L-765,314 Suppresses Melanin Synthesis by Regulating Tyrosinase Activity.

Although melanin production is a key self-defense mechanism against ultraviolet radiation (UVR)-induced skin damage, uneven or excessive deposition of melanin causes hyperpigmentary disorders. Currently available whitening agents are unsatisfactory because of issues with efficacy and safety. To develop more effective depigmenting agents, we performed high-throughput melanin content assay screening using the B16F10 melanoma cell line and identified L-765,314 as a drug that suppressed melanin production in cultured melanocytes in a dose-dependent manner as well as cAMP- or 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated melanin production without cytotoxicity. Interestingly, melanogenic gene expression was not altered by L-765,314. Rather, diminished melanin production by L-765,314 appeared to be caused by downregulation of tyrosinase activity via inhibition of protein kinase C (PKC). Because L-765,314 did not show any adverse effect in melanocytes, altogether our data suggest that L-765,314 could be a potential therapeutic candidate for skin hyperpigmentary disorders and further discovery of selective inhibitors targeting PKC might be a promising strategy for the development of depigmenting agents to treat hyperpigmentary disorders.

Inhibitory effects of N,N,N-trimethyl phytosphingosine-iodide on melanogenesis via ERK activation-mediated MITF degradation.

N,N,N-trimethyl phytosphingosine-iodide (TMP) was recently developed as an antitumor agent. We examined the effects of TMP on melanogenesis and its related signaling pathways in normal human melanocytes. Our results showed that melanin is significantly reduced in a dose-dependent manner in both cells following liposomal TMP treatment. We also investigated changes in the phosphorylation of extracellular signal-regulated kinase (ERK), which is related to the degradation of microphthalmia-associated transcription factor (MITF). Our results indicated that liposomal TMP treatment leads to the phosphorylation of ERK, which reduces both MITF and tyrosinase protein levels. Treatment with PD98059, an ERK pathway-specific inhibitor, restored liposomal TMP-induced reductions in melanin, abrogated reductions in tyrosinase activity, and downregulated MITF and tyrosinase protein. In conclusion, these results suggest that the inhibitory effects of TMP on melanogenesis are due to MITF and tyrosinase downregulation via ERK activation.

Low vitamin D levels are associated with atopic dermatitis, but not allergic rhinitis, asthma, or IgE sensitization, in the adult Korean population.

BACKGROUND: The effect of vitamin D on allergic conditions is unclear. In particular, large-scale, population-based studies examining this relationship in adult Asian populations are lacking. OBJECTIVE: To evaluate the association between serum vitamin D levels and allergic conditions in the general adult Korean population. METHODS: A cross-sectional study was performed by using data collected from 15,212 individuals 19 years or older who participated in the Korean National Health and Nutrition Examination Survey from 2008 to 2010. The confounder-adjusted mean serum 25-hydroxyvitamin D (25[OH]D) levels of participants with and without allergic conditions (including atopic dermatitis, asthma, allergic rhinitis, and increased total and allergen-specific serum IgE) were compared by using multiple linear regression analyses. Multiple logistic regression analyses with confounder adjustment estimated the odds ratios (ORs) for developing each condition according to adequate, inadequate, or deficient serum 25(OH)D levels. RESULTS: After adjusting for potential confounders, mean serum 25(OH)D levels were significantly lower in participants diagnosed with atopic dermatitis than in those without this diagnosis (mean ± SE, 18.58 ± 0.29 ng/mL vs 19.20 ± 0.15 ng/mL; P = .02). Compared with participants with adequate vitamin D levels (≥20 ng/mL), confounder-adjusted ORs of atopic dermatitis were significantly higher in those with inadequate (12-19.99 ng/mL) or deficient (<12 ng/mL) levels (OR [95% CI], 1.50 [1.10-2.06] and 1.48 [1.04-2.12], respectively; P = .02). This relationship was not observed in participants with the other allergic conditions. CONCLUSION: Vitamin D-insufficient adult individuals within the general Korean population have an increased likelihood of atopic dermatitis, but not asthma, allergic rhinitis, or IgE sensitization.

Saponins of Korean Red Ginseng May Protect Human Skin from Adipokine-Associated Inflammation and Pigmentation Resulting from Particulate Matter Exposure.

BACKGROUND: Exposure to airborne particulate matter (PM) is an ever-increasing concern worldwide. Strategies to counter the detrimental effects that follow cutaneous exposure to PM, such as induction of pigmentation, inflammation, and alterations in adipokine profile, need to be investigated further. Korean red ginseng (KRG) extracts and individual ingredients have been demonstrated to play an effective role in suppression of ROS, inflammation, and resultant skin aging. In addition, recent investigations revealed that Rg3 and Rf saponins work as antimelanogenic agents. In this study, we investigated whether saponins of KRG can protect against or reverse the PM-induced detrimental effects. METHODS: The biological effects of PM and saponins were evaluated both in vitro and ex vivo. Cell viability and intracellular ROS levels were determined in normal human epidermal melanocytes (NHMs), human epidermal keratinocytes (NHKs), and their cocultures. Experiments to demonstrate the protective properties of saponins against consequences of exposure to PM were performed. Melanin assay, quantitative real-time PCR, and Western blotting were carried out to determine the effects on melanogenesis and the implicated molecular signaling pathways. RESULTS: Exposure to PM resulted in decreased keratinocyte viability, which was coupled with augmented oxidative stress. These changes were attenuated by treatment with saponins. PM exposure resulted in increased expression of leptin, which was reduced by saponins. Moreover, PM exposure led to increased melanin production in a coculture model, which was mitigated by treatment with saponins. Treatment with saponins resulted in a decrease in matrix metalloproteinase (MMP) levels after exposure to PM. CONCLUSION: Saponins of KRG can protect the skin from the harmful effects of PM exposure by reducing levels of ROS, leptin, inflammatory cytokines, and melanin.

Clinicoprognostic and Histopathological Features of Guttate and Plaque Psoriasis Based on PD-1 Expression.

Several studies have determined the correlation between programmed cell death protein-1 (PD-1) and chronic plaque psoriasis (CPP). However, limited studies have assessed the association between PD-1 expression and the clinicoprognostic and distinct clinicopathological characteristics of CPP and guttate psoriasis (GP). Twenty-nine patients with skin biopsy-confirmed CPP were recruited at the Asan Medical Center between January 2018 and June 2020, and 33 patients with biopsy-confirmed GP were enrolled between January 2002 and June 2020. The clinicoprognostic and histopathological characteristics were analyzed according to immunohistochemical PD-1 expression in the epidermal or dermal inflammatory infiltrates. The CPP and GP lesions were divided into PD-1-low and PD-1-high groups. The CPP epidermal PD-1-high group had typical histopathological changes and significantly higher psoriasis area and severity index scores (p = 0.014) and disease duration (p = 0.009) than the epidermal PD-1-low group. In patients with GP, compared with the dermal PD-1-high group, the dermal PD-1-low group exhibited significantly higher disease duration (p = 0.002) and relapse rate of plaque psoriasis (p = 0.005) and significantly lower relapse-free survival (p = 0.016). Upregulated epidermal PD-1 expression was correlated with the chronicity and severity of CPP, while downregulated dermal PD-1 expression was correlated with poor prognosis of GP.

CRTC3, a sensor and key regulator for melanogenesis, as a tunable therapeutic target for pigmentary disorders.

Background: Although CREB phosphorylation is known to be essential in UVB/cAMP-stimulated melanogenesis, CREB null mice did not show identifiable pigmentation phenotypes. Here, we show that CREB-regulated transcription co-activator 3 (CRTC3) quantitatively regulates and orchestrates melanogenesis by directly targeting microphthalmia-associated transcription factor (MITF) and regulating the expression of most key melanogenesis-related genes. Methods: We analyzed CRTC3-null, KRT14-SCF transgenic, and their crossover mice. The molecular basis of CRTC3 effects on pigmentation was investigated by histology, melanin/tyrosinase assay, immunoblotting, shRNA, promoter assay, qRT-PCR, and subcellular localization. These analyses were carried out in primary cultured melanocytes, mouse cell lines, normal human cells, co-cultures, and ex vivo human skin. CRTC/CREB activity screening was performed to identify candidate agents for the regulation of melanogenesis. Results: The coat and skin color of CRTC3-null mice was paler due to a reduction in melanin deposition. Melanogenesis-related genes were reduced in CRTC3-deficient cultured melanocytes and tail skin of CRTC3-null mice. Notably, basal levels of MITF present in CRTC3-null mice were sufficient for melanocytic differentiation/survival. Thus CRTC3-null mice showed a comparable number of epidermal melanocytes compared to control mice. Stem cell factor (SCF) introduction by crossing with KRT14-SCF mice increased epidermal melanocytes and melanin deposition in control and CRTC3-null mice, but the skin color remained still light on the CRTC3-null background. Furthermore, we identified the therapeutic potential of altiratinib to inhibit melanogenesis in human melanocytes and human skin effectively and safely. Conclusion: CRTC3 appears to be a key sensor for melanogenesis and can be used as a reversible and tunable tool for selectively regulating melanogenesis without affecting melanocyte integrity. Thus, CRTC3 can also serve as a screening tool for the discovery of ideal melanogenesis-modulating small molecules.

TC1 (C8orf4) enhances the Wnt/beta-catenin pathway by relieving antagonistic activity of Chibby.

The Wnt/beta-catenin pathway has been implicated in human cancers. Here, we show that TC1 (C8orf4), a small protein present in vertebrates, functions as a positive regulator of the pathway. TC1 interacts with Chibby (Cby) and thereby enhances the signaling pathway by relieving the antagonistic function of Cby on the beta-catenin-mediated transcription. Upon coexpression in mammalian cells, TC1 redistributes from nucleolus to nuclear speckles, where it colocalizes with Cby. TC1 up-regulates the expression of beta-catenin target genes that are implicated in invasiveness and aggressive behavior of cancers, such as metalloproteinases, laminin gamma2, and others. Our data indicate that TC1 is a novel upstream regulator of the Wnt/beta-catenin pathway that enhances aggressive behavior of cancers.

TC1(C8orf4) correlates with Wnt/beta-catenin target genes and aggressive biological behavior in gastric cancer.

PURPOSE: We have recently reported that TC1(C8orf4), a small protein present in vertebrates, functions as a novel regulator of the Wnt/beta-catenin pathway. TC1 up-regulates beta-catenin target genes that are implicated in the aggressive behavior of cancers. Our aim was to investigate the clinical and pathobiological relevance of TC1 in gastric cancer. EXPERIMENTAL DESIGN: The expression of TC1 was analyzed using tissue microarray in correlation with clinicopathologic variables and beta-catenin target genes in 299 gastric cancers. The biological effects of TC1 on Matrigel invasiveness and the proliferation of cancer cells were analyzed. TC1 expression was analyzed in gastric cancer cells after serial peritoneal implantation in nude mice. RESULTS: TC1 expression was present in 111 carcinomas (37.1%), correlating with tumor stage (P < 0.002), poor differentiation (P < 0.001), lymphatic infiltration (P < 0.005), and lymph node metastasis (P < 0.006). TC1 also correlated with poor survival in diffuse type carcinomas (P < 0.0001), and even in patients with lymph node metastasis (P < 0.0014). TC1 also correlated with the expression of beta-catenin target genes including laminin gamma2, metalloproteinase-7 and metalloproteinase-14, cyclin D1, c-Met, and CD44. TC1 enhanced Matrigel invasiveness and proliferation, supporting its role in the aggressive biological behavior of cancers. The expression of TC1 increased in MKN45 cells after serial peritoneal seeding in nude mice. CONCLUSIONS: Our data suggests that TC1 coordinates the up-regulation of Wnt/beta-catenin target genes that are implicated in the aggressive biological behavior of cancers. The strong clinical relevance, even in patients with lymph node metastasis, suggested that TC1 could be a potential therapeutic target of advanced gastric cancers.

Novel regulation of melanogenesis by adiponectin via the AMPK/CRTC pathway.

Several studies observed that adiponectin, an important adipokine that improves glucose metabolism by regulating AMP-activated protein kinase (AMPK) signaling, is dermatologically beneficial. In our recent microarray data, we found that adiponectin expression was lower in lesional skin than in non-lesional skin of melasma patients. Given that AMPK is a key adiponectin signaling mediator, we investigated the role of adiponectin and AICAR, a cell-permeable AMPK activator, in melanogenesis. We herein showed that adiponectin and AICAR downregulated MITF, tyrosinase, TRP-1, and DCT expression and reduced melanin content in normal human and mouse melanocytes. The depigmenting effect of adiponectin was mediated via AMPK activation, which induced the inhibitory phosphorylation of CREB-regulated transcription co-activators (CRTCs) and subsequent suppression of the novel CRTC/CREB pathway in melanocytes. These findings suggest that adiponectin and its analogs are useful as a clinical strategy for treating hyperpigmentation disorders.

TC1(C8orf4) is upregulated by IL-1beta/TNF-alpha and enhances proliferation of human follicular dendritic cells.

Follicular dendritic cells (FDC) play crucial roles in immune regulation. TNF-alpha has been shown to be essential to the FDC network. However, the molecular regulation of FDC proliferation has not been characterized. Here, we show that TC1(C8orf4), a novel positive regulator of the Wnt/beta-catenin pathway in vertebrates, is upregulated by IL-1beta and TNF-alpha in the human FDC-like line HK. TC1 enhances HK cell proliferation, while TC1-knockdown inhibits the proliferation induced by IL-1beta, suggesting a role of TC1 as a regulator of FDC proliferation. The regulation by pro-inflammatory cytokines suggests that TC1 might be implicated in linking local inflammation to immune response by stimulating FDC.

Differential expression in normal-adenoma-carcinoma sequence suggests complex molecular carcinogenesis in colon.

The majority of colon cancers develop from pre-existing adenomas. We analyzed the expression profiles in the sequence of normal colon crypts, adenomas and early-stage carcinomas using microdissected cells from tubular adenomas with foci of malignant transformation. Differentially expressed genes were detected between normal-adenoma and adenoma-carcinoma, and were grouped according to the patterns of expression changes in the sequence. Down-regulated genes in the sequence included PLA2G2A, TSPAN1, PDCD4, FCGBP, AATK, EPLIN, FABP1, AGR2, MTUS1, TSC1, galectin 4 and MT1F. PLA2G2A has been shown to suppress colon tumorigenesis in mice, but the pathobiological role in humans has been controversial. Our data showed continuous down-regulation of PLA2G2A in the sequence supporting an implication in human colon cancer. Tumor suppressor and/ or proapoptotic activities have also been reported in other genes. Up-regulated genes included ribosomal proteins, IER3 and TPR. TGF-beta2 and matrix metalloproteinase 23B were up-regulated in carcinoma but not in adenoma, supporting the pathobiological roles in malignant transformation. Differentially expressed genes partly coincided with those in the adenoma-carcinoma sequence of the stomach, which was published previously, suggesting a partial overlap between the adenoma-carcinoma sequences of the colon and stomach.

Expression profiling and subtype-specific expression of stomach cancer.

The expression profiling and molecular grouping of stomach cancers has been a challenging task because of their complexity and variation. We have analyzed gene expression profiles of 22 gastric cancer/nontumor mucosa couples using 14K cDNA microarray chips designed for gastric cancer analysis. Upon pairwise analysis of the individual couples at the false significance rate 0.91%, 79 and 398 genes were reported to be up-regulated and down-regulated in tumors, respectively. Tumors were clustered into two groups having high and low inflammatory infiltration, respectively. The latter consisted of three subgroups, including diffuse type carcinomas and intestinal types with distinct pathological characteristics of aggressive behavior. When the pooled tumor was hybridized against the pooled nontumor mucosa samples, more genes were detected to express differentially than those detected by the pairwise analysis at the same threshold level. However, they did not render satisfactory clustering of individual tumors. Our data showed that stomach cancers could be clustered effectively using stomach-specific microarrays and pairwise analysis of tumor/nontumor mucosa couples. It is suggested that the application of specific goal-oriented experimental designing would be advantageous for efficient analysis of expression profiles of such a complex disease as gastric cancer.

1-Phenyl-3-(2-thiazolyl)-2-thiourea inhibits melanogenesis via a dual-action mechanism.

1-Phenyl-3-(2-thiazolyl)-2-thiourea (PTTU) is a well-characterized dopamine ß-hydroxylase inhibitor that prevents 6-hydroxydopamine-induced degenerative neuronal disease. However, the effect of PTTU on melanogenesis has not been reported. In this study, we examined the effect of PTTU on melanogenesis and studied its mechanism of action. We found that PTTU decreased melanin biosynthesis in a dose-dependent manner in normal human epidermal melanocytes (NHEMs). PTTU also inhibited tyrosinase catalytic activity in NHEMs. Moreover, PTTU treatment led to reduced protein levels of tyrosinase in NHEMs, while the protein levels of tyrosinase-related protein-1, tyrosinase-related protein-2, and microphthalmia-associated transcription factor were not affected. However, PTTU treatment did not affect the mRNA expression of tyrosinase. We found that PTTU-accelerated tyrosinase degradation via the ubiquitin-dependent proteasome pathway. In summary, we found that PTTU decreased melanin biosynthesis by decreasing the enzymatic activity and stability of tyrosinase. Our results indicate that PTTU could be used as a depigmentation agent for hyperpigmentation disorder.

Gene expression profiling reveals sequential changes in gastric tubular adenoma and carcinoma in situ.

AIM: To analyze the expression profiles of premalignant and/or preclinical lesions of gastric cancers. METHODS: We analyzed the expression profiles of normal gastric pit, tubular adenoma and carcinoma in situ using microdissected cells from routine gastric biopsies. For the DNA microarray analysis of formalin-fixed samples, we developed a simple and reproducible RNA extraction and linear amplification procedure applying two polymerase-binding sites. The amplification procedure took only 8 h and yielded comparable DNA microarray data between formalin-fixed tissues and unfixed controls. RESULTS: In comparison with normal pit, adenoma/carcinoma showed 504 up-regulated and 29 down-regulated genes at the expected false significance rate 0.15%. The differential expression between adenoma and carcinoma in situ was subtle: 50 and 22 genes were up-, and down-regulated in carcinomas at the expected false significance rate of 0.61%, respectively. Differentially expressed genes were grouped according to patterns of the sequential changes for the 'tendency analysis' in the gastric mucosa-adenoma-carcinoma sequence. CONCLUSION: Groups of genes are shown to reflect the sequential expression changes in the early carcinogenic steps of stomach cancer. It is suggested that molecular carcinogenic pathways could be analyzed using routinely processed biopsies.

Usefulness of Skin Explants for Histologic Analysis after Fractional Photothermolysis.

BACKGROUND: Fractional laser resurfacing treatment has been extensively investigated and is widely used. However, the mechanism underlying its effects is poorly understood because of the ethical and cosmetic problems of obtaining skin biopsies required to study the changes after laser treatment. OBJECTIVE: To evaluate the usefulness of human skin explants for the investigation of fractional photothermolysis. METHODS: Full-thickness discarded skin was treated in 4 ways: no treatment (control), fractional carbon dioxide laser, fractional Er:YAG laser, and fractional 1,550-nm erbium-doped fiber laser. Both treated and non-treated skin samples were cultured ex vivo at the air-medium interface for 7 days. Frozen tissue was sectioned and stained with hematoxylin & eosin for histologic examination and nitro blue tetrazolium chloride for viability testing. RESULTS: Skin explants cultured for up to 3 days exhibited histologic changes similar to those observed in in vivo studies, including microscopic treatment zones surrounded by a thermal coagulation zone, re-epithelialization, and formation of microscopic epidermal necrotic debris. However, the explant structure lost its original form within 7 days of culture. The viability of skin explants was maintained for 3 days of culture but was also lost within 7 days. CONCLUSION: The skin explant model may be a useful tool for investigating the immediate or early changes following fractional photothermolysis, but further improvements are required to evaluate the long-term and dermal changes.

TRF2 is in neuroglial cytoplasm and induces neurite-like processes.

TRF2 is a ubiquitous protein that protects telomeres in the nucleus. We found that TRF2 was present at the peripheral nerve axons and the brain neuroglial cell processes extensively. It was in the cytoplasmic membrane as well as nuclear fractions, but not in the soluble cytoplasmic fraction of SH-SY5Y neuroblastoma cells. TRF2 was up-regulated in P19 embryonal carcinoma cells at the early stage of induced neural differentiation with retinoic acid treatment. Upon transfection, TRF2-expressing COS cells often produced neurite-like long cytoplasmic processes. TRF2 is a component of neuroglial cells and appears to be involved in the cytoplasmic process formation that is necessary for neural differentiation.