotion of tumor differentiation by acting both directly on tumor cells and indirectly through tumor reoxygenation.

Cistanche deserticola polysaccharide induces melanogenesis in melanocytes and reduces oxidative stress via activating NRF2/HO-1 pathway.

As a main part of pigmentation disorders, skin depigmentation diseases such as vitiligo and achromic naevus are very common and get more attention now. The pathogenesis of depigmentation includes melanocyte dysfunction and loss, which are possibly caused by heredity, autoimmunity and oxidative stress. Among them, oxidative stress plays a key role; however, few clinical treatments can deal with oxidative stress. As reported, Cistanche deserticola polysaccharide (CDP) is an effective antioxidant; based on that, we evaluated its role in melanocyte and further revealed the mechanisms. In this study, we found that CDP could promote melanogenesis in human epidermal melanocytes (HEMs) and mouse melanoma B16F10 cells, it also induced pigmentation in zebrafish. Furthermore, CDP could activate mitogen‐activated protein kinase (MAPK) signal pathway, then up‐regulated the expression of microphthalmia‐associated transcription factor (MITF) and downstream genes TYR, TRP1, TRP2 and RAB27A. Otherwise, we found that CDP could attenuate H2O2‐induced cytotoxicity and apoptosis in melanocytes. Further evidence revealed that CDP could enhance NRF2/HO‐1 antioxidant pathway and scavenge intracellular ROS. In summary, CDP can promote melanogenesis and prevent melanocytes from oxidative stress injury, suggesting that CDP helps maintain the normal status of melanocytes. Thus, CDP may be a novel drug for the treatment of depigmentation diseases.

Substance P Administered after Myocardial Infarction Upregulates Microphthalmia-Associated Transcription Factor, GATA4, and the Expansion of c-Kit+ Cells.

Microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper transcription factor, can govern gene expression by binding to E box elements in the promoter region of its target gene. Although high levels of MITF have been observed in cardiomyocytes and the heart, the role of MITF after myocardial infarction (MI) remains unclear. We investigated the association between substance P (SP)/neurokinin-1 receptor (NK1R) signaling and MITF expression after MI. Male Sprague-Dawley rats (8 weeks) were randomly divided in two groups: ischemia/reperfusion injury (I/R) and SP injection (5 nmol/kg, SP+I/R). At the end of 7 days, the left ventricle (LV; LV7daysI/R, LV7daysSP+I/R) and infarct-related areas (IA; IA7daysI/R, IA7daysSP+I/R) from the hearts were collected. Immunofluorescence staining demonstrated that the LV7daysSP+I/R had a larger population of c-Kit+ GATA4high cells, which markedly upregulated MITF, c-Kit, and GATA4. c-Kit+ cells in the explant-derived cells (EDCs) derived from IA7daysSP+I/R migrated more widely than EDCs IA7daysI/R. Immunofluorescence staining, western blot analysis, and qRT-PCR assay showed that SP-treated c-Kit+ cells exhibited a high expression of c-Kit, GATA4, and MITF. FTY720 (a MITF inhibitor), RP67580 (NK1R inhibitor), or both inhibited the migration and proliferation of c-Kit+ cells increased by SP and blocked the upregulation of c-Kit, GATA4, and MITF. Overall, we suggest that MITF might be a potential regulator in SP-mediated c-Kit+ cell expansion post-MI via c-Kit and GATA4.

Expression and contribution of microphthalmia-associated transcription factor to the melanin deposition in Liancheng white ducks

The present study investigates the expression of microphthalmia-associated transcription factor (MITF) and its contribution to the melanin deposition in Liancheng white ducks. Nested PCR was used to clone the MITF gene sequence from the skin tissue of female Liancheng white ducks. Ultraviolet spectrophotometry was used to detect the melanin deposition. MITF mRNA expression and melanin deposition in different tissues and organs were detected and their correlation was analyzed. The MITF gene (GenBank number: MG516570) was 1 323 bp in length, contains a complete CDS region (34–1 323 bp) and codes 429 amino acids with 100% homology to the MITF of Anas platyrhynchos and over 95% homology to those of Gallus gallus and Coturnix japonica. Genetic evolution analysis reveals a close relationship of Liancheng white ducks with A. platyrhynchos, and also to lesser extents with Anser cygnoides, silky fowl and G. gallus, as well as Sus scrofa, Ovis aries and other mammals. Real-time quantitative PCR (qPCR) analysis demonstrated that MITF was expressed in skin, gizzard, liver, kidney and muscle, and of these tissues, its expression was the highest in the skin tissue (skin>gizzard>liver>kidney>muscle). Ultraviolet spectrophotometry showed that melanin deposition was positively correlated with the MITF expression level in these five tissues and organs (P<0.05). Together, these results demonstrated a tissue-specific pattern of MITF expression and a positive correlation between MITF expression and melanin deposition, indicating that MITF expression may contribute to the melanin deposition in Liancheng white ducks.

MiRNA-183∼96∼182 regulates melanogenesis, cell proliferation and migration in B16 cells

Melanoma is a highly invasive malignant skin tumor having high metastatic rate and poor prognosis. The biology of melanoma is controled by miRNAs. The miRNA-183 cluster, which is composed of miRNA-183∼96∼182 genes, plays an important roles in tumor development. In order to investigate the role and action of miRNA-183 cluster in B16 cells, we overexpressed and knocked down miRNA-183 cluster in B16 cells. Using bioinformatics analysis, we predicted that the key framscript factor of melangenic genes. Microphthalmia-associated transcription factor (MITF) is one of the targets of miRNA-183 cluster. The results of Luciferase activity assays confirmed that MITF was targeted by miRNA-183 cluster. Overexpression and knockdown of miRNA-183 cluster in B16 cells resulted in down and up regulation of MITF expression, respectively at both mRNA and protein levels. Furthmore, overexpression and knockdown of the miRNA-183 cluster in B16 cells decreased and increased the expression of mRNA and protein of melangenic genes tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP1), dopachrome-tautomerase (DCT), as well as the production of melanins and eumelanin production, respectively. On the proliferation and migration pathway, overexpression and knockdown of miRNA-183 cluster increased and decreased, respectively the expression of mRNA and protein of mitogen-activated protein kinase 1 (MEK1), extracellular regulated protein kinases1/2 (ERK1/2) and cAMP-responsive-element binding protein (CREB). These results indicated that miRNA-183 cluster regulated melanogenesis in B16 cells as well as cell proliferation and migration by directly targeting MITF through migration pathway.

Melochia corchorifolia extract inhibits melanogenesis in B16F10 mouse melanoma cells via activation of the ERK signaling.

Numerous researches have focused on discovering available inhibitors of melanogenesis from natural medicinal plants with stable efficacy and safety to resolve cutaneous hyperpigmentary problems. Melochia corchorifolia Linn. (MC) has been used as folk medicine to treat various diseases. However, the effect of MC on melanogenesis remains unknown. In this study, we investigated the effect of MC extract on melanogenesis and its underlying mechanisms in B16F10 mouse melanoma cells. B16F10 cells were treated with MC extract, and then, cell viability, melanin content, and tyrosinase activity were analyzed. The mRNA and protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF) were evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Phosphorylated or total protein levels in MC extract-induced signaling pathways were analyzed by Western blotting. Treatment of B16F10 cells with MC extract inhibited melanin synthesis and intracellular tyrosinase activity in a dose-dependent manner with no cytotoxicity. Protein and mRNA expressions of tyrosinase and MITF were also significantly decreased by MC extract treatment. In addition, phosphorylated level of extracellular signal-regulated kinase (ERK) was obviously increased by MC extract, but AKT pathway was not activated. Inhibited ERK phosphorylation by pretreatment with a selective ERK inhibitor PD98059 significantly reversed the decreased melanin content induced by treatment with MC extract in B16F10 cells. MC extract inhibits melanogenesis in B16F10 mouse melanoma cells through suppression of MITF-tyrosinase signaling pathway by ERK activation.

Antioxidant and anti-melanogenic activities of ultrasonic extract from Stichopus japonicus

Objective: To investigate the antioxidant and anti-melanogenesis activities of an ultrasonic extract of red sea cucumber, Stichopus japonicus, collected from Jeju Island. Methods: Antioxidant activity experiments were assessed by an electron spin resonance system and a cellular model of immortalized human keratinocytes (HaCaT) to determine its radical scavenging activity and protective effects against 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress. Anti-melanogenic activity of the ultrasonic extract of red sea cucumber was also examined using the melanoma cell model B16F10 and mushroom tyrosinase. Following the induction by α-melanocyte-stimulating hormone, the effects of the ultrasonic extract of red sea cucumber on intracellular tyrosinase activity, melanin content and the melanogenic protein expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related proteins (TRP-1, and TRP-2) were examined. Results: The ultrasonic extract of red sea cucumber significantly scavenged 2,2-diphenyl-1-picrylhydrazyl and alkyl radicals [IC50: (0.924±0.035) and (0.327±0.006) mg/mL, respectively], as well as showed a protective effect against oxidative stress and attenuated generation of intracellular reactive oxygen species on AAPH- induced HaCaT cells, with no cytotoxicity (12.5-400 μg/mL). The ultrasonic extract of red sea cucumber also exhibited a tyrosinase inhibitory effect [IC50: (2.750±0.006) mg/mL]. On α-melanocyte-stimulating hormone-stimulated B16F10 melanoma cells, the ultrasonic extract of red sea cucumber (25-200 μg/mL) significantly inhibited not only melanin synthesis and tyrosinase activity, but also protein expressions of microphthalmia-associated transcriptional factor, tyrosinase, TRP-1, and TRP-2. Conclusions: The ultrasonic extract of red sea cucumber shows antioxidant and anti-melanogenic potential and may be a natural candidate for anti-aging as well as a whitening agent in the cosmeceuticals industry.

Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells.

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.

Melanogenic Effects of Maclurin Are Mediated through the Activation of cAMP/PKA/CREB and p38 MAPK/CREB Signaling Pathways

Melanogenesis is the biological process which the skin pigment melanin is synthesized to protect the skin against ultraviolet irradiation and other external stresses. Abnormal biology of melanocytes is closely associated with depigmented skin disorders such as vitiligo. In this study, we examined the effects of maclurin on melanogenesis and cytoprotection. Maclurin enhanced cellular tyrosinase activity as well as cellular melanin levels. We found that maclurin treatment increased the expression of microphthalmia-associated transcription factor (MITF), tyrosinase-related protein- (TRP-) 1, TRP-2, and tyrosinase. Mechanistically, maclurin promoted melanogenesis through cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein-dependent upregulation of MITF. CREB activation was found to be mediated by p38 mitogen-activated protein kinase (MAPK) or cAMP-protein kinase A (PKA) signaling. In addition, maclurin-induced CREB phosphorylation was mediated through the activation of both the cAMP/PKA and the p38 MAPK signaling pathways. Maclurin-induced suppression of p44/42 MAPK activation also contributed to its melanogenic activity. Furthermore, maclurin showed protective effects against H2O2 treatment and UVB irradiation in human melanocytes. These findings indicate that the melanogenic effects of maclurin depend on increased MITF gene expression, which is mediated by the activation of both p38 MAPK/CREB and cAMP/PKA/CREB signaling. Our results thus suggest that maclurin could be useful as a protective agent against hypopigmented skin disorders.

Inhibitory effects on melanogenesis by thymoquinone are mediated through the β‑catenin pathway in B16F10 mouse melanoma cells.

Thymoquinone (TQ) is a component found in the seeds of Nigella sativa, an annual plant growing on the Mediterranean coast, and is known for its anticancer and anti‑inflammatory effects. However, to date, at least to the best of our knowledge, limited studies are available examining the molecular mechanisms through which TQ inhibits melanogenesis. Accordingly, this study aimed to treat B16F10 mouse melanoma cells with TQ to investigate its apparent effects and its molecular regulatory mechanisms. Treatment of the B16F10 cells with 10, 15and20µM of TQ for 48h resulted in a dose‑dependent decrease in the expression of microphthalmia‑associated transcription factor (MITF), tyrosinase expression and tyrosinase activity, and these treatments simultaneously led to a decrease in the protein expression and transcription of β‑catenin, a Wnt signaling pathway protein. Pre‑treatment of the cells with the proteasome inhibitor, MG132, to confirm the inhibition of melanogenesis through the β‑catenin pathway by TQ treatment resulted in an increase in the expression of β‑catenin that was initially reduced by TQ, and the expression and activity of MITF and tyrosinase also increased. Pre‑treatment with LiCl, which is known to inactivate glycogen synthase kinase3β (GSK3β) by inducing the phosphorylation of the Ser‑9 site, resulted in an increased phospho‑GSK3β expression accompanied by β‑catenin that was initially reduced by TQ, and the recovery of the expression and activity of tyrosinase was also confirmed. The transfection of S37A cDNA into B16F10 cells that overexpress β‑catenin resulted in the recovery of β‑catenin expression that was initially reduced by TQ, and this treatment also recovered the expression and activity of tyrosinase. When zebrafish eggs were treated with 1, 2.5and5µM of TQ at 10h following fertilization, their melanin content decreased in a dose‑dependent manner. On the whole, these findings demonstrated that the inhibition of melanogenesis in B16F10 mouse melanoma cells by TQ treatment resulted from the inhibition of the β‑catenin pathway and confirmed that TQ treatment inhibited melanogenesis in zebrafish.

Anthocyanins from Hibiscus syriacus L. Inhibit Melanogenesis by Activating the ERK Signaling Pathway.

Hibiscus syriacus L. exhibited promising potential as a new source of food and colorants containing various anthocyanins. However, the function of anthocyanins from H. syriacus L. has not been investigated. In the current study, we evaluated whether anthocyanins from the H. syriacus L. varieties Pulsae and Paektanshim (PS and PTS) inhibit melanin biogenesis. B16F10 cells and zebrafish larvae were exposed to PS and PTS in the presence or absence of α-melanocyte-stimulating hormone (α-MSH), and melanin contents accompanied by its regulating genes and proteins were analyzed. PS and PTS moderately downregulated mushroom tyrosinase activity in vitro, but significantly decreased extracellular and intracellular melanin production in B16F10 cells, and inhibited α-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. PS and PTS also attenuated pigmentation in α-MSH-stimulated zebrafish larvae. Furthermore, PS and PTS activated the phosphorylation of extracellular signal-regulated kinase (ERK), whereas PD98059, a specific ERK inhibitor, completely reversed PS- and PTS-mediated anti-melanogenic activity in B16F10 cells and zebrafish larvae, which indicates that PS- and PTS-mediated anti-melanogenic activity is due to ERK activation. Moreover, chromatography data showed that PS and PTS possessed 17 identical anthocyanins as a negative regulator of ERK. These findings suggested that anthocyanins from PS and PTS inhibited melanogenesis in vitro and in vivo by activating the ERK signaling pathway.

Pigmentation Effect of Rice Bran Extract in Hair Follicle-Like Tissue and Organ Culture Models.

Melanogenesis is a biological process resulting in the production of melanin pigment, which plays an important role in the prevention of sun-induced skin injury and determines the hair and skin color. Melanin has the ability to block ultraviolet radiation and scavenge free oxygen radicals, thus protecting the skin from their harmful effects. Agents that increase melanin synthesis in melanocytes may reduce the risk of photodamage and skin cancer. Hence, various approaches have been proposed to increase the synthesis of melanin. The current study aimed to develop a three-dimensional hair follicle-like tissue (HFLT) model with human dermal papilla, melanocytes, and outer root sheaths cells. This model showed enhanced melanogenesis-related protein expression after rice bran ash extract (RBE) treatment. Next, we investigated the melanogenic effect of RBE in the HFLT and compared the results to those of hair follicle (HF) organ culture model. RBE was found to significantly increase the expression of microphthalmia-associated transcription factor, a key transcription factor involved in melanin production, in both HFLT and organ culture models. Results showed that melanogenesis-related protein expression levels were higher in the RBE group compared to those in the control group. Similar results were obtained by immunohistochemistry. Our data suggested that RBE promotes melanin biosynthesis. Taken together, this simple in vitro HFLT model system has the potential to provide significant insights into the underlying molecular mechanisms of HF melanogenesis, and hence can be used for controlled evaluation of the efficacy of new materials for melanogenesis.

Flumequine-Mediated Upregulation of p38 MAPK and JNK Results in Melanogenesis in B16F10 Cells and Zebrafish Larvae.

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo. According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

Possible role of BMP-4 in the hyper-pigmentation of psoriatic plaques after anti-TNF-α treatment.

Psoriasis over-expresses several inflammatory mediators, which impacts the activity of melanocytes. Tyrosinase (Tyr) and microphthalmia-associated transcription factor (MITF) are the primary regulators of melanogenesis. Furthermore, bone morphogenetic proteins (BMPs) modulate various pathobiologic processes including inflammation, melanogenesis and melanomagenesis. To determine the association between psoriasis and melanogenesis, psoriatic lesional skin was screened through gene expression, immunohistochemistry, immunogold staining and melanin content assays. The present study detected a decreased expression of Tyr, MITF and BMP-4 in psoriatic lesional skin compared with healthy skin. Tyr, BMP-4 and melanin content were also evaluated in the psoriatic lesional skin of patients receiving adalimumab therapy, before and after 16 weeks of treatment. TNF-α blockade modulated the Tyr, BMP-4 and melanin content of the patient skin lesions, which supported the hypothesis that hyper-pigmentation may occur in areas of psoriatic plaque after biological treatment. The present study confirmed the influence of the psoriatic pro-inflammatory network on melanogenesis, exerting an inhibitory effect mediated by TNF-α. Furthermore, the results regarding BMP-4 in the present study add another important element to the mechanism of psoriasis.

8-Methoxycoumarin enhances melanogenesis via the MAPKase signaling pathway.

8-Methoxycoumarin (8-methoxy-chromen-2-one), isolated from R. graveolens L., is able to alleviate arthritis by inhibition of proinflammatory cytokines. However, its effects on melanogenesis have largely remained unreported. The present study examined the effects of 8-methoxycoumarin on melanogenesis in B16F10 murine cells, together with its effect on the mechanism of melanin synthesis. The cells were treated with different concentrations of 8-methoxycoumarin; α-MSH was used as the positive control. We found 8-methoxycoumarin to significantly increase the melanin content of the cells without exerting any cytotoxicity. In addition, it significantly upregulated the expression of tyrosinase and tyrosinase-related protein-1 and 2 via inducing the expression of microphthalmia-associated transcription factor. Furthermore, we demonstrated the involvement of mitogen-activated protein kinase (MAPK) pathway-mediated phosphorylation of p38 and c-Jun N-terminal kinase (JNK), and inhibition of phosphorylation of extracellular signal-regulated kinase (ERK) to be responsible for enhanced melanin production. Use of SB203580 (p38 inhibitor) and SP600125 (p-JNK inhibitor) corroborated these findings. Additionally, we investigated the effects of 8-methoxycoumarin on protein kinase B (AKT) phosphorylation and protein kinase A (PKA) signaling pathway (using H89, a PKA inhibitor). These results suggested that 8-methoxycoumarin increases melanogenesis via the MAPK signaling pathway. Based on these findings, we conclude that 8-methoxycoumarin could serve as a potential compound for treating hypopigmentation disorders. It could also serve as a promising chemical for hair depigmentation treatment in the cosmetic industry.

Comparison of miRNA-101a-3p and miRNA-144a-3p regulation with the key genes of alpaca melanocyte pigmentation

BackgroundMany miRNA functions have been revealed to date. Single miRNAs can participate in life processes by regulating more than one target gene, and more than one miRNA can also simultaneously act on one target mRNA. Thus, a complex regulatory network involved in many processes can be formed. Herein, the pigmentation regulation mechanism of miR-101a-3p and miR-144a-3p was studied at the cellular level by the overexpression and equal overexpression of miR-101a-3p and miR-144a-3p.ResultsResults revealed that miR-101a-3p and miR-144a-3p directly regulated the expression of microphthalmia-associated transcription factor, thereby affecting melanin synthesis.ConclusionsThe two miRNAs with the same binding site in the same gene independently excreted each other’s function. However, the inhibitory effect of miR-144a-3p was stronger than that of miR-101-3p in alpaca melanocytes, although both decreased melanin production.

Downregulation of microRNA-218 is cardioprotective against cardiac fibrosis and cardiac function impairment in myocardial infarction by binding to MITF.

Objective: This study is intended to figure out the function of microRNA-218 (miR-218) together with microphthalmia-associated transcription factor (MITF) on the cardiac fibrosis and cardiac function impairment in rat models of myocardial infarction (MI).Results: The rats with MI exhibited cardiac function impairment, cardiac fibrosis, oxidative stress, cardiomyocyte apoptosis, as well as inflammatory injury. Additionally, upregulated miR-218 and downregulated MITF were detected in cardiac tissues of MI rats. MI rats injected with miR-218 inhibitors or overexpressed MITF exhibited elevated MITF expression, improved cardiac function, and diminished pathological damages, infarct size, cardiomyocyte apoptosis, cardiac fibrosis, oxidative stress as well as inflammatory injury in cardiac tissues. Furthermore, downregulated miR-218 and MITF aggravated the conditions than downregulation of miR-218 alone in MI rats.Methods: MI models were performed in rats, and then the rats were injected with miR-218 inhibitors and/or MITF overexpression plasmid to elucidate the role of miR-218 and/or MITF on the cardiac function, pathological damage, cardiac fibrosis, angiogenesis, oxidative stress and inflammatory injury of cardiac tissues in MI rats by performing a series of assays.Conclusion: Collectively, we found that the suppression of miR-218 alleviates cardiac fibrosis and cardiac function impairment, and stimulates angiogenesis in MI rats through inhibiting MITF.

Differentiation of adipose-derived stem cells to functional CD105neg CD73low melanocyte precursors guided by defined culture condition

BackgroundThe generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo. Despite the important efforts exerted to obtain melanin-producing cells from stem cells, pre-clinical results still lack the safety and scalability characteristics essential for their translational application.MethodsHere, we report a rapid and efficient protocol based on defined culture conditions capable of differentiating adult adipose-derived stem cells (ADSC) to scalable amounts of proliferative melanocyte precursors (PreMel) within 30 days. PreMel were characterized in vitro through qPCR, Western blot, flow cytometry, biochemical assays, and in vivo assays in immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, or NSG).ResultsAfter 30 days of differentiation, the stem cell-derived PreMel were defined as CD105neg CD73low according to immunophenotypic changes in comparison with parental stem cell markers. In addition, expression of microphthalmia-associated transcription factor (MITF), active tyrosinase (TYR), and the terminal differentiation-involved premelanosome protein (PMEL) were detected. Furthermore, PreMel had the potential to synthesize melanin and package it into melanosomes both in vitro and in vivo in NSG mice skin.ConclusionsThis study proposes a rapid and scalable protocol for the generation of proliferative melanocyte precursors (PreMel) from ADSC. These PreMel display the essential functional characteristics of bona fide HEM, opening a new path for an autologous cellular therapy for vitiligo patients.

Resveratrol-mediated inhibition of cyclooxygenase-2 in melanocytes suppresses melanogenesis through extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase/Akt signalling

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), has been reported to exert a variety of important pharmacological effects including anti-inflammatory, anticancer, and direct inhibition of tyrosinase. This study aimed to examine the expression of melanogenic molecules following down-regulation of cyclooxygenase (COX)-2 expression by resveratrol and the related signal transduction pathways in mouse B16F10 melanoma cells and zebrafish larvae. We report that resveratrol suppressed COX-2 in melanocytes and decreased the expressions of tyrosinase and microphthalmia-associated transcription factor (MITF). Furthermore, inhibition of COX-2 with NS398 enhanced resveratrol-reduced tyrosinase and MITF expression. Resveratrol also induced phosphorylation of extracellular signal-regulated 1/2 (ERK1/2) and phosphoinositide-3 (PI-3)-kinase/Akt. Inhibition of ERK1/2 or PI-3K/Akt by PD98059 and LY294002 restored the decreased tyrosinase activity and MITF expression via resveratrol-mediated down-regulation of COX-2. Additionally, resveratrol inhibited body pigmentation in zebrafish. These results indicated that resveratrol inhibited melanogenesis by down-regulating COX-2 via ERK1/2 and PI-3K/Akt pathways in B16F10 cells.

Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma.

Melanocortin 1 receptor (MC1R) is under investigation as a target for drug delivery for metastatic melanoma therapy and imaging. The purpose of this study was to determine the potential of using BRAF inhibitors (BRAFi) and histone deacetylase inhibitors (HDACi) to enhance the delivery of MC1R-targeted radiolabeled peptide ([212Pb]DOTA-MC1L) by pharmacologically upregulating the MC1R expression in metastatic melanoma cells and tumors. MC1R expression was analyzed in de-identified melanoma biopsies by immunohistochemical staining. Upregulation of MC1R expression was determined in BRAFV600E cells (A2058) and BRAF wild-type melanoma cells (MEWO) by quantitative real-time polymerase chain reaction, flow cytometry, and receptor-ligand binding assays. The role of microphthalmia-associated transcription factor (MITF) in the upregulation of MC1R was also examined in A2058 and MEWO cells. The effectiveness of [212Pb]DOTA-MC1L α-particle radiotherapy in combination with BRAFi and/or HDACi was determined in athymic nu/nu mice bearing A2058 and MEWO human melanoma xenografts. High expression of MC1R was observed in situ in clinical melanoma biopsies. BRAFi and HDACi significantly increased the MC1R expression (up to 10-fold in mRNA and 4-fold in protein levels) via MITF-dependent pathways, and this increase led to enhanced ligand binding on the cell surface. Inhibition of MITF expression antagonized the upregulation of MC1R in both BRAFV600E and BRAFWT cells. Combining [212Pb]DOTA-MC1L with BRAFi and/or HDACi improved the tumor response by increasing the delivery of 212Pb α-particle emissions to melanoma tumors via augmented MC1R expression. These data suggest that FDA-approved HDACi and BRAFi could improve the effectiveness of MC1R-targeted therapies by enhancing drug delivery via upregulated MC1R.