Discovery of PPARγ and glucocorticoid receptor dual agonists to promote the adiponectin and leptin biosynthesis in human bone marrow mesenchymal stem cells.

Adiponectin and leptin are major adipocytokines that control crosstalk between adipose tissue and other organ systems. Hypoadiponectinemia and hypoleptinemia are associated with human metabolic diseases. Compounds with adipocytokine biosynthesis-stimulating activities could be developed as therapeutics against diverse metabolic conditions. In phenotypic screening with human bone marrow mesenchymal stem cells (hBM-MSCs), (E)-4-hydroxy-3-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)-6-methyl-2H-pyran-2-one (1) was identified to increase adiponectin biosynthesis during adipogenesis and simultaneously to stimulate leptin production. Using the compound 1 structure, the structure-activity relationship study was performed to discover more potent compounds stimulating both adiponectin and leptin production. (E)-3-(3-(2-fluoropyridin-4-yl)acryloyl)-4-hydroxy-6-methyl-2H-pyran-2-one (11) exhibited the most potent adiponectin (EC50, 2.87 µM) and leptin (EC50, 2.82 µM) biosynthesis-stimulating activities in hBM-MSCs. In a target identification study, compound 11 was characterized as a dual modulator binding to both peroxisome proliferator-activated receptor (PPAR) γ and glucocorticoid receptor (GR). This study provides a novel pharmacophore for PPARγ/GR dual modulators with therapeutic potential against human metabolic diseases.

3,4,5-Trimethoxycinnamate thymol ester inhibits melanogenesis in normal human melanocytes and 3D human epidermal equivalents via the PGC-1α-independent PPARγ partial agonism.

BACKGROUND: 3,4,5-Trimethoxycinnamate thymol ester (TCTE), an anti-melanogenic cosmetic agent prescribed currently, promotes adiponectin synthesis during adipogenesis in human bone marrow mesenchymal stem cells (hBM-MSCs). Adiponectin inhibits melanin biosynthesis and its biosynthesis is directly regulated by peroxisome proliferator-activated receptor (PPAR) γ. In this regard, TCTE may potentially affect PPARγ activity. However, contradicting effects of PPARγ agonists with different chemical structures on human melanogenesis have been reported. OBJECTIVE: A molecular target of TCTE was investigated to elucidate the association of both adiponectin and PPARγ with anti-melanogenic activity. METHODS: The adiponectin secretion-promoting activity of TCTE was tested in an adipogenesis model of hBM-MSCs. A molecular target of TCTE for adiponectin secretion was evaluated via time-resolved fluorescence resonance energy transfer-based receptor binding and transactivation of PPARs. RESULTS: TCTE significantly promoted adiponectin secretion (EC50, 27.9 µM) during adipogenesis in hBM-MSCs and directly bound to PPARγ (Ki, 13.2 µM). The TCTE-bound PPARγ increased the recruitment of SRC-1, SRC-3, and TRAP220/DRIP-1 coactivator peptides without affecting PGC-1α coactivation. In the docking analysis, the optimal ligand binding mode of TCTE exhibited typical ligand-receptor interactions of PPARγ partial agonists. The PPARγ partial agonism of TCTE was established experimentally and the anti-melanogenic activity of TCTE was decreased by treatment with a PPARγ antagonist in cultured normal human melanocytes and a 3D model of human epidermis. CONCLUSION: The anti-melanogenic activity of TCTE was associated with a PGC-1α-independent PPARγ partial agonism.

Sunscreen filter octocrylene is a potential obesogen by acting as a PPARγ partial agonist.

Octocrylene (OC) is an extensively prescribed organic ultraviolet B filter used in sunscreen products. Due to its extensive use, a significant level of OC is detected in marine and freshwater environments. Notably, the bioaccumulation of OC in aquatic biota may affect human health. In this study, the effect of OC on metabolism was investigated using the adipogenesis model of human bone marrow mesenchymal stem cells (hBM-MSCs). OC promoted adiponectin production during adipogenesis in hBM-MSCs compared to the vehicle-treated control (EC50, 29.6 µM). In target identification, OC directly bound to peroxisome proliferator-activated receptor (PPAR) γ (Ki, 37.8 µM). OC-bound PPARγ also significantly recruited nuclear receptor coactivator proteins SRC-1 (EC50, 54.1 µM) and SRC-2 (EC50, 58.6 µM). In the molecular docking simulation study, the optimal ligand-binding mode of OC suggested that OC is a PPARγ partial agonist. A competitive analysis with a PPARγ full agonist pioglitazone revealed that OC acted as a PPARγ partial agonist. OC altered the gene transcription profile of lipid-metabolism associated enzymes in normal human keratinocytes, primarily exposed human cells after the application of sunscreens. In conclusion, OC is a potential metabolic disrupting obesogen.

Diallyl Biphenyl-Type Neolignans Have a Pharmacophore of PPARα/γ Dual Modulators.

Adiponectin secretion-promoting compounds have therapeutic potentials in human metabolic diseases. Diallyl biphenyl-type neolignan compounds, magnolol, honokiol, and 4-O-methylhonokiol, from a Magnolia officinalis extract were screened as adiponectin- secretion promoting compounds in the adipogenic differentiation model of human bone marrow mesenchymal stem cells (hBM-MSCs). In a target identification study, magnolol, honokiol, and 4-O-methylhonokiol were elucidated as PPARα and PPARγ dual modulators. Diallyl biphenyl-type neolignans affected the transcription of lipid metabolism-associated genes in a different way compared to those of specific PPAR ligands. The diallyl biphenyl-type neolignan structure provides a novel pharmacophore of PPARα/γ dual modulators, which may have unique therapeutic potentials in diverse metabolic diseases.

Benzophenone-3 and benzophenone-8 exhibit obesogenic activity via peroxisome proliferator-activated receptor γ pathway.

Benzophenone-3 (BP-3) and benzopenone-8 (BP-8) are commonly used ultraviolet (UV) filter ingredients in diverse sunscreen products. Recently, the obesogenic activity of avobenzone, a long wave UV A filter, was elucidated in the adipogenesis model of human bone marrow mesenchymal stem cells (hBM-MSCs). In this study, the obesogenic potentials of BP-3 and BP-8 were investigated because of their chemical similarity to avobenzone. During the adipogenesis in hBM-MSCs, BP-3 and BP-8 (EC50, 25.05 and 43.20 µM, respectively) potently promoted adiponectin secretion than avobenzone (EC50, 72.69 µM). In target identification, both BP-3 and BP-8 directly bound to peroxisome proliferator-activated receptor γ (PPARγ), which was associated with the recruitment of steroid receptor coactivator-2 (SRC-2). BP-3 functioned as a PPARγ full agonist whereas BP-8 was a PPARγ partial agonist. In addition, BP-3 and BP-8 significantly increased the gene transcription of PPARα, PPARγ, and major lipid metabolism-associated enzymes in human epidermal keratinocytes, a major target site of UV filters in human skin. This study suggests that BP-3 and BP-8 are obesogenic environmental chemicals similar to phthalates, bisphenols, and organotins.

CXCL14 downregulation in human keratinocytes is a potential biomarker for a novel in vitro skin sensitization test.

To elucidate the roles of epidermal keratinocytes in the toxicological outcomes of chemically induced contact dermatitis, genome-scale transcriptional analyses were performed using normal human keratinocytes (NHKCs) treated with 10 µM sodium lauryl sulfate (SLS) or 5 µM urushiol. In Gene Ontology (GO) enrichment analyses, SLS- and urushiol-induced upregulated DEGs are commonly associated with the regulation of pro-inflammatory responses and epidermal differentiation processes in NHKCs whereas cellular protein metabolic process was also identified as a commonly downregulated DEG signature. Among the downregulated DEGs, CXCL14 was investigated as a potential biomarker for a new in vitro skin sensitization test using OECD TG429 reference chemicals. CXCL14 was significantly downregulated in NHKCs in response to 62.5% of the OECD TG429 sensitizers in a concentration-dependent manner. When the sensitizer-induced upregulation of chemokine CXCL8 was included in the analysis, 87.5% of the OECD TG429 reference sensitizing chemicals significantly induced either CXCL8 upregulation or CXCL14 downregulation in NHKCs. Only one OECD TG429 reference non-sensitizer changed the constitutive CXCL14 expression in NHKCs whereas five out of six non-sensitizers altered CXCL8 production. The reference irritating non-sensitizer SLS caused a false-positive outcome. The downregulation of constitutively expressed CXCL14 was regulated by both the MAPK/ERK and JAK3/STAT6 pathways in NHKCs. CXCL14 can be used as a mechanism-based biomarker in the development of in vitro skin sensitization tests and may help improve the distinction between allergenic sensitizers and non-sensitizers.

Keratinocyte-derived IL-36γ plays a role in hydroquinone-induced chemical leukoderma through inhibition of melanogenesis in human epidermal melanocytes.

Chemical leukoderma is an acquired type of vitiligo that can be initiated by various exogenous chemicals such as hydroquinone (HQ), rhododendrol (RD), or 4-tertiary butyl phenol (4-TBP). Despite the importance of epidermal keratinocytes in diverse dermatological conditions, their toxicological role in chemical leukoderma is poorly understood. To elucidate their role in the pathogenesis of chemical leukoderma, genome-scale transcriptional analysis was performed in human epidermal keratinocytes (HEKs) treated with a sub-cytotoxic HQ concentration (10 µM). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based functional enrichment analysis of HQ-induced differentially expressed genes (DEGs) revealed that HQ significantly upregulated DEGs related to the IL-17 signaling pathway and significantly downregulated DEGs associated with melanogenesis in HEKs. The meta-analysis between the HQ-induced and cytokine-induced transcriptional data (GSE53751) showed that 58 DEGs were commonly upregulated between HQ- and IL-17A-treated HEKs. Notably, the expression of IL36G was significantly increased in HEKs in response to both HQ and IL-17A. IL-36γ (2 µg/ml) directly inhibits melanin biosynthesis in cultured human epidermal melanocytes (HEMs) and downregulates the gene transcription of key enzymes in the melanogenesis pathway including TYR, DCT, and TYRP1. Moreover, IL-36γ autocrinally regulated keratinocyte function to produce the proinflammatory cytokines IL-36γ, IL-6, and CXCL8/IL-8 in a concentration-dependent manner, suggesting that IL-36γ may stimulate the amplification cycle of cutaneous inflammation. In this regard, hydroquinone-induced IL-36γ from human keratinocytes plays a pivotal role in the development of chemical leukoderma by autocrinally or paracrinally modulating the crosstalk between keratinocytes and melanocytes.

A long-wave UVA filter avobenzone induces obesogenic phenotypes in normal human epidermal keratinocytes and mesenchymal stem cells.

Avobenzone is the most commonly used ultraviolet (UV) A filter ingredient in sunscreen. To investigate the biological activity of avobenzone in normal human epidermal keratinocytes (NHEKs), the genome-scale transcriptional profile of NHEKs was performed. In this microarray study, we found 273 up-regulated and 274 down-regulated differentially expressed genes (DEGs) in NHEKs treated with avobenzone (10 µM). Gene Ontology (GO) enrichment analysis showed that avobenzone significantly increased the DEGs associated with lipid metabolism in NHEKs. In addition, avobenzone increased the gene transcription of peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid binding protein 4 in NHEKs, implicating that avobenzone may be one of the metabolic disrupting obesogens. To confirm the obesogenic potential, we examined the effect of avobenzone on adipogenesis in human bone marrow mesenchymal stem cells (hBM-MSCs). Avobenzone (EC50, 14.1 µM) significantly promoted adipogenesis in hBM-MSCs as its positive control obesogenic chemicals. Avobenzone (10 µM) significantly up-regulated mRNA levels of PPARγ during adipogenesis in hBM-MSCs. However, avobenzone did not directly bind to PPARγ and the avobenzone-induced adipogenesis-promoting activity was not affected by PPARγ antagonists T0070907 and GW9662. Therefore, avobenzone promoted adipogenesis in hBM-MSCs through a PPARγ-independent mechanism. This study suggests that avobenzone functions as a metabolic disrupting obesogen.

Kojyl cinnamate esters are peroxisome proliferator-activated receptor α/γ dual agonists.

Adiponectin is an adipocytokine with insulin-sensitizing, anti-inflammatory, anti-atherosclerotic, and anti-aging properties. Compounds with the ability to promote adiponectin secretion are of interest for the development of anti-aging drugs to improve skin-aging phenotypes. In the phenotypic assay to measure adiponectin secretion during adipogenesis in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs), kojyl cinnamate ester derivatives increased adiponectin secretion. A target identification study showed that the kojyl cinnamate ester derivatives competitively bound to peroxisome proliferator-activated receptor α/γ (PPARα/γ). The upregulation of adiponectin production induced by kojyl cinnamate ester derivatives was significantly correlated with PPARα and PPARγ binding activities. Kojyl cinnamate ester derivatives significantly increased the transcription of genes encoding cholesterol and fatty acid synthesizing enzymes in hAT-MSCs. Notably, the kojyl cinnamate esters upregulated the gene transcription of lipid metabolic enzymes in human epidermal keratinocytes, which are important in the integrity of skin permeability barrier. In addition, the kojyl cinnamate esters that function as PPARα/γ dual modulators inhibited ultraviolet B irradiation-induced inflammation in human epidermal keratinocytes. Therefore, kojyl cinnamate ester derivatives are a novel class of PPARα/γ dual agonists with the potential to improve skin-aging phenotypes.

Leptin regulates the pro-inflammatory response in human epidermal keratinocytes.

The role of leptin in cutaneous wound healing process has been suggested in genetically obese mouse studies. However, the molecular and cellular effects of leptin on human epidermal keratinocytes are still unclear. In this study, the whole-genome-scale microarray analysis was performed to elucidate the effect of leptin on epidermal keratinocyte functions. In the leptin-treated normal human keratinocytes (NHKs), we identified the 151 upregulated and 53 downregulated differentially expressed genes (DEGs). The gene ontology (GO) enrichment analysis with the leptin-induced DEGs suggests that leptin regulates NHKs to promote pro-inflammatory responses, extracellular matrix organization, and angiogenesis. Among the DEGs, the protein expression of IL-8, MMP-1, fibronectin, and S100A7, which play roles in which is important in the regulation of cutaneous inflammation, was confirmed in the leptin-treated NHKs. The upregulation of the leptin-induced proteins is mainly regulated by the STAT3 signaling pathway in NHKs. Among the downregulated DEGs, the protein expression of nucleosome assembly-associated centromere protein A (CENPA) and CENPM was confirmed in the leptin-treated NHKs. However, the expression of CENPA and CENPM was not coupled with those of other chromosome passenger complex like Aurora A kinase, INCENP, and survivin. In cell growth kinetics analysis, leptin had no significant effect on the cell growth curves of NHKs in the normal growth factor-enriched condition. Therefore, leptin-dependent downregulation of CENPA and CENPM in NHKs may not be directly associated with mitotic regulation during inflammation.

Phosphodiesterase 4B plays a role in benzophenone-3-induced phototoxicity in normal human keratinocytes.

Benzophenone-3 (BP-3), which is extensively used in organic sunscreen, has phototoxic potential in human skin. Phosphodiesterase 4B (PDE4B) has a well-established role in inflammatory responses in immune cells. Currently, it is unknown if PDE4B is associated with BP-3-induced phototoxicity in normal human keratinocytes (NHKs). We found that BP-3 significantly increased PDE4B expression in ultraviolet B (UVB)-irradiated NHKs. Notably, BP-8, a sunscreen agent that shares the 2-hydroxy-4-methoxyphenyl methanone moiety with BP-3, also upregulated PDE4B expression in NHKs. Upon UVB irradiation, BP-3 upregulated the expression of pro-inflammatory factors, such as prostaglandin endoperoxide synthase 2, tumor necrosis factor α, interleukin 8, and S100A7, and downregulated the level of cornified envelope associated proteins, which are important in the development of the epidermal permeability barrier. The additive effects of UVB-activated BP-3 on the expression of both pro-inflammatory mediators and cornified envelope associated proteins were antagonized by treatment with the PDE4 inhibitor rolipram. The BP-3 and UVB co-stimulation-induced PDE4B upregulation and its association with the upregulation of pro-inflammatory mediators and the downregulation of epidermal differentiation markers were confirmed in a reconstituted three dimensional human epidermis model. Therefore, PDE4B has a role in the mechanism of BP-3-induced phototoxicity.

Polypharmacology of N6-(3-Iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and Related A3 Adenosine Receptor Ligands: Peroxisome Proliferator Activated Receptor (PPAR) γ Partial Agonist and PPARδ Antagonist Activity Suggests Their Antidiabetic Potential.

A3 adenosine receptor (AR) ligands including A3 AR agonist, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (1a, IB-MECA) were examined for adiponectin production in human bone marrow mesenchymal stem cells (hBM-MSCs). In this model, 1a significantly increased adiponectin production, which is associated with improved insulin sensitivity. However, A3 AR antagonists also promoted adiponectin production in hBM-MSCs, indicating that the A3 AR pathway may not be directly involved in the adiponectin promoting activity. In a target deconvolution study, their adiponectin-promoting activity was significantly correlated to their binding activity to both peroxisome proliferator activated receptor (PPAR) γ and PPARδ. They functioned as both PPARγ partial agonists and PPARδ antagonists. In the diabetic mouse model, 1a and its structural analogues A3 AR antagonists significantly decreased the serum levels of glucose and triglyceride, supporting their antidiabetic potential. These findings indicate that the polypharmacophore of these compounds may provide therapeutic insight into their multipotent efficacy against various human diseases.

Cystathionine metabolic enzymes play a role in the inflammation resolution of human keratinocytes in response to sub-cytotoxic formaldehyde exposure.

Low-level formaldehyde exposure is inevitable in industrialized countries. Although daily-life formaldehyde exposure level is practically impossible to induce cell death, most of mechanistic studies related to formaldehyde toxicity have been performed in cytotoxic concentrations enough to trigger cell death mechanism. Currently, toxicological mechanisms underlying the sub-cytotoxic exposure to formaldehyde are not clearly elucidated in skin cells. In this study, the genome-scale transcriptional analysis in normal human keratinocytes (NHKs) was performed to investigate cutaneous biological pathways associated with daily life formaldehyde exposure. We selected the 175 upregulated differentially expressed genes (DEGs) and 116 downregulated DEGs in NHKs treated with 200µM formaldehyde. In the Gene Ontology (GO) enrichment analysis of the 175 upregulated DEGs, the endoplasmic reticulum (ER) unfolded protein response (UPR) was identified as the most significant GO biological process in the formaldeyde-treated NHKs. Interestingly, the sub-cytotoxic formaldehyde affected NHKs to upregulate two enzymes important in the cellular transsulfuration pathway, cystathionine γ-lyase (CTH) and cystathionine-ß-synthase (CBS). In the temporal expression analysis, the upregulation of the pro-inflammatory DEGs such as MMP1 and PTGS2 was detected earlier than that of CTH, CBS and other ER UPR genes. The metabolites of CTH and CBS, l-cystathionine and l-cysteine, attenuated the formaldehyde-induced upregulation of pro-inflammatory DEGs, MMP1, PTGS2, and CXCL8, suggesting that CTH and CBS play a role in the negative feedback regulation of formaldehyde-induced pro-inflammatory responses in NHKs. In this regard, the sub-cytotoxic formaldehyde-induced CBS and CTH may regulate inflammation fate decision to resolution by suppressing the early pro-inflammatory response.

Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD.

Human melanocytes form a PAX3-expressing melanocyte cluster on Matrigel by the cell migration process.

BACKGROUND: The interactions between human epidermal melanocytes and their cellular microenvironment are important in the regulation of human melanocyte functions or in their malignant transformation into melanoma. Although the basement membrane extracellular matrix (BM-ECM) is one of major melanocyte microenvironments, the effects of BM-ECM on the human melanocyte functions are not fully explained at a molecular level. OBJECTIVE: This study was aimed to characterize the molecular and cellular interactions between normal human melanocytes (NHMs) and BM-ECM. METHODS: We investigated cell culture models of normal human melanocytes or melanoma cells on three-dimensional (3D) Matrigel to understand the roles of the basement membrane microenvironment in human melanocyte functions. Melanogenesis and melanobast biomarker expression in both primary human melanocytes and melanoma cells on 3D Matrigel were evaluated. RESULTS: We found that NHMs migrated and formed reversible paired box 3 (PAX3) expressing cell clusters on three-dimensional (3D) Matrigel. The melanogenesis was significantly decreased in the PAX3 expressing cell cluster. The expression profile of PAX3, SOX10, and MITF in the melanocyte cluster on 3D Matrigel was similar to that of melanoblasts. Interestingly, PAX3 and SOX10 showed an inverse expression profile in NHMs, whereas the inverse expression pattern of PAX3 and SOX10 was disrupted in melanoma MNT1 and WM266-4 cells. CONCLUSION: The human melanocyte culture on 3D Matrigel provides an alternative model system to study functions of human melanoblasts. In addition, this system will contribute to the elucidation of PAX3-related tumorigenic mechanisms to understand human melanoma.

Keratinocyte-derived IL-24 plays a role in the positive feedback regulation of epidermal inflammation in response to environmental and endogenous toxic stressors.

Keratinocytes are the major cellular components of human epidermis and play a key role in the modulating cutaneous inflammation and toxic responses. In human chronic skin diseases, the common skin inflammatory phenotypes like skin barrier disruption and epidermal hyperplasia are manifested in epidermal keratinocytes by interactions with T helper (Th) cells. To find a common gene expression signature of human keratinocytes in chronic skin diseases, we performed a whole genome microarray analysis on normal human epidermal keratinocytes (NHKs) treated with IFNγ, IL-4, IL-17A or IL-22, major cytokines from Th1, Th2, Th17 or Th22 cells, respectively. The microarray results showed that the four genes, IL-24, PDZK1IP1, H19 and filaggrin, had common expression profiles in NHKs exposed to Th cell cytokines. In addition, the acute phase pro-inflammatory cytokines, IL-1ß, IL-6 and TNFα, also change the gene transcriptional profile of IL-24, PDZK1IP1, H19, and filaggrin in NHKs as those of Th cytokines. Therefore, the signature gene set, consisting of IL-24, PDZK1IP1, H19, and filaggrin, provides essential insights for understanding the process of cutaneous inflammation and toxic responses. We demonstrate that environmental toxic stressors, such as chemical irritants and ultraviolet irradiation stimulate the production of IL-24 in NHKs. IL-24 stimulates the JAK1-STAT3 and MAPK pathways in NHKs, and promotes the secretion of pro-inflammatory mediators IL-8, PGE2, and MMP-1. These results suggest that keratinocyte-derived IL-24 participates in the positive feedback regulation of epidermal inflammation in response to both endogenous and environmental toxic stressors.

IL-4 inhibits the melanogenesis of normal human melanocytes through the JAK2-STAT6 signaling pathway.

Skin diseases can be characterized by their predominant CD4-positive T-helper (Th) cell profiles. Chronic dermatological conditions often give rise to abnormal skin pigmentation. To understand the role of Th cells in pigmentation, the effects of the major Th cell cytokines, IFNγ, IL-4, and IL-17A, on melanogenesis were evaluated using cultured normal human melanocytes (NHMs) instead of relying on transformed melanoma cell lines. IL-4 directly inhibited melanogenesis in NHMs and downregulated both transcription and translation of melanogenesis-associated genes, such as microphthalmia-associated transcription factor (MITF) and dopachrome tautomerase. Despite the lack of a direct inhibition of melanin pigment synthesis, IFNγ and IL-17A increased the synthesis of an antimelanogenic cytokine IL-6 in NHMs. IFNγ activated signal transducers and activators of transcription 1 (STAT1) and STAT3 phosphorylation in NHMs, and IL-4 increased the STAT3 and STAT6 phosphorylation. The differential phosphorylation profile of STAT proteins between IFNγ and IL-4 may explain the difference in their effect on melanogenesis in NHMs. The IL-4-induced downregulation of melanogenesis was inhibited by treating NHMs with a JAK2 inhibitor AG490 or STAT6 siRNA. In conclusion, the involvement of the IL-4-induced JAK2-STAT6 signaling and the IFNγ- or IL-17A-dependent antimelanogenic IL-6 production should be considered as one of the mechanisms explaining the association with hypopigmention in skin diseases.

Kojic acid-induced IL-6 production in human keratinocytes plays a role in its anti-melanogenic activity in skin.

BACKGROUND: Kojic acid is a fungal metabolite widely used in medicinal and cosmetic formulations as a skin-lightening agent based on its de-pigmenting activity. Although in human clinical studies kojic acid has been shown to be effective in the treatment of hyper-pigmentation disorders such as melasma, the reasons for its apparent lack of anti-melanogenic activity in cultured mammalian melanocytes are unclear. OBJECTIVES: This study was aimed to elucidate pharmacological mechanisms of the in vivo anti-melanogenic activity of kojic acid in human skin. METHODS: A primary human melanocyte and keratinocyte co-culture system was used to evaluate whether kojic-acid-induced changes in keratinocytes were associated with anti-melanogenic activities in melanocytes. The cytokine secretion profiles in response to kojic acid were analyzed. RESULTS: Kojic acid increased interleukin (IL)-6 and IL-8 production in melanocyte/keratinocyte co-cultures; however, IL-6 directly inhibited melanogenesis whereas IL-8 did not. In melanocyte monocultures, kojic acid did not increase IL-6 production whereas in keratinocyte monocultures it significantly up-regulated IL-6 gene and protein expression. Therefore, the up-regulation of IL-6 in melanocyte/keratinocyte co-cultures seems to be originated from kojic acid-induced changes in keratinocytes. Anti-IL-6 antibody treatment antagonized the anti-melanogenic effect of kojic acid on the co-cultures. CONCLUSIONS: The pharmacological mechanism of kojic acid to explain clinically effective anti-melanogenic activity on hyper-pigmented skin is associated with the kojic acid-induced IL-6 production in keratinocytes. The cross-talk between melanocytes and keratinocytes should be determined in future studies on the pharmacological mechanisms of clinically effective dermatological drugs acting on the epidermis.

The retinoic acid-induced up-regulation of insulin-like growth factor 1 and 2 is associated with prolidase-dependent collagen synthesis in UVA-irradiated human dermal equivalents.

BACKGROUND: Ultraviolet (UV) A irradiation causes the degeneration of extracellular matrix in the skin dermis, mainly due to disrupted collagen homeostasis, resulting in the photo-aging of human skin. All-trans retinoic acid (ATRA) improves photo-aged human skin in vivo. OBJECTIVES: Although the effects of ATRA on collagen synthesis and MMP regulation are well known, the effects of ATRA on other collagen homeostasis-associated genes have not been elucidated. This study was aimed to study the factors that are pharmacologically associated with the effect of ATRA on collagen homeostasis. METHODS: The gene transcription profile of collagen homeostasis-associated genes was systematically evaluated in three-dimensional human dermal equivalents (HDEs) following UVA-irradiation and/or ATRA treatment. RESULTS: In addition to the expected changes in MMPs and collagen synthesis in HDEs in response to ATRA, prolidase, an important enzyme in the recycling of proline and hydroxyproline from degraded collagen molecules, was significantly decreased by UVA irradiation, and its down-regulation was antagonized by ATRA. Transfection with a prolidase-specific siRNA led to a significant decrease in procollagen synthesis in human fibroblasts. ATRA inhibited the UVA irradiation-induced decrease in prolidase activity through an insulin-like growth factor (IGF) receptor signaling pathway in HDEs. ARTA increased IGF1 and IGF2 production in HDEs, and neutralizing IGFs with anti-IGF antibodies abolished the effect of ATRA on proliase activity. CONCLUSIONS: These data demonstrate that ATRA regulates prolidase activity in HDEs via IGF receptor signaling, suggesting one of the pharmacological mechanisms by which improves photo-aged human skin.

Activation of Toll-like Receptors 1, 2, 4, 5, and 7 on Human Melanocytes Modulate Pigmentation.

Human melanocytes are not simply pigment-producing cells. It may be part of the inflammatory response, during which the pigmentary system may produce more melanin or suppress melanization. Toll-like receptors (TLRs) have been implicated in both innate host defense against pathogens and inflammatory response. Therefore, it may be possible that activation of TLRs in melanocytes may play a role in the modulation of melanogenesis. In this study, we investigated whether normal human melanocytes expressed TLRs and analyzed pigmentation changes upon TLR stimulation. The expression of TLR1~10 mRNA in cultured human melanocyte was analyzed using RT-PCR, Western blotting and immunocytochemistry. Human melanocytes constitutively express mRNA and protein for TLR2, 3, 4, 5, 7, 9 and 10. Stimulation of TLR1/2 and 4 with Pam3CSK4 and lipopolysaccharide induced pigmentation of melanocytes. Activation of TLR5 and 7 with flagellin and imiquimod treatments reduced pigmentation of melanocytes and zebrafish. In summary, the results provided evidence for TLRs expression in normal human melanocytes. It is speculated that a response of melanocyte to TLR ligands may play a role in the pigmentary change in the skin.