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Disruption of the autophagy-lysosome pathway is involved in hypopigmented macules in patients with tuberous sclerosis complex
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
the appropriate amount of sunscreen for a long period in Japanese people, similar to Caucasians.
Category 13 (P13): Pigmentation and Melanoma
http://dx.doi.org/10.1016/j.jdermsci.2016.08.509
Novel non-cell autonomous role of epidermal polarity protein Par3 in melanocyte homeostasis and melanomagenesis
P12-18[O3-23] Up-regulation of expression of heme oxygenase-1 by Psidium guajava extract Yuumi Horibe 2,∗ , Yoshiki Okita 1 , Keiko Usui 1 , Keiko Inoue 2 , Kenichi Hamada 2 , Tohru Mizushima 1 1
Department of Drug Discovery and Development, Faculty of Pharmacy, Keio University, Tokyo, Japan 2 Saishunkan Phamaceutical Co., Ltd., Japan In addition to changes with aging, the skin is damaged by various environmental stressors, particularly solar ultraviolet (UV) radiation. UV light induces skin disorders (such as wrinkle formation and hyperpigmentation) through induction of inflammation and apoptosis, leading to cosmetic problems, for which a number of hypopigmenting and anti-wrinkle cosmetic agents have been developed. When cells are exposed to various stressors, they induce a number of proteins, so-called stress proteins, in order to protect themselves against such stressors and heat shock proteins (HSPs) are representative stress proteins. We previously reported that HSPs protect skin against UV-induced wrinkle formation and hyperpigmentation and developed cosmetic agents with HSP-inducing ability. Heme oxygenase-1 (HO-1) is another type of stress protein. Various stressors such as UV irradiation have been reported to induce HO-1 production. HO-1 degrades heme to carbon monoxide (CO), free iron and biliverdin and biliverdin are potent antioxidants and CO has anti-apoptotic activity. Thus, HO-1 has attracted considerable attention as a novel approach to skin photoprotection. In the present study, we searched for HO-1inducers from a library of herbal extracts and selected an extract of Psidium guajava (P. guajava), based on its high HO-1-inducing activity and low cytotoxicity. Treatment of cultured mouse keratinocytes with P. guajava extract up-regulated the expression of HO-1 in a dose-dependent manner. The up-regulation was observed from 3 to 12 h after the initiation of treatment. We are now examining the effect of P. guajava extract on melanin production, UV-induced apoptosis and collagen synthesis to address its beneficial effects against UV-induced skin disorders. http://dx.doi.org/10.1016/j.jdermsci.2016.08.510
e173
P13-01[II-2]
Melina Mescher 1,2 , Matthias Ruebsam 1,3 , Peter Jeong 1 , Marina Kranen 1 , Jennifer Landsberg 4 , Max Schlaak 3 , Cornelia Mauch 3 , Carien M. Niessen 1,2,3 , Thomas Tueting 4 , Sandra Iden 1,2,∗ 1
CECAD Cluster of Excellence, University of Cologne, Cologne, Germany 2 Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 3 Department of Dermatology, University of Cologne, Cologne, Germany 4 Department of Dermatology and Allergy, University of Bonn, Bonn, Germany The Par3-aPKC-Par6 polarity complex controls a variety of polarization processes during development and tissue homeostasis. We recently identified both pro-oncogenic and tumor-suppressive roles of mammalian Par3 in epidermal tumors. Here we report a non-cell autonomous Par3 function in skin cancer. Using the HGF-Cdk4R24C mouse melanoma model, Par3 loss in the epidermal environment (Par3eKO) resulted in increased melanoma multiplicity and lung metastasis, suggesting that polarity proteins modulate the cross-talk of keratinocytes (KCs) and melanocytes (MCs) and serve to suppress melanoma. MCs cocultured with Par3KO KCs showed increased proliferation and less-differentiated morphology, changes also observed in Par3eKO mice. As this required direct KC-MC contacts, we asked for adhesion molecules involved. P-cadherin localized at KC-MC contacts and was elevated in Par3-KO KCs, suggesting that epidermal Par3 controls P-cadherin stability and function. Inhibition of P-but not E-cadherin rescued the morphology of MCs cocultured with Par3-KO KCs. Aberrant P-cadherin-based adhesion upon Par3 loss may thus provide a permissive niche for MC dedifferentiation and transformation. In human melanoma epidermal Par3 expression was reduced in tumor-adjacent areas and negatively correlated with melanoma progression. P-cadherin instead was robustly expressed in melanoma and localized to heterologous adhesions, indicating that P-cadherin contributes to KC-MC cross-talk also in the context of human disease. Together, we show that epidermal polarity proteins control heterologous communication of key skin-resident cell populations through a subset of classical cadherins, and identified a novel non-cell autonomous tumor-suppressor role of Par3 in the microenvironment of malignant melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.511 P13-02[III-3] Disruption of the autophagy-lysosome pathway is involved in hypopigmented macules in patients with tuberous sclerosis complex Lingli Yang ∗ , Fei Yang, Mari Wataya-Kaneda, Ichiro Katayama Department of dermatology, Osaka University, Japan Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomatous lesions present in many organs. Hypopigmented macules are always
e174
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
observed in most patients with TSC. However, the pathogenesis is still unknown. Recently, autophagy-lysosome pathway has been known as a novel pathway involved in skin pigmentation. Objective: To investigate whether the autophagy-lysosome pathway was involved in TSC-associated hypopigmentation. Method: Using skin tissues from hypopigmented macules of TSC, the activity of autophagy-lysosome pathway was assessed by immunohistochemical staining. Furthermore, we investigated both the pigmentation and autophagy-lysosome pathway in TSC2 knockdown (KD) human primary melanocytes and keratinocytes, by real-time PCR, western blotting analyses and intracellular immunofluorescence staining. Results: Compared to healthy donors, the number of melanocytes was not changed, but autophagy-lysosome activity was dramatically decreased in skin tissue of hypopigmentation macules from TSC. Furthermore, in TSC2 KD melanocytes, melanin contents decreased strikingly, and decreased expression levels of melanogenesis-related proteins were observed. Besides, we found that expression levels of LC3, LAMP1 and the number of autophagosomes and lysosomes were significantly decreased in TSC2 KD melanocytes. Furthermore, to clarify whether the reduction of autophagy was related to hypopigmentation, ATG7 siRNA was transfected into melanocytes. Very interestingly, we found that melanogenesis was significantly inhibited in ATG7 KD melanocytes. Conclusions: These results suggest that dysfunction of autophagy-lysosome pathway might be involved in the pathogenesis of hypopigmentation in patients with TSC. http://dx.doi.org/10.1016/j.jdermsci.2016.08.512 P13-03[C11-08] TLR3 agonist Poly (I:C) enhance Rab27A and melanosome transportation through TLR3 and MAVS pathway in human epidermal melanocytes Saaya Koike ∗ , Kenshi Yamasaki, Takeshi Yamauchi, Mai Inoue, Kenichiro Tsuchiyama, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Japan Innate immune stimuli restlessly influence epidermis where human melanocytes reside. We have examined how innate immunity affects pigmentation and observed that TLR3 agonist poly (I:C) increased melanin release from melanocytes. Poly (I:C) increased Rab27A expression and cell peripheral accumulation, which facilitate melanosome transportation to cell membrane. Knockdown of Rab27A abrogated melanosome transfer to keratinocytes by poly (I:C). Since MITF is known to increase Rab27A transcription, we examined if MITF is involved in Rab27A induction by poly (I:C). Poly (I:C) increased MITF mRNA, but siRNA for MITF did not suppress Rab27A increase, perimembranous localization of Gp100 and Rab27A, and extracellular melanin release by poly (I:C). These suggested that poly (I:C) induces Rab27A and melanosome transportation independent of MITF function. We next knocked down TRIF and MAVS, the molecules involved in TLR3 pathway. We observed that siTRIF and siMAVS suppressed Rab27A induction and melanosome transport to neighboring keratinocytes by poly (I:C) stimulation. We also observed that poly (I:C) increased expression of MAVS mRNA about 7-fold, but poly (I:C) did not altered the mRNA level of TRIF and its downstream molecules IRF3 and TBK1. These indicated that TLR3 agonist poly (I:C) activates melanosome trans-
port by increase Rab27A expression through the TLR3 and MAVS pathway. http://dx.doi.org/10.1016/j.jdermsci.2016.08.513 P13-04[C11-07] IFN- modulates chemokines from tumor-associated macrophages to enhance the therapeutic effect of anti-PD-1 antibodies in B16F10 melanoma Aya Kakizaki 1,∗ , Taku Fujimura 1 , Sadanori Furudate 1 , Yumi Kambayashi 1 , Takeshi Yamauchi 1 , Hideo Yagita 2 , Setsuya Aiba 1 1 Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan 2 Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
Tumor-associated macrophages (TAMs) play roles in maintaining the immunosuppressive microenvironment by recruiting regulatory T cells (Tregs). Since IFN- and anti-PD-1 Ab have been clinically used for the treatment of malignant melanoma, we investigated their immunomodulatory effects on melanoma using the B16F10 mouse melanoma model. B16F10 melanoma cells were subcutaneously injected into C57BL/6 mice, and when the tumor had established, we administered IFN-. The peritumoral administration of IFN- significantly decreased the mRNA expression of Tregs-related chemokines (CCL17, CCL22), which led to the suppressed recruitment of Tregs in B16F10 melanoma. Moreover, to validate the production of chemokines, we isolated CD11b+ TAMs by MACS, analyzed the chemokine production, and confirmed the decrease of CCL22 in the IFN- treated group. Furthermore, the culture supernatant from CD11b+ TAMs significantly increased the chemotactic activity for TRP-2-specific CD4+ T cells, which might suggest that chemotactic factors from CD11b+ TAMs recruit B16F10 melanoma-specific T cells into the tumor microenvironment. Since the administration of IFN- augments the expression of PD-1 on TILs, the co-administration of anti-PD-1 Ab augmented the therapeutic effect of IFN- on the B16F10 melanoma. As in the mouse model, in human, IFN- decreased the production of Th2related chemokines and augmented the production of Th1-related chemokines from monocyte- derived M2 macrophages. Since these immunomodulatory effects of IFN- on macrophages were also observed in the lesional skin of human in- transit melanoma, our present data suggest one of the possible immunomodulatory effects of IFN- and support the use of IFN- in combination with anti-PD1 Ab for the treatment of melanoma patients. http://dx.doi.org/10.1016/j.jdermsci.2016.08.514 P13-05[C11-06] Hair shaft miRNA-221 levels as a new tumor marker of melanoma Satoshi Fukushima ∗ , Taisuke Inada, Masayuki Murai, Masatoshi Jinnin, Azusa Miyashita, Satoshi Nakahara, Aki Tokuzumi, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan Micro RNA-221 (miR-221) is known to be abnormally expressed in many human cancers. We have reported that the serum levels
the appropriate amount of sunscreen for a long period in Japanese people, similar to Caucasians.
Category 13 (P13): Pigmentation and Melanoma
http://dx.doi.org/10.1016/j.jdermsci.2016.08.509
Novel non-cell autonomous role of epidermal polarity protein Par3 in melanocyte homeostasis and melanomagenesis
P12-18[O3-23] Up-regulation of expression of heme oxygenase-1 by Psidium guajava extract Yuumi Horibe 2,∗ , Yoshiki Okita 1 , Keiko Usui 1 , Keiko Inoue 2 , Kenichi Hamada 2 , Tohru Mizushima 1 1
Department of Drug Discovery and Development, Faculty of Pharmacy, Keio University, Tokyo, Japan 2 Saishunkan Phamaceutical Co., Ltd., Japan In addition to changes with aging, the skin is damaged by various environmental stressors, particularly solar ultraviolet (UV) radiation. UV light induces skin disorders (such as wrinkle formation and hyperpigmentation) through induction of inflammation and apoptosis, leading to cosmetic problems, for which a number of hypopigmenting and anti-wrinkle cosmetic agents have been developed. When cells are exposed to various stressors, they induce a number of proteins, so-called stress proteins, in order to protect themselves against such stressors and heat shock proteins (HSPs) are representative stress proteins. We previously reported that HSPs protect skin against UV-induced wrinkle formation and hyperpigmentation and developed cosmetic agents with HSP-inducing ability. Heme oxygenase-1 (HO-1) is another type of stress protein. Various stressors such as UV irradiation have been reported to induce HO-1 production. HO-1 degrades heme to carbon monoxide (CO), free iron and biliverdin and biliverdin are potent antioxidants and CO has anti-apoptotic activity. Thus, HO-1 has attracted considerable attention as a novel approach to skin photoprotection. In the present study, we searched for HO-1inducers from a library of herbal extracts and selected an extract of Psidium guajava (P. guajava), based on its high HO-1-inducing activity and low cytotoxicity. Treatment of cultured mouse keratinocytes with P. guajava extract up-regulated the expression of HO-1 in a dose-dependent manner. The up-regulation was observed from 3 to 12 h after the initiation of treatment. We are now examining the effect of P. guajava extract on melanin production, UV-induced apoptosis and collagen synthesis to address its beneficial effects against UV-induced skin disorders. http://dx.doi.org/10.1016/j.jdermsci.2016.08.510
e173
P13-01[II-2]
Melina Mescher 1,2 , Matthias Ruebsam 1,3 , Peter Jeong 1 , Marina Kranen 1 , Jennifer Landsberg 4 , Max Schlaak 3 , Cornelia Mauch 3 , Carien M. Niessen 1,2,3 , Thomas Tueting 4 , Sandra Iden 1,2,∗ 1
CECAD Cluster of Excellence, University of Cologne, Cologne, Germany 2 Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 3 Department of Dermatology, University of Cologne, Cologne, Germany 4 Department of Dermatology and Allergy, University of Bonn, Bonn, Germany The Par3-aPKC-Par6 polarity complex controls a variety of polarization processes during development and tissue homeostasis. We recently identified both pro-oncogenic and tumor-suppressive roles of mammalian Par3 in epidermal tumors. Here we report a non-cell autonomous Par3 function in skin cancer. Using the HGF-Cdk4R24C mouse melanoma model, Par3 loss in the epidermal environment (Par3eKO) resulted in increased melanoma multiplicity and lung metastasis, suggesting that polarity proteins modulate the cross-talk of keratinocytes (KCs) and melanocytes (MCs) and serve to suppress melanoma. MCs cocultured with Par3KO KCs showed increased proliferation and less-differentiated morphology, changes also observed in Par3eKO mice. As this required direct KC-MC contacts, we asked for adhesion molecules involved. P-cadherin localized at KC-MC contacts and was elevated in Par3-KO KCs, suggesting that epidermal Par3 controls P-cadherin stability and function. Inhibition of P-but not E-cadherin rescued the morphology of MCs cocultured with Par3-KO KCs. Aberrant P-cadherin-based adhesion upon Par3 loss may thus provide a permissive niche for MC dedifferentiation and transformation. In human melanoma epidermal Par3 expression was reduced in tumor-adjacent areas and negatively correlated with melanoma progression. P-cadherin instead was robustly expressed in melanoma and localized to heterologous adhesions, indicating that P-cadherin contributes to KC-MC cross-talk also in the context of human disease. Together, we show that epidermal polarity proteins control heterologous communication of key skin-resident cell populations through a subset of classical cadherins, and identified a novel non-cell autonomous tumor-suppressor role of Par3 in the microenvironment of malignant melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.511 P13-02[III-3] Disruption of the autophagy-lysosome pathway is involved in hypopigmented macules in patients with tuberous sclerosis complex Lingli Yang ∗ , Fei Yang, Mari Wataya-Kaneda, Ichiro Katayama Department of dermatology, Osaka University, Japan Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomatous lesions present in many organs. Hypopigmented macules are always
e174
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
observed in most patients with TSC. However, the pathogenesis is still unknown. Recently, autophagy-lysosome pathway has been known as a novel pathway involved in skin pigmentation. Objective: To investigate whether the autophagy-lysosome pathway was involved in TSC-associated hypopigmentation. Method: Using skin tissues from hypopigmented macules of TSC, the activity of autophagy-lysosome pathway was assessed by immunohistochemical staining. Furthermore, we investigated both the pigmentation and autophagy-lysosome pathway in TSC2 knockdown (KD) human primary melanocytes and keratinocytes, by real-time PCR, western blotting analyses and intracellular immunofluorescence staining. Results: Compared to healthy donors, the number of melanocytes was not changed, but autophagy-lysosome activity was dramatically decreased in skin tissue of hypopigmentation macules from TSC. Furthermore, in TSC2 KD melanocytes, melanin contents decreased strikingly, and decreased expression levels of melanogenesis-related proteins were observed. Besides, we found that expression levels of LC3, LAMP1 and the number of autophagosomes and lysosomes were significantly decreased in TSC2 KD melanocytes. Furthermore, to clarify whether the reduction of autophagy was related to hypopigmentation, ATG7 siRNA was transfected into melanocytes. Very interestingly, we found that melanogenesis was significantly inhibited in ATG7 KD melanocytes. Conclusions: These results suggest that dysfunction of autophagy-lysosome pathway might be involved in the pathogenesis of hypopigmentation in patients with TSC. http://dx.doi.org/10.1016/j.jdermsci.2016.08.512 P13-03[C11-08] TLR3 agonist Poly (I:C) enhance Rab27A and melanosome transportation through TLR3 and MAVS pathway in human epidermal melanocytes Saaya Koike ∗ , Kenshi Yamasaki, Takeshi Yamauchi, Mai Inoue, Kenichiro Tsuchiyama, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Japan Innate immune stimuli restlessly influence epidermis where human melanocytes reside. We have examined how innate immunity affects pigmentation and observed that TLR3 agonist poly (I:C) increased melanin release from melanocytes. Poly (I:C) increased Rab27A expression and cell peripheral accumulation, which facilitate melanosome transportation to cell membrane. Knockdown of Rab27A abrogated melanosome transfer to keratinocytes by poly (I:C). Since MITF is known to increase Rab27A transcription, we examined if MITF is involved in Rab27A induction by poly (I:C). Poly (I:C) increased MITF mRNA, but siRNA for MITF did not suppress Rab27A increase, perimembranous localization of Gp100 and Rab27A, and extracellular melanin release by poly (I:C). These suggested that poly (I:C) induces Rab27A and melanosome transportation independent of MITF function. We next knocked down TRIF and MAVS, the molecules involved in TLR3 pathway. We observed that siTRIF and siMAVS suppressed Rab27A induction and melanosome transport to neighboring keratinocytes by poly (I:C) stimulation. We also observed that poly (I:C) increased expression of MAVS mRNA about 7-fold, but poly (I:C) did not altered the mRNA level of TRIF and its downstream molecules IRF3 and TBK1. These indicated that TLR3 agonist poly (I:C) activates melanosome trans-
port by increase Rab27A expression through the TLR3 and MAVS pathway. http://dx.doi.org/10.1016/j.jdermsci.2016.08.513 P13-04[C11-07] IFN- modulates chemokines from tumor-associated macrophages to enhance the therapeutic effect of anti-PD-1 antibodies in B16F10 melanoma Aya Kakizaki 1,∗ , Taku Fujimura 1 , Sadanori Furudate 1 , Yumi Kambayashi 1 , Takeshi Yamauchi 1 , Hideo Yagita 2 , Setsuya Aiba 1 1 Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan 2 Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
Tumor-associated macrophages (TAMs) play roles in maintaining the immunosuppressive microenvironment by recruiting regulatory T cells (Tregs). Since IFN- and anti-PD-1 Ab have been clinically used for the treatment of malignant melanoma, we investigated their immunomodulatory effects on melanoma using the B16F10 mouse melanoma model. B16F10 melanoma cells were subcutaneously injected into C57BL/6 mice, and when the tumor had established, we administered IFN-. The peritumoral administration of IFN- significantly decreased the mRNA expression of Tregs-related chemokines (CCL17, CCL22), which led to the suppressed recruitment of Tregs in B16F10 melanoma. Moreover, to validate the production of chemokines, we isolated CD11b+ TAMs by MACS, analyzed the chemokine production, and confirmed the decrease of CCL22 in the IFN- treated group. Furthermore, the culture supernatant from CD11b+ TAMs significantly increased the chemotactic activity for TRP-2-specific CD4+ T cells, which might suggest that chemotactic factors from CD11b+ TAMs recruit B16F10 melanoma-specific T cells into the tumor microenvironment. Since the administration of IFN- augments the expression of PD-1 on TILs, the co-administration of anti-PD-1 Ab augmented the therapeutic effect of IFN- on the B16F10 melanoma. As in the mouse model, in human, IFN- decreased the production of Th2related chemokines and augmented the production of Th1-related chemokines from monocyte- derived M2 macrophages. Since these immunomodulatory effects of IFN- on macrophages were also observed in the lesional skin of human in- transit melanoma, our present data suggest one of the possible immunomodulatory effects of IFN- and support the use of IFN- in combination with anti-PD1 Ab for the treatment of melanoma patients. http://dx.doi.org/10.1016/j.jdermsci.2016.08.514 P13-05[C11-06] Hair shaft miRNA-221 levels as a new tumor marker of melanoma Satoshi Fukushima ∗ , Taisuke Inada, Masayuki Murai, Masatoshi Jinnin, Azusa Miyashita, Satoshi Nakahara, Aki Tokuzumi, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan Micro RNA-221 (miR-221) is known to be abnormally expressed in many human cancers. We have reported that the serum levels