Efficacy of horse oil on lipopolysaccharide-induced inflammation in human keratinocyte.

OBJECTIVE: To investigate the efficacy of horse oil on lipopolysaccharide (LPS)-induced inflammation in human keratinocytes. METHODS: Western blot analysis was performed to measure the expression of cyclooxygenase-2 (COX-2) and IκBα. ELISA was used to analyze prostaglandin E2 (PGE2) levels. RESULTS: Horse oil decreased LPS-induced COX-2 and PGE2 levels in a dose-dependent manner. Nuclear factor-kappa B (NF-κB) plays a key role in the expression of inflammatory cytokines and mediators. Therefore, we investigated the influence of horse oil on the NF-κB signaling pathways. Horse oil inhibited translocation of NF-κB from the cytosol to the nucleus. Furthermore, LPS-induced degradation of IκBα was recovered by horse oil. The activation of p38 mitogen-activated protein kinase (MAPK) reportedly induces degradation of IκBα In agreement with this, LPS activated p38 MAPK and caused IκBα degradation. Conversely, horse oil inhibited LPS-induced p38 MAPK activation and IκBα degradation. In addition, a specific p38 MAPK inhibitor, SB203580, blocked IκBα degradation. CONCLUSION: Horse oil decreased COX-2 and PGE2 by inhibiting p38 MAPK activation, IκBα degradation, and the translocation of NF-κB.

LGI3 is secreted and binds to ADAM22 via TRIF-dependent NF-κB pathway in response to LPS in human keratinocytes.

Recently, we reported that HaCaT human keratinocytes secreted leucine-rich repeat LGI family member 3 (LGI3) protein after exposure to ultraviolet B (UVB) irradiation. In the present study, we aimed to determine whether LGI3 is also released in response to stimulation by lipopolysaccharides (LPS), membrane components of gram-negative bacteria. Our results showed that LGI3 was indeed secreted by LPS-stimulated HaCaT cells. We also found that LPS potently stimulated the induction of cycloxygenase-2 (COX-2), which is involved in the inflammatory response. In addition, LPS-induced LGI3 secretion and COX-2 expression were blocked by NS-398, a selective COX-2 inhibitor. Moreover, LPS activated nuclear factor-κB (NF-κB) via a TRIF-dependent pathway, and activated NF-κB led to LGI3 production in HaCaT cells. For the first time, we predicted the LGI3 promoter sequence and demonstrated that NF-κB bound to the LGI3 gene promoter region. LPS treatment also increased the expression of a disintegrin and metalloproteinase domain-containing protein 22 (ADAM22), a candidate LGI3 receptor. Furthermore, co-immunoprecipitation, flow cytometry, and immunocytochemistry revealed that LGI3 associated with ADAM22 in LPS-treated keratinocytes. Thus, ADAM22 may be an LGI3 receptor in human keratinocytes. Taken together, these data suggest that the TRIF-dependent pathway is a novel regulator of LGI3 secretion in response to LPS stimulation in HaCaT cells and that keratinocyte-derived LGI3 interacts with ADAM22 and mediates LPS-induced inflammation.

Leucine rich repeat LGI family member 3: Integrative analyses reveal its prognostic association with non-small cell lung cancer.

Leucine rich repeat LGI family member 3 (LGI3) is a member of the LGI protein family. Our previous studies reported that LGI3 was expressed in adipose tissues, brain and skin, where it served roles as a multifunctional cytokine and pro-inflammatory adipokine. It was hypothesized that LGI3 may be involved in cytokine networks in cancer. The present study aimed to analyze differentially expressed genes in non-small cell lung cancer (NSCLC) tissues and NSCLC cohort data, to evaluate the prognostic role of LGI3. Expression microarray and NSCLC cohort data were statistically analyzed by bioinformatic methods, and protein-protein interactions, functional enrichment and pathway, gene coexpression network (GCN) and prognostic association analyses were performed. The results demonstrated that the expression levels of LGI3 and its receptor a disintegrin and metalloproteinase domain-containing protein 22 were significantly decreased in NSCLC tissues. A total of two upregulated genes and 11 downregulated genes in NSCLC tissues were identified as LGI3-regulated genes. Protein-protein interaction network analysis demonstrated that all LGI3-regulated genes that were altered in NSCLC were involved in a protein-protein interaction network cluster. Functional enrichment, Kyoto Encyclopedia of Genes and Genomes pathway and GCN analyses demonstrated the association of these genes with the immune and inflammatory responses, angiogenesis, the tumor necrosis factor pathway, and chemokine and peroxisome proliferator-activated receptor signaling pathways. Analysis of NSCLC cohorts revealed that low expression levels of LGI3 was significantly associated with poor prognosis of NSCLC. Analysis of the somatic mutations of the LGI3 gene in NSCLC revealed that the amino acid residues altered in NSCLC included two single nucleotide polymorphism sites and three phylogenetically coevolved amino acid residues. Taken together, these results suggest that LGI3 may be a potential prognostic marker of NSCLC.

LGI3 promotes human keratinocyte differentiation via the Akt pathway.

Leucine-rich repeat LGI family member 3 (LGI3), a member of the LGI family, is a secreted protein that is expressed not only in the brain and adipose tissues, but also in various skin cells. We previously reported that LGI3 was secreted after exposure to ultraviolet B and promoted the migration of HaCaT human keratinocytes. In the present study, we investigated whether LGI3 influences the differentiation of keratinocytes. The results show that the expression of involucrin, a keratinocyte differentiation marker, was reduced in tissue from LGI3-knockout mice. Those results indicate that LGI3 plays an important role in keratinocyte differentiation. Therefore, we treated HaCaT cells with LGI3 to examine its effect on keratinocyte differentiation. Protein levels of various differentiation markers were enhanced by treatment with LGI3. Furthermore, expression of differentiation markers was inhibited when keratinocytes were transfected with an siRNA for LGI3. LGI3 strongly activated Akt, whereas it had no apparent effect on extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, or the c-Jun N-terminal kinase. A specific inhibitor of phosphoinositide 3-kinase, LY294002, reduced LGI3-induced expression of differentiation markers in HaCaT cells. Taken together, these results suggest that LGI3 promotes keratinocyte differentiation and could be used as a therapeutic agent to recover skin barrier function in epidermal barrier disruption.

Laminin peptide YIGSR enhances epidermal development of skin equivalents.

Since the use of animal experimentation is restricted with regard to cosmetic materials, alternative in vitro models such as skin equivalents (SEs) are needed. Laminin is one of the major non-collagenous glycoproteins. The pentapeptide YIGSR (Tyr-Ile-Gly-Ser-Arg) is a functional motif of laminin that binds to the laminin receptor. In the present study, we examined whether YIGSR could improve the reconstruction of SEs. YIGSR has no effects on monolayer cell proliferation of CCD25-Sk fibroblasts or HaCaT keratinocytes. Interestingly, YIGSR decreased TGF-ß1 levels, although it promoted type Ι collagen synthesis in CCD25-Sk cells. In HaCaT cells, YIGSR decreased the expression of involucrin and loricrin, which are differentiation markers. Furthermore, YIGSR increased levels of proliferating cell nuclear antigen (PCNA), p63, and integrin α6, and decreased involucrin in SE models. In addition, two models containing YIGSR (mixed with dermal equivalents or added into media) did not show any differences in expression levels of PCNA, p63, integrin α6, and involucrin. Therefore, YIGSR is a useful agent for reconstruction of SEs, independent of its method of application. These results indicate that YIGSR stimulates epidermal proliferation and basement membrane formation while inhibiting keratinocyte differentiation of SEs. Taken together, these results indicate that YIGSR promotes the reconstruction of SEs, potentially via decreased TGF-ß1 levels and consequent inhibition of epidermal differentiation.

Leucine-rich glioma inactivated 3: Integrative analyses reveal its potential prognostic role in cancer.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein in vertebrates, which belongs to the LGI family. In our previous study, LGI3 was found to be expressed in brain, adipose tissues and the skin, where it functions as a multifunctional cytokine. In the present study, we used bioinformatic tools to perform data mining, phylogenetics and prognostic association analysis to investigate the prognostic role of LGI3 in cancers. The sequences of LGI3 orthologues were analyzed from various species, and it was found that LGI3 was highly conserved in mammals and that the subsets of amino acid residues were phylogenetically coevolved in four major clusters. Single nucleotide polymorphisms (SNPs) of the human LGI3 gene included 228 functionally relevant variants (missense, nonsense and frameshift) in a total of 1,042 SNPs. Four missense SNPs had a global minor allele frequency ≥0.001. Somatic mutations in cancer with functional relevance were found in various types of cancer, including uterine, stomach and lung cancer. In addition, five amino acid residues with cancer mutations were shown to be coevolved in the vertebrate phylogeny, suggesting their importance in protein dysfunctions in cancer. One conserved amino acid and three SNPs were found to be mutated in stomach cancer and melanoma. Analysis of expression microarray data demonstrated that the expression of LGI3 was significantly associated with the prognosis of brain, colorectal and lung cancer. Taken together, these results suggested that the genetic variations and expression levels of LGI3 have potential value in cancer prognosis.

Avian Collagen Is Useful for the Construction of Skin Equivalents.

As a result of restrictions on animal experimentation, improved skin equivalents (SEs) are needed as alternative test models. This work investigated the effects of avian collagen on the construction of SEs, and to the best of our knowledge is the first study to do so. Hematoxylin and eosin and immunohistochemical staining were used to analyze the SEs. In models containing avian collagen as a dermal equivalent (DE) ingredient, fibroblast proliferation increased by about 60% relative to the control model. Immunohistochemical staining showed that the expression of proliferating cell nuclear antigen (PCNA) and p63 increased in the avian collagen models, while the expression of involucrin, integrin α6, and integrin ß1 remained unchanged. Next, DEs were cryopreserved to allow the easier creation of SEs. Keratinocytes were seeded on thawed DEs, and SEs were constructed. Avian collagen increased the viability of DEs relative to the control. Furthermore, avian collagen increased the expression of PCNA and p63 in keratinocytes on thawed DEs. The results indicate that DEs containing avian collagen can be thawed as needed after cryopreservation. Avian collagen can improve the construction of SEs and be used as part of a dermal kit for SE construction.

Leucine-rich glioma inactivated 3: integrative analyses support its prognostic role in glioma.

BACKGROUND: Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 was expressed in brain, adipose tissues and skin, where it played roles as a multifunctional cytokine. We postulated that LGI3 may be involved in cytokine network in cancers. AIM: This study aimed to analyze differentially expressed genes in glioma tissues and glioma cohort data to investigate the prognostic role of LGI3 and its receptors. MATERIALS AND METHODS: Expression microarray data from Gene Expression Omnibus and glioma cohort data were analyzed using bioinformatic tools for statistical analysis, protein-protein interactions, functional enrichment and pathway analyses and prognostic association analysis. RESULTS: We found that LGI3 and its receptors, ADAM22 and ADAM23, were significantly downregulated in glioma tissues. Eleven upregulated genes and two downregulated genes in glioma tissues were found to be the previously reported LGI3-regulated genes. Protein-protein interaction network analysis showed that 85% of the LGI3-regulated and glioma-altered genes formed a cluster of interaction network. Functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed the association of these genes with hypoxia responses, p53 and Akt signaling and various cancer-related pathways including glioma. Analysis of expression microarray data of glioma cohorts demonstrated that low expression levels of LGI3, ADAM22 and ADAM23 were significantly associated with poor prognosis of glioma. CONCLUSION: These results propose that LGI3 and its receptors may play a prognostic role in glioma.

Dual hypopigmentary effects of punicalagin via the ERK and Akt pathways.

Punicalagin is a phenolic compound with antioxidant properties. However, the effects of punicalagin on melanin synthesis have been poorly evaluated. Therefore, we investigated the effects of punicalagin on melanogenesis in Mel-Ab cells. Punicalagin significantly inhibited melanin synthesis in a dose-dependent manner. In accordance with the melanin content, punicalagin also dose-dependently decreased tyrosinase activity. Punicalagin did not directly inhibit tyrosinase in a cell-free system but did downregulate the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Therefore, we examined the effects of punicalagin on melanogenesis-related signaling pathways. Punicalagin induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation but had no effect on ß-catenin level. We measured melanin content and MITF expression in the presence of the ERK pathway inhibitor PD98059 and/or the Akt pathway inhibitor LY294002. Cotreatment with PD98059 and LY294002 almost completely restored punicalagin-induced hypopigmentation. These data indicate that punicalagin inhibits melanin synthesis through ERK and Akt phosphorylation, with subsequent downregulation of MITF and tyrosinase.

Leucine-rich glioma inactivated 3: Integrative analyses support its role in the cytokine network.

Leucine-rich glioma inactivated (LGI)3 is a secreted protein member of LGI family. We previously repo-rted that LGI3 was upregulated in adipose tissues from obese mice and suppressed adipogenesis through its receptor, a disintegrin and metalloproteinase domain-containing protein 23 (ADAM23). We demonstrated that LGI3 regulated tumor necrosis factor-α and adiponectin, and proposed that LGI3 may be a pro-inflammatory adipokine involved in adipose tissue inflammation. In this study, we analyzed adipokine and cytokine profiles in LGI3 knockout mice and demonstrated that multiple factors were increased or decreased in the adipose tissues and plasma of the LGI3 knockout mice. Phosphoprotein array analysis revealed increases in the phosphorylation levels of Akt, AMP-activated protein kinase (AMPK), Bad, extracellular signal-regulated kinase (Erk)1/2, glycogen synthase kinase 3α (GSK3α), phosphatase and tensin homolog (PTEN) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the LGI3-treated 3T3-L1 pre-adipocytes. Treatment with LGI3 increased the expression of various inflammatory genes in pre-adipocytes, adipocytes and macrophages. Integrative functional enrichment analysis for all LGI3-regulated gene products suggested their involvement in a number of biological processes, including cancer, inflammatory response, response to wounding, as well as cell proliferation and differentiation. Protein interaction network analysis of LGI3­regulated gene products revealed that 94% of the gene products formed a cluster of interaction networks. Taken together, these results support the critical involvement of LGI3 in the cytokine network by interplaying with multiple adipokines, cytokines and signaling proteins.

Geranylgeranylacetone induces apoptosis via the intrinsic pathway in human melanoma cells.

The aim of this study was to test the anti-cancer effects of geranylgeranylacetone (GGA), an isoprenoid compound, on human melanoma cells. Human melanoma cell lines G361, SK-MEL-2, and SK-MEL-5 were treated with GGA at various doses (1-100µM). Cell viability was measured by crystal violet assay. Western blot analysis was adopted to detect marker proteins of apoptosis. GGA significantly reduced the viability of G361, SK-MEL-2, and SK-MEL-5 human melanoma cells at concentrations above 10µM. Western blot analysis showed the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) after GGA treatment, as well as activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) cleavage. GGA also induced p53 and Bax expression, but did not affect expression of Bcl-2 and MITF. These findings suggest that GGA induces apoptosis through the intrinsic pathway. Accordingly, GGA should be considered for further development as a potential agent for melanoma.

The DNA methylation inhibitor 5-azacytidine decreases melanin synthesis by inhibiting CREB phosphorylation.

Here we examined the effects of a DNA methylation inhibitor, 5-azacytidine, on melanogenesis in Mel-Ab cells. We found that 5-azacytidine decreased the melanin content and tyrosinase activity in these cells in a dose-dependent manner; importantly, 5-azacytidine was not cytotoxic at the concentrations used in these experiments. On the other hand, 5-azacytidine did not affect tyrosinase activity in a cell-free system, indicating that 5-azacytidine is not a direct tyrosinase inhibitor. Instead, 5-azacytidine decreased the protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Thus, we investigated the effects of 5-azacytidine on signal transduction pathways related to melanogenesis. However, 5-azacytidine did not have any effect on either Akt or glycogen synthase kinase 3ß (GSK3ß) phosphorylation. The phosphorylation of cAMP response element-binding protein (CREB) is well known to regulate MITF expression, thereby also regulating tyrosinase expression. We found that 5-azacytidine decreased the phosphorylation of CREB. Therefore, we propose that 5-azacytidine may decrease melanin synthesis by downregulating MITF and tyrosinase via CREB inactivation.

Baicalin-induced Akt activation decreases melanogenesis through downregulation of microphthalmia-associated transcription factor and tyrosinase.

Scutellaria baicalensis has been used topically to treat inflammatory skin diseases in traditional East Asian medicine. Because post-inflammatory hyperpigmentation of the skin is difficult to manage, we investigated the effects of baicalin, a major component of S. baicalensis, on melanin synthesis in Mel-Ab cells. Our data showed that baicalin significantly inhibited melanin production and tyrosinase activity in a dose-dependent fashion, but it did not directly influence tyrosinase activity. Moreover, baicalin treatment triggered decreases in both mRNA and protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Although AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) activation were induced in baicalin-treated Mel-Ab cells, they were not responsible for baicalin-induced hypopigmentation. Because the Akt pathway is also known to be involved in regulation of melanogenic protein expression and melanin synthesis, we examined the effects of baicalin on the Akt pathway. Our results showed that baicalin treatment stimulated Akt activation. Treatment with LY294002, a specific Akt inhibitor, restored baicalin-induced melanogenesis inhibition and abolished MITF and tyrosinase downregulation by baicalin. Taken together, our data suggest that Akt activation by baicalin inhibits melanin production via downregulation of MITF and tyrosinase in Mel-Ab cells.

Methyl gallate from Acer barbinerve decreases melanin synthesis in Mel-Ab cells.

Methyl gallate (MG) was isolated from the bark of Acer barbinerve, which has traditionally been used in Oriental medicine. In the present study, we examined the effects of MG on melanin synthesis in Mel-Ab melanocyte cells. MG decreased melanin pigmentation in a concentration-dependent manner, but did not directly inhibit tyrosinase activity. Further analysis showed that MG had no effect on extracellular signal-regulated kinase (ERK) activation, but induced phosphorylation of glycogen synthase kinase (GSK)3ß, which is known to increase ß-catenin accumulation. Accordingly, the ß-catenin level was increased by MG. However, a specific GSK3ß inhibitor did not rescue the MG-induced inhibition of melanogenesis. Additionally, MG decreased the protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase, which regulate melanin synthesis. Based on these results, we conclude that MG inhibits melanogenesis by decreasing the expression of MITF and tyrosinase.

Leucine-rich glioma inactivated 3 and tumor necrosis factor-α regulate mutually through NF-κB.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 increased in obese adipose tissues and suppressed adipogenesis through its receptor, ADAM23. We proposed that LGI3 may be a pro-inflammatory adipokine secreted predominantly by preadipocytes and macrophages. In this study, we showed that LGI3 and tumor necrosis factor-α (TNF-α) upregulated each other in 3T3-L1 cells. Treatment of 3T3-L1 preadipocytes with LGI3 protein increased TNF-α mRNA and protein. LGI3 treatment led to NF-κB activation and binding to an NF-κB binding site (-523 to -514) in TNF-α promoter. TNF-α treatment increased mRNA and protein expression of LGI3 and ADAM23. TNF-α increased NF-κB binding to a predicted binding site (-40 to -31) in LGI3 promoter. High fat diet-fed mice showed that LGI3 and TNF-α were increased and colocalized in adipose tissue inflammation. Taken together, these results suggested that mutual upregulation of LGI3 and TNF-α may play a role in adipose tissue inflammation in obesity.

ERK Activation by Fucoidan Leads to Inhibition of Melanogenesis in Mel-Ab Cells.

Fucoidan, a fucose-rich sulfated polysaccharide derived from brown seaweed in the class Phaeophyceae, has been widely studied for its possible health benefits. However, the potential of fucoidan as a possible treatment for hyperpigmentation is not fully understood. This study investigated the effects of fucoidan on melanogenesis and related signaling pathways using Mel-Ab cells. Fucoidan significantly decreased melanin content. While fucoidan treatment decreased tyrosinase activity, it did not do so directly. Western blot analysis indicated that fucoidan downregulated microphthalmia-associated transcription factor and reduced tyrosinase protein expression. Further investigation showed that fucoidan activated the extracellular signal-regulated kinase (ERK) pathway, suggesting a possible mechanism for the inhibition of melanin synthesis. Treatment with PD98059, a specific ERK inhibitor, resulted in the recovery of melanin production. Taken together, these findings suggest that fucoidan inhibits melanogenesis via ERK phosphorylation.

Fucoidan promotes the reconstruction of skin equivalents.

In this study we investigated the effects of fucoidan on the proliferation of fibroblasts and the reconstruction of a skin equivalent (SE). Fucoidan significantly stimulated the proliferation of CCD-25Sk human fibroblasts and Western blot analysis demonstrated that fucoidan markedly increased the expression of cyclin D1 and decreased the expression of p27. Fucoidan was used to reconstruct SE. Immunohistochemical staining showed that the addition of fucoidan to dermal equivalents increased expression of proliferating cell nuclear antigen (PCNA) and p63. In addition, expression of α6-integrin was significantly increased by fucoidan, whereas expression of ß1-integrin, type 1 collagen, elastin, fibronectin did not markedly change. These results suggest that fucoidan has positive effects on epidermal reconstruction and will therefore be beneficial in the reconstruction of SE.

KHG26792 Inhibits Melanin Synthesis in Mel-Ab Cells and a Skin Equivalent Model.

The purpose of this study is to characterize the effects of KHG26792 (3-(naphthalen-2-yl(propoxy) methyl)azetidine hydrochloride), a potential skin whitening agent, on melanin synthesis and identify the underlying mechanism of action. Our data showed that KHG26792 significantly reduced melanin synthesis in a dose-dependent manner. Additionally, KHG26792 downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase, the rate-limiting enzyme in melanogenesis, although tyrosinase was not inhibited directly. KHG26792 activated extracellular signal-regulated kinase (ERK), whereas an ERK pathway inhibitor, PD98059, rescued KHG26792-induced hypopigmentation. These results suggest that KHG26792 decreases melanin production via ERK activation. Moreover, the hypopigmentary effects of KHG26792 were confirmed in a pigmented skin equivalent model using Cervi cornus Colla (deer antler glue), in which the color of the pigmented artificial skin became lighter after treatment with KHG26792. In summary, our findings suggest that KHG26792 is a novel skin whitening agent.

Leucine-rich glioma inactivated 3 is a melanogenic cytokine in human skin.

Recently, we demonstrated that leucine-rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the effects of LGI3 on melanocytes remain unknown. The present study demonstrated that LGI3 can serve to stimulate melanogenesis without affecting cell viability. To determine the effects of LGI3 on melanin synthesis, normal human melanocytes and Mel-Ab cells were treated with recombinant LGI3 and melanin content was measured. Our results showed that LGI3 promoted melanin synthesis in both cell types. Moreover, upregulation of microphthalmia-associated transcription factor (MITF) and tyrosinase was observed at both the mRNA and protein levels via RT-PCR and Western blotting, respectively. Furthermore, immunohistochemical staining showed that the expression of LGI3 increased in the basal layer of melasma skin samples, whereas it decreased slightly in vitiligo samples. These results suggest that LGI3 may play a role as a melanogenic cytokine in human skin.

Myriocin, a serine palmitoyltransferase inhibitor, increases melanin synthesis in Mel-Ab cells and a skin equivalent model.

The purpose of this study was to investigate effects of myriocin, an inhibitor of serine palmitoyltransferase, on melanogenesis. It was found that myriocin increased melanin synthesis in a concentration-dependent manner. Moreover, myriocin up-regulated microphthalmia-associated transcription factor (MITF) and tyrosinase expression via phosphorylation of CREB, but it did not directly activate tyrosinase, a rate-limiting melanogenic enzyme. Furthermore, we demonstrated increased melanin synthesis with myriocin on a pigmented skin equivalent model established using Cervi cornus Colla (deer antler glue). One and 5 microM of myriocin darkened the color of the skin equivalent. These results suggest that myriocin may have potential effects for the treatment of hypopigmentary skin diseases like vitiligo or for sunless tanning.