- Email: [email protected]
Gli3 expression
Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
and fibronectin type III domains. TNC is localized in the extracellular matrix and plays a role as a ligand of several types of integrin. TNC also increases mRNA and protein levels of type I collagen in hepatic stellate cells and foreskin fibroblasts. We confirmed that expression of the TnC gene is down-regulated in aged mouse dorsal skin and aged human buttock skin at RNA and protein levels by RT-PCR, immunohistochemistry, and immunofluorescence analyses. In this regards, we propose that down-regulation of the TnC gene is an important characteristic of intrinsically aged skin in mice and humans. http://dx.doi.org/10.1016/j.jdermsci.2017.02.271 L-14[O1-53] Leucine-rich alpha-2-glycoprotein 1 modulates expression of matrix metalloproteinase-1 and type 1 collagen in human dermal fibroblasts So Yun Ahn 1,∗ , Dong Hun Lee 2 , Jin Ho Chung 2 , Seung-Taek Lee 1 1
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea 2 Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea Differentially expressed genes of RNA sequencing database from dorsal skin tissues of young and old mice were analyzed to identify intrinsic factors of skin aging. We found that expression of Lrg1 gene encoding leucine-rich alpha-2-glycoprotein 1 (LRG1) was significantly downregulated in skin tissues of the old group. Although LRG1 is known as a positive regulator of transforming growth factor-beta signaling pathway, its function and detailed mechanism in skin aging has not been studied well. Our results showed that Lrg1 mRNA level using RT-PCR and LRG1 protein level by western blot and immunohistochemistry markedly decreased in old group compared with young group. His-tagged recombinant human LRG1 (rhLRG1) was expressed and purified from HEK 293 cells. Treatment of rhLRG1 increased secretion of type I collagen and decreased secretion of matrix metalloproteinase (MMP)-1 in human dermal fibroblasts. Moreover, LRG1-induced type I collagen upregulation and MMP-1 down-regulation involves activation of TGF-beta signaling pathway. Taken together, our findings indicate that downregulation of LRG1 promotes skin aging due to increased degradation and decreased synthesis of extracellular matrix components such as type I collagen. http://dx.doi.org/10.1016/j.jdermsci.2017.02.272
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L-15[O3-53] Evaluation of 14 Thai herbs with free radical scavenging activity and enzyme inhibitory behavior Moragot Chatatikun 1,∗ , Anchalee Chiabchalard 2 1
Ph.D. Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand 2 Center for Excellence in Omics-Nano Medical Technology Development Project, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand Overexposure to sunlight induces overexpression of reactive oxygen species (ROS) is problematic resulting in skin aging, hyperpigmentation and cancer. Development of novel whitening and anti-wrinkle compounds from natural products to address premature aging is important. The purpose of this study was to find products that reduce the reactively of ROS. To determine the anti-aging effects of antioxidant activity, anti-tyrosinase activity and anti-collagenase activity we compared extracts of 14 Thai plant using different solvents (petroleum ether, dichloromethane and ethanol). Scavenging activity ranged from 7.11 ± 0.59% SC to 96.17 ± 0.05% SC by DPPH assay and 8.03 ± 0.54% SC to 99.84 ± 0.07% SC by ABTS assay. The ethanol leaf extract of Ardisia elliptica (Thunb) had the highest antioxidant activity and collagenase inhibition. Interestingly, three plants extracts, which were ethanol fraction of Annonasquamosa L., Ardisiaelliptica Thunb. and Senna alata (L.), Roxb, with high antioxidant activity significantly inhibited both tyrosinase and collagenase. Our finding, the ethanol fractions of Annona squamosa L., Ardisia elliptica Thunb. and Senna alata (L.) had the highest anti-oxidant activity, suggests that this compound is a promising ingredient in cosmetic products as an anti-wrinkle agents as well as useful in skin whitening creams and lotions. http://dx.doi.org/10.1016/j.jdermsci.2017.02.273 L-16[O1-54] Basal cell carcinoma response to imiquimod is associated with the ratio of Gli1/Gli3 expression Yukihiko Kato 1,2,∗ , Ayano Kanzaki 1,3 , Yoshihiro Umebayashi 2 , Ryoji Tsuboi 3 1 Department of Dermatology, Tokyo Metropolitan Tama Medical Center, Japan 2 Department of Dermatology, Tokyo Medical University Hachioji Medical Center, Japan 3 Department of Dermatology, Tokyo Medical University, Japan Introduction: Imiquimod is used to treat a superficial type of basal cell carcinoma (BCC) in the USA. The anti-tumor effect of this toll-like receptor (TLR) 7 agonist is thought to occur via stimulation of TLR, which activates a Th1 immune response. Wolf reported that imiquimod directly repress hedgehog (HH) signaling by negatively modulating GLI activity in a murine model. Here we discussed 4 cases of BCC treated with imiquimod, and examined the relationship between the BCC response to the drug and the expression of the Gli1-related genes. Materials and methods: The subjects were 4 adults, a male and a female in their fifties, and a male and a female in their eighties. Imiquimod cream was applied 3-5 times weekly to their lesion, which was then examined histopathologically. The expression of
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Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
Gli1 as well as its repressor, Gli3, was examined in the lesions before and after imiquimod treatment. GLi1 and GLi3 expression in sites showing remission of the lesions was compared to that in sites with remnant lesions using quantitative PCR with paraffin-embedded tissue. Results: The post-treatment Gli1/Gli3 ratio clearly declined in comparison with the pre-treatment ratio. Similarly, the posttreatment ratio in the remission sites was much lower than in the remnant lesions. Discussion: The Gli1/Gli3 ratio could serve as an index of the BCC response to imiquimod treatment in a ligand-independent manner. http://dx.doi.org/10.1016/j.jdermsci.2017.02.274 L-17[O3-54] TPA inhibits melanoma growth through inactivation of STAT3 through protein tyrosine phosphatases Tetsushi Iwasaki 1,2,3,∗ , Miwa Yamauchi 1 , Zhu Liang 2 , Ayano Itai 3 , Masanobu Sakaguchi 4 , Taiki Nagano 1 , Shinji Kamada 1,2,3 , Masahiro Oka 4 1
Biosignal Research Center, Kobe University, Japan Department of Biology, Graduate School of Science, Kobe University, Japan 3 Department of Biology, Faculty of Science, Kobe University, Japan 4 Divisions of Dermatology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Japan The signal transducer and activator of transcription 3 (STAT3) is a transcription factor involved in the expression of variety of genes. STAT3 is normally activated by tyrosine 705 phosphorylation in the molecule when adequate ligands bind cell surface receptors. However, constitutive activation of STAT3, namely constitutive tyrosine 705 phosphorylation, has been observed in melanoma cells. It has been elucidated that the constitutive activation of STAT3 plays a crucial role in the growth and metastasis in these cells. Thus, STAT3 has been considered to be a strong candidate for melanoma therapy. We have previously found that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits both the proliferation and the DNA synthesis in melanoma cells concomitant with inactivation of STAT3 through decreasing the phosphorylation level at Tyr705 of STAT3. In this study, we investigated the molecular mechanisms by which TPA inhibits melanoma growth through dephosphorylating Tyr705 phosphorylation by pharmaceutical and biochemical analysis using various melanoma cell lines. The growth inhibition of melanoma cells was disabled by introduction of a small interference RNA (siRNA) for STAT3 or several protein tyrosine phosphatase(s) (PTPs) including TC-PTP and SH-PTP2. TPA-induced STAT3 dephosphorylation was also blocked by siRNAs for the PTPs. These data suggest that the TPA-induced growth inhibition of melanoma cells is due to the dephosphorylation of STAT3 and that multiple PTPs contribute to the dephosphorylation of STAT3. 2
http://dx.doi.org/10.1016/j.jdermsci.2017.02.275
L-18[O1-55] The expression of serine protease inhibitors are induced by TNF-␣ and IL-17A in skin inflammatory diseases Satoru Sugihara ∗ , Shin Morizane, Saeko Sugimoto, Mina Kobashi, Keiji Iwatsuki Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Background: The serine protease activity of the skin is tightly regulated by proteases including kallikrein (KLK) 5 and KLK7 and the inhibitors, which include lympho-epithelial Kazal-type related inhibitor (LEKTI), secretory leukocyte peptidase inhibitor (SLPI), and elafin. Previously our group reported that TH2 cytokines, IL-4 and IL-13, but not TH1 or TH17 cytokines increase KLK7 expression and function in keratinocytes. However, the effect of the cytokines on serine protease inhibitors in keratinocytes are not well-studied. Methods: We stimulated normal human epidermal keratinocytes (NHEKs) with TNF-␣, IL-4, -13, -17A, -22, -33, or IFN-␥ (50 ng/ml). The expression of SPINK5, SLPI and PI3 mRNA were analyzed by real-time PCR. LEKTI protein was analyzed by ELISA. Protease activities in the culture media were monitored using the specific substrates. Immunohistochemical staining for LEKTI was performed using skin samples from normal skin (NS) and the lesions of psoriasis (PS) and atopic dermatitis (AD). Results: TNF-␣ and IL-17A significantly induced the expression of SPINK 5, SLPI and PI3 mRNA in NHEKs. ELISA analysis revealed that LEKTI protein was similarly induced by these cytokines in NHEKs. Unexpectedly, both trypsin- and chymotrypsin-like protease activities in the culture media were also increased by TNF-␣ and IL-17A. Immunohistochemical analysis showed that LEKTI expression in PS lesions was significantly higher than that of AD and NS. http://dx.doi.org/10.1016/j.jdermsci.2017.02.276 L-19[O3-55] Can skin microbes predispose you to eczema? Kern Rei Ng 1 , Angeline Tay 2 , Chanhao Li 1 , Amanda Ng 1 , Bani Kaur Suri 3 , Sri Anusha Matta 3 , Colin Wong 2 , Andreas Wilm 1 , Birgit Lane 2 , Fook Tim Chew 3 , Niranjan Nagaranjan 1 , John Common 2,∗ 1
Genome Institute of Singapore, Singapore Institute of Medical Biology, Singapore 3 National University of Singapore, Singapore Atopic dermatitis (AD) is an inflammatory skin disorder affecting up to 20% of the population in developed countries, with increasing incidence and significant healthcare burden worldwide. Together with strong genetic predisposition to AD from filaggrin mutations, the disease is suspected to have a microbial component. Whole skin metagenome analysis has the potential to reveal functional triggers of the disease, including possible viral, bacterial and eukaryotic pathogens, but issues of cost, robustness and sampling efficacy have limited its application. Using whole metagenomics analysis we identified distinct nonflare, skin microbiome signatures with significant enrichment and depletion of specific microbe genera on AD-susceptible skin versus normal healthy skin. Bacterial differences were complemented by perturbations in the eukaryotic community (such as changes in various Malassezia species that have been implicated 2
and fibronectin type III domains. TNC is localized in the extracellular matrix and plays a role as a ligand of several types of integrin. TNC also increases mRNA and protein levels of type I collagen in hepatic stellate cells and foreskin fibroblasts. We confirmed that expression of the TnC gene is down-regulated in aged mouse dorsal skin and aged human buttock skin at RNA and protein levels by RT-PCR, immunohistochemistry, and immunofluorescence analyses. In this regards, we propose that down-regulation of the TnC gene is an important characteristic of intrinsically aged skin in mice and humans. http://dx.doi.org/10.1016/j.jdermsci.2017.02.271 L-14[O1-53] Leucine-rich alpha-2-glycoprotein 1 modulates expression of matrix metalloproteinase-1 and type 1 collagen in human dermal fibroblasts So Yun Ahn 1,∗ , Dong Hun Lee 2 , Jin Ho Chung 2 , Seung-Taek Lee 1 1
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea 2 Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea Differentially expressed genes of RNA sequencing database from dorsal skin tissues of young and old mice were analyzed to identify intrinsic factors of skin aging. We found that expression of Lrg1 gene encoding leucine-rich alpha-2-glycoprotein 1 (LRG1) was significantly downregulated in skin tissues of the old group. Although LRG1 is known as a positive regulator of transforming growth factor-beta signaling pathway, its function and detailed mechanism in skin aging has not been studied well. Our results showed that Lrg1 mRNA level using RT-PCR and LRG1 protein level by western blot and immunohistochemistry markedly decreased in old group compared with young group. His-tagged recombinant human LRG1 (rhLRG1) was expressed and purified from HEK 293 cells. Treatment of rhLRG1 increased secretion of type I collagen and decreased secretion of matrix metalloproteinase (MMP)-1 in human dermal fibroblasts. Moreover, LRG1-induced type I collagen upregulation and MMP-1 down-regulation involves activation of TGF-beta signaling pathway. Taken together, our findings indicate that downregulation of LRG1 promotes skin aging due to increased degradation and decreased synthesis of extracellular matrix components such as type I collagen. http://dx.doi.org/10.1016/j.jdermsci.2017.02.272
e93
L-15[O3-53] Evaluation of 14 Thai herbs with free radical scavenging activity and enzyme inhibitory behavior Moragot Chatatikun 1,∗ , Anchalee Chiabchalard 2 1
Ph.D. Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand 2 Center for Excellence in Omics-Nano Medical Technology Development Project, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand Overexposure to sunlight induces overexpression of reactive oxygen species (ROS) is problematic resulting in skin aging, hyperpigmentation and cancer. Development of novel whitening and anti-wrinkle compounds from natural products to address premature aging is important. The purpose of this study was to find products that reduce the reactively of ROS. To determine the anti-aging effects of antioxidant activity, anti-tyrosinase activity and anti-collagenase activity we compared extracts of 14 Thai plant using different solvents (petroleum ether, dichloromethane and ethanol). Scavenging activity ranged from 7.11 ± 0.59% SC to 96.17 ± 0.05% SC by DPPH assay and 8.03 ± 0.54% SC to 99.84 ± 0.07% SC by ABTS assay. The ethanol leaf extract of Ardisia elliptica (Thunb) had the highest antioxidant activity and collagenase inhibition. Interestingly, three plants extracts, which were ethanol fraction of Annonasquamosa L., Ardisiaelliptica Thunb. and Senna alata (L.), Roxb, with high antioxidant activity significantly inhibited both tyrosinase and collagenase. Our finding, the ethanol fractions of Annona squamosa L., Ardisia elliptica Thunb. and Senna alata (L.) had the highest anti-oxidant activity, suggests that this compound is a promising ingredient in cosmetic products as an anti-wrinkle agents as well as useful in skin whitening creams and lotions. http://dx.doi.org/10.1016/j.jdermsci.2017.02.273 L-16[O1-54] Basal cell carcinoma response to imiquimod is associated with the ratio of Gli1/Gli3 expression Yukihiko Kato 1,2,∗ , Ayano Kanzaki 1,3 , Yoshihiro Umebayashi 2 , Ryoji Tsuboi 3 1 Department of Dermatology, Tokyo Metropolitan Tama Medical Center, Japan 2 Department of Dermatology, Tokyo Medical University Hachioji Medical Center, Japan 3 Department of Dermatology, Tokyo Medical University, Japan Introduction: Imiquimod is used to treat a superficial type of basal cell carcinoma (BCC) in the USA. The anti-tumor effect of this toll-like receptor (TLR) 7 agonist is thought to occur via stimulation of TLR, which activates a Th1 immune response. Wolf reported that imiquimod directly repress hedgehog (HH) signaling by negatively modulating GLI activity in a murine model. Here we discussed 4 cases of BCC treated with imiquimod, and examined the relationship between the BCC response to the drug and the expression of the Gli1-related genes. Materials and methods: The subjects were 4 adults, a male and a female in their fifties, and a male and a female in their eighties. Imiquimod cream was applied 3-5 times weekly to their lesion, which was then examined histopathologically. The expression of
e94
Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
Gli1 as well as its repressor, Gli3, was examined in the lesions before and after imiquimod treatment. GLi1 and GLi3 expression in sites showing remission of the lesions was compared to that in sites with remnant lesions using quantitative PCR with paraffin-embedded tissue. Results: The post-treatment Gli1/Gli3 ratio clearly declined in comparison with the pre-treatment ratio. Similarly, the posttreatment ratio in the remission sites was much lower than in the remnant lesions. Discussion: The Gli1/Gli3 ratio could serve as an index of the BCC response to imiquimod treatment in a ligand-independent manner. http://dx.doi.org/10.1016/j.jdermsci.2017.02.274 L-17[O3-54] TPA inhibits melanoma growth through inactivation of STAT3 through protein tyrosine phosphatases Tetsushi Iwasaki 1,2,3,∗ , Miwa Yamauchi 1 , Zhu Liang 2 , Ayano Itai 3 , Masanobu Sakaguchi 4 , Taiki Nagano 1 , Shinji Kamada 1,2,3 , Masahiro Oka 4 1
Biosignal Research Center, Kobe University, Japan Department of Biology, Graduate School of Science, Kobe University, Japan 3 Department of Biology, Faculty of Science, Kobe University, Japan 4 Divisions of Dermatology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Japan The signal transducer and activator of transcription 3 (STAT3) is a transcription factor involved in the expression of variety of genes. STAT3 is normally activated by tyrosine 705 phosphorylation in the molecule when adequate ligands bind cell surface receptors. However, constitutive activation of STAT3, namely constitutive tyrosine 705 phosphorylation, has been observed in melanoma cells. It has been elucidated that the constitutive activation of STAT3 plays a crucial role in the growth and metastasis in these cells. Thus, STAT3 has been considered to be a strong candidate for melanoma therapy. We have previously found that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits both the proliferation and the DNA synthesis in melanoma cells concomitant with inactivation of STAT3 through decreasing the phosphorylation level at Tyr705 of STAT3. In this study, we investigated the molecular mechanisms by which TPA inhibits melanoma growth through dephosphorylating Tyr705 phosphorylation by pharmaceutical and biochemical analysis using various melanoma cell lines. The growth inhibition of melanoma cells was disabled by introduction of a small interference RNA (siRNA) for STAT3 or several protein tyrosine phosphatase(s) (PTPs) including TC-PTP and SH-PTP2. TPA-induced STAT3 dephosphorylation was also blocked by siRNAs for the PTPs. These data suggest that the TPA-induced growth inhibition of melanoma cells is due to the dephosphorylation of STAT3 and that multiple PTPs contribute to the dephosphorylation of STAT3. 2
http://dx.doi.org/10.1016/j.jdermsci.2017.02.275
L-18[O1-55] The expression of serine protease inhibitors are induced by TNF-␣ and IL-17A in skin inflammatory diseases Satoru Sugihara ∗ , Shin Morizane, Saeko Sugimoto, Mina Kobashi, Keiji Iwatsuki Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Background: The serine protease activity of the skin is tightly regulated by proteases including kallikrein (KLK) 5 and KLK7 and the inhibitors, which include lympho-epithelial Kazal-type related inhibitor (LEKTI), secretory leukocyte peptidase inhibitor (SLPI), and elafin. Previously our group reported that TH2 cytokines, IL-4 and IL-13, but not TH1 or TH17 cytokines increase KLK7 expression and function in keratinocytes. However, the effect of the cytokines on serine protease inhibitors in keratinocytes are not well-studied. Methods: We stimulated normal human epidermal keratinocytes (NHEKs) with TNF-␣, IL-4, -13, -17A, -22, -33, or IFN-␥ (50 ng/ml). The expression of SPINK5, SLPI and PI3 mRNA were analyzed by real-time PCR. LEKTI protein was analyzed by ELISA. Protease activities in the culture media were monitored using the specific substrates. Immunohistochemical staining for LEKTI was performed using skin samples from normal skin (NS) and the lesions of psoriasis (PS) and atopic dermatitis (AD). Results: TNF-␣ and IL-17A significantly induced the expression of SPINK 5, SLPI and PI3 mRNA in NHEKs. ELISA analysis revealed that LEKTI protein was similarly induced by these cytokines in NHEKs. Unexpectedly, both trypsin- and chymotrypsin-like protease activities in the culture media were also increased by TNF-␣ and IL-17A. Immunohistochemical analysis showed that LEKTI expression in PS lesions was significantly higher than that of AD and NS. http://dx.doi.org/10.1016/j.jdermsci.2017.02.276 L-19[O3-55] Can skin microbes predispose you to eczema? Kern Rei Ng 1 , Angeline Tay 2 , Chanhao Li 1 , Amanda Ng 1 , Bani Kaur Suri 3 , Sri Anusha Matta 3 , Colin Wong 2 , Andreas Wilm 1 , Birgit Lane 2 , Fook Tim Chew 3 , Niranjan Nagaranjan 1 , John Common 2,∗ 1
Genome Institute of Singapore, Singapore Institute of Medical Biology, Singapore 3 National University of Singapore, Singapore Atopic dermatitis (AD) is an inflammatory skin disorder affecting up to 20% of the population in developed countries, with increasing incidence and significant healthcare burden worldwide. Together with strong genetic predisposition to AD from filaggrin mutations, the disease is suspected to have a microbial component. Whole skin metagenome analysis has the potential to reveal functional triggers of the disease, including possible viral, bacterial and eukaryotic pathogens, but issues of cost, robustness and sampling efficacy have limited its application. Using whole metagenomics analysis we identified distinct nonflare, skin microbiome signatures with significant enrichment and depletion of specific microbe genera on AD-susceptible skin versus normal healthy skin. Bacterial differences were complemented by perturbations in the eukaryotic community (such as changes in various Malassezia species that have been implicated 2