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451 Anacardic acid reduces lipogenesis in human differentiated adipocytes via inhibition of histone acetylation
Inflammation, Immunity and Infection | ABSTRACTS 448
449
PRINS long non-coding RNA directly binds to the mRNA of IL-6 leading to its destabilization J Danis2,1, A Go¨blo¨s2,1, Z Bata-Cso¨rgo¨1,2, L Keme´ny1,2 and M Sze´ll2,3 1 Department of Dermatology and Allergology, university of Szeged, Szeged, Hungary, 2 MTA-SZTE Dermatological Research Group, Szeged, Hungary and 3 Department of Medical Genetics, University of Szeged, Szeged, Hungary Cytosolic DNA fragments represent pathogen and danger associated molecular patterns and induces a cascade of innate immune responses in the cells. Excessive cytosolic DNA can enhance chronic inflammation predominantly by activating inflammasomes therefore contributing to the pathogenesis of chronic diseases such as psoriasis. Psoriasis associated non-protein coding RNA induced by stress (PRINS) is a long non-coding RNA, which has already been associated with psoriasis susceptibility and cellular stress response; however its precise mechanism was less studied. The aim of this study was to identify the role of PRINS in psoriasis associated inflammatory reactions, which could explain the importance of its high expression in psoriatic uninvolved epidermis. The synthetic DNA analogue poly(dA:dT) transfection was used to induce inflammatory reactions in normal human epidermal keratinocytes (NHEKs), and expression of inflammatory cytokines was measured by real-time RTPCR and ELISA. Poly(dA:dT) transfection induced the expression and secretion of IL-1a, IL-1b, IL-6 and TNF-a, while decreased PRINS expression was detected. To study the possible role of PRINS in the poly(dA:dT) induced cytokine production of NHEKs we forced its expression by vector based method. Overexpression of PRINS reduced the poly(dA:dT)-induced IL-6 production, but did not affect the production of the other investigated cytokines. In silico analysis revealed a putative interaction site between PRINS and the mRNA of IL-6 and the interaction was confirmed by an in vitro binding assay. On cellular level, destruction of the IL-6 mRNA binding site in the PRINS sequence lead to the loss of PRINS’ ability to inhibit IL-6 production. These results show a restrictive effect of PRINS in inflammatory processes, and indicate the role of its higher expression in psoriatic uninvolved epidermis.
Novel function of heparinoid as an anti-inflammatory agent R Utsunomiya1, H Okazaki3, X Dai1, M Murakami1, K Masuda1, H Mori2, K Shiraishi1, M Tohyama1 and K Sayama1 1 Dermatology, Ehime university, Matsuyama, Japan, 2 Dermatology, Ehime prefectural central hospital, Matsuyama, Japan and 3 Dermatology, Ehime prefectural central hospital, Ehime, Japan Heparinoid was established as an analog of heparin, which is a sulfated linear-polysaccharide derived from animal tissue. The molecular structure of heparinoid permits considerable hydrogen bonding with adjacent water molecules, which leads to effective hydration of the surrounding tissue. Today, heparinoid ointment is regarded as an effective moisturizer and is used commonly for dry skin. In addition to this effect, we have found that heparinoid was acting not only as a moisturizer, but also as a suppressant of the triggering of skin inflammation. To assess the inhibitory effect of heparinoid on IL-1b, cultured normal human keratinocytes were pre-treated with heparinoid and then stimulated with house dust mites (HDM) allergens. Then, interleukin IL-1b release from keratinocytes and IL-1b mRNA expression in keratinocytes were evaluated. HDM allergen-induced IL-1b release from keratinocytes was inhibited significantly by 0.01% heparinoid pretreatment for 1 hour without affecting cell viability. However, heparinoid did not affect caspase-1 release, suggesting that heparinoid did not affect HDM allergen-induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro-IL-1b, but also suppressed IL-1b mRNA expression in keratinocytes. We evaluated the activation of extracellular signalregulated kinase (ERK), c-jun N-terminal kinase p38 MAPK and NF-kB pathways. Among them, the active state of ERK and p38 pathways, which are required for IL-1b mRNA expression in keratinocytes, were inhibited by 0.01% heparinoid treatment. The phosphorylation of ERK was strongly reduced at 1 minutes and lasted for 24 hours. The activation of p38 MAPK was reduced from 1 hour to 24 hours. From these results, we conclude that heparinoid blocks the initiation of keratinocyte-mediated skin inflammation and it could work as an anti-inflammatory agent.
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Etretinate as a therapeutic option for pseudolymphoma Y Iwahashi1, Y Kasa1, Y Saruta2, H Uno1 and T Nakada1 1 Dermatology, Showa University Fujigaoka Hospital, Yokohama, Japan and 2 Dermatology, Showa University, Tokyo, Japan Although pseudolymphoma does not have a guarded prognosis, no standard treatment exists. It is not rare to have a difficulty to treat this clinical behavior. Patient: A 57-year-old man presented Dermatology Department with 1.5-year-history of cutaneous lesions of the face. Past medical history included diabetes mellitus, hypertension, blood pressure, hyperlipidosis, and goat. Physical examination revealed dark reddish, infiltrative erythematous lesions on forehead, both lateral eyelids, and preauricular regions. CBC and soluble interleukin-2 receptor (sIL-2R) were within normal limits: WBC 7400/ml (59.4% neuts, 30.2% lymphs, 6.1% monos, 3.8% eos, 0.5% basos), sIL-2R 326U/ml. Histopathological findings demonstrated dense infiltrate of lymphoid cells in the dermis. Follicular arrangements were noted, and follicular center cells included atypical lymphocytes and tingible body macrophages. Phenotype of lymphoid cells were CD20+, CD79a+, and follicular center cells were CD10+, bcl-2-. We diagnosed as pseudolymphoma on the basis of findings described above. Although the patient received topical corticosteroid therapy and nUVB (narrow-band ultraviolet B: total 13.09 J/cm2) therapy, these were not effective. Indometacin and minocycline had to be stopped by adverse effects, and systemic corticosteroid was believed unavailable because of his complications. Etretinate, 40mg per day, was started two years after the initial visit. After the administration, cutaneous lesions were improved, and recurrences were prevented. We concluded that etretinate, a vitamin A derivative, has been used as an adjunctive agent for malignant lymphoma. It may have a potential as a therapeutic option for pseudolymphoma.
Anacardic acid reduces lipogenesis in human differentiated adipocytes via inhibition of histone acetylation M Kim, E Kim, K Cho, D Lee and J Chung Department of Dermatology, Seoul National University College of Medicine, SNUH Biomedical Research Institute, Seoul, Korea (the Republic of) Here we investigated effects of anacardic acid (AA) on the regulation of lipogenesis in differentiated human adipocytes, and elucidated possible epigenetic mechanisms via p300 histone acetyltransferase activity. To investigate the role of histone acetylation in lipogenesis regulation, we evaluated triglyceride (TG) contents, expression of key lipogenic enzyme acetyl-CoA carboxylase (ACC) and sterol regulatory element binding protein 1c (SREBP-1c) in primary cultured adipocytes isolated from subcutaneous adipose tissues. Treatment of AA or knockdown of p300 by using transient transfection of p300 siRNA led to significant reduction of TG contents, SREBP-1c and ACC expression, indicating that p300 mediates SREBP-1, ACC expression, and corollary lipid production. While p300 overexpression by p300WT was associated with significantly enhanced activity of SREBP1-908luc promoter, the SREBP1908luc promoter activity was significantly reduced in the presence of p300DHAT. In addition, we performed a promoter assay using HEK293T cells treated with AA or TSA. While the SREBP1-908luc promoter activity was significantly decreased by AA, but significantly increased by TSA treatment. These findings suggest that histone acetyltransferase activity of p300, not a p300 expression per se, is critical for the transcriptional regulation of SREBP-1 and p300HAT inhibitors such as AA could be employed as anti-obesity modalities.
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Housekeeping gene selection and validation for qPCR analysis of psoriasis biopsies A Zolotarenko, EV Chekalin, A Prelovskaya and S Bruskin Laboratory of functional genomics, Vavilov Institute of General Genetics RAS, Moscow, Russian Federation Real-time PCR is the “gold standard” of the existing methods for assessing the gene expression. However, despite the accuracy of the methodology as a whole, the results obtained by this method can vary greatly depending on the selected normalization method. Despite the general stability, the expression profiles of the housekeeping genes can vary greatly depending on the type of sample being analyzed. Thus, for each specific task - for example, different types of cells and tissues - it is necessary to select the optimal housekeeping genes that could be used as the reference ones. In this study, based on the data obtained from the RNA-seq of 14 pairs of biopsies taken from lesional and non-lesional skin of psoriasis patients, 14 genes were selected as the candidate reference genes in the analysis of expression profiles. This were BABAM1, GAPDH, ANAPC5, UIMC1, TAX1BP1, POLS3C, OS9, NRD1, XRCC1, USP16, SAP18, NMT1, GGNBP2, CES. Expression profiles of the selected genes were evaluated by the quantitative real-time PCR at an additional panel of 8 pairs of biopsies taken from lesional and non-lesional skin of psoriasis patients. The resulting expression data was analyzed using Bestkeeper, GeNorm, Normfinder services in order to identify the most stable reference genes for psoriatic tissues. Based on the results of the analysis, the genes NMT1, BABAM1, XRCC1 proved to be the most stable, and the most variable ones were TAX1BP1, NRD1 and GAPDH -the gene widely used in the analysis of qPCR data. The data obtained highlights the importance of choosing the right housekeeping genes for each specific cell or tissue type prior to the performance of the expression studies.
The role of purinergic signaling in development of irritant dermatitis of acrodermatitis enteropathica T Okamoto, Y Ogawa, S Shimada and T Kawamura Department of Dermatology, University of Yamanashi, Yamanashi, Japan Purinergic signaling is crucial to maintain homeostasis of inflammation. We previously elucidated that disruption of the homeostasis of skin inflammation by Zn deficiency-mediated ablation of Langerhans cells (LCs) that expressed CD39 (E-NTPDase1) was causative for development of dermatitis in acrodermatitis enteropathica. ATP was hydrolyzed into AMP by E-NTPDases (Entpd), E-NPPs (Enpp), and tissue non-specific alkaline phosphatases (TNAP). It has been well known that LCs express Entpd1 (CD39) and keratinocytes (KCs) do not express it in both mice and humans. However, the expression of other ATP-hydrolyzing molecules such as Entpd 2-3, Enpp 1-3, and TNAP in LCs and KCs is not addressed so far. The purpose of this study is 1. to determine the expression of these ATP-hydrolyzing molecules in LCs and KCs of both mice and humans by qPCR, and 2. to examine the alteration of expression of these ATP-hydrolyzing molecules under Zn deficient condition by qPCR. Normal murine KCs expressed Entpd2, Enpp1, and low levels of Enpp2 and TNAP, whereas normal human epidermal KCs (NHEKs) expressed Entpd2, Entpd3, and Enpp1. Normal murine LCs expressed Entpd1 (CD39), Entpd2, and low level of Enpp1, whereas normal human LCs expressed Entpd1 (CD39), and low levels of Enpp2 and Enpp3. KCs from Zn-deficient diet mice showed increased expression of Entpd2 and Enpp1, and loss of expression of Enpp2 and TNAP. Finally, we examined the impact of LCs in ATP hydrolysis. ATP hydrolysis activity was determined by measuring released phosphate. ATP hydrolysis activity was impaired up to 82% in LC-depleted murine epidermis compared with LC-existing murine epidermis. These data suggested that the expression of ATP-hydrolyzing molecules in LCs and KCs were different between mice and humans. Additionally, Zn deficiency altered the expression of ATP-hydrolyzing molecules in KCs. Lastly, Entpd1 (CD39)- and Entpd2-expressing LCs assumed about 80% of ATP hydrolysis, whereas Entpd2- and Enpp1-expressing KCs assumed the remaining 20% ATP hydrolysis in murine epidermis.
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449
PRINS long non-coding RNA directly binds to the mRNA of IL-6 leading to its destabilization J Danis2,1, A Go¨blo¨s2,1, Z Bata-Cso¨rgo¨1,2, L Keme´ny1,2 and M Sze´ll2,3 1 Department of Dermatology and Allergology, university of Szeged, Szeged, Hungary, 2 MTA-SZTE Dermatological Research Group, Szeged, Hungary and 3 Department of Medical Genetics, University of Szeged, Szeged, Hungary Cytosolic DNA fragments represent pathogen and danger associated molecular patterns and induces a cascade of innate immune responses in the cells. Excessive cytosolic DNA can enhance chronic inflammation predominantly by activating inflammasomes therefore contributing to the pathogenesis of chronic diseases such as psoriasis. Psoriasis associated non-protein coding RNA induced by stress (PRINS) is a long non-coding RNA, which has already been associated with psoriasis susceptibility and cellular stress response; however its precise mechanism was less studied. The aim of this study was to identify the role of PRINS in psoriasis associated inflammatory reactions, which could explain the importance of its high expression in psoriatic uninvolved epidermis. The synthetic DNA analogue poly(dA:dT) transfection was used to induce inflammatory reactions in normal human epidermal keratinocytes (NHEKs), and expression of inflammatory cytokines was measured by real-time RTPCR and ELISA. Poly(dA:dT) transfection induced the expression and secretion of IL-1a, IL-1b, IL-6 and TNF-a, while decreased PRINS expression was detected. To study the possible role of PRINS in the poly(dA:dT) induced cytokine production of NHEKs we forced its expression by vector based method. Overexpression of PRINS reduced the poly(dA:dT)-induced IL-6 production, but did not affect the production of the other investigated cytokines. In silico analysis revealed a putative interaction site between PRINS and the mRNA of IL-6 and the interaction was confirmed by an in vitro binding assay. On cellular level, destruction of the IL-6 mRNA binding site in the PRINS sequence lead to the loss of PRINS’ ability to inhibit IL-6 production. These results show a restrictive effect of PRINS in inflammatory processes, and indicate the role of its higher expression in psoriatic uninvolved epidermis.
Novel function of heparinoid as an anti-inflammatory agent R Utsunomiya1, H Okazaki3, X Dai1, M Murakami1, K Masuda1, H Mori2, K Shiraishi1, M Tohyama1 and K Sayama1 1 Dermatology, Ehime university, Matsuyama, Japan, 2 Dermatology, Ehime prefectural central hospital, Matsuyama, Japan and 3 Dermatology, Ehime prefectural central hospital, Ehime, Japan Heparinoid was established as an analog of heparin, which is a sulfated linear-polysaccharide derived from animal tissue. The molecular structure of heparinoid permits considerable hydrogen bonding with adjacent water molecules, which leads to effective hydration of the surrounding tissue. Today, heparinoid ointment is regarded as an effective moisturizer and is used commonly for dry skin. In addition to this effect, we have found that heparinoid was acting not only as a moisturizer, but also as a suppressant of the triggering of skin inflammation. To assess the inhibitory effect of heparinoid on IL-1b, cultured normal human keratinocytes were pre-treated with heparinoid and then stimulated with house dust mites (HDM) allergens. Then, interleukin IL-1b release from keratinocytes and IL-1b mRNA expression in keratinocytes were evaluated. HDM allergen-induced IL-1b release from keratinocytes was inhibited significantly by 0.01% heparinoid pretreatment for 1 hour without affecting cell viability. However, heparinoid did not affect caspase-1 release, suggesting that heparinoid did not affect HDM allergen-induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro-IL-1b, but also suppressed IL-1b mRNA expression in keratinocytes. We evaluated the activation of extracellular signalregulated kinase (ERK), c-jun N-terminal kinase p38 MAPK and NF-kB pathways. Among them, the active state of ERK and p38 pathways, which are required for IL-1b mRNA expression in keratinocytes, were inhibited by 0.01% heparinoid treatment. The phosphorylation of ERK was strongly reduced at 1 minutes and lasted for 24 hours. The activation of p38 MAPK was reduced from 1 hour to 24 hours. From these results, we conclude that heparinoid blocks the initiation of keratinocyte-mediated skin inflammation and it could work as an anti-inflammatory agent.
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451
Etretinate as a therapeutic option for pseudolymphoma Y Iwahashi1, Y Kasa1, Y Saruta2, H Uno1 and T Nakada1 1 Dermatology, Showa University Fujigaoka Hospital, Yokohama, Japan and 2 Dermatology, Showa University, Tokyo, Japan Although pseudolymphoma does not have a guarded prognosis, no standard treatment exists. It is not rare to have a difficulty to treat this clinical behavior. Patient: A 57-year-old man presented Dermatology Department with 1.5-year-history of cutaneous lesions of the face. Past medical history included diabetes mellitus, hypertension, blood pressure, hyperlipidosis, and goat. Physical examination revealed dark reddish, infiltrative erythematous lesions on forehead, both lateral eyelids, and preauricular regions. CBC and soluble interleukin-2 receptor (sIL-2R) were within normal limits: WBC 7400/ml (59.4% neuts, 30.2% lymphs, 6.1% monos, 3.8% eos, 0.5% basos), sIL-2R 326U/ml. Histopathological findings demonstrated dense infiltrate of lymphoid cells in the dermis. Follicular arrangements were noted, and follicular center cells included atypical lymphocytes and tingible body macrophages. Phenotype of lymphoid cells were CD20+, CD79a+, and follicular center cells were CD10+, bcl-2-. We diagnosed as pseudolymphoma on the basis of findings described above. Although the patient received topical corticosteroid therapy and nUVB (narrow-band ultraviolet B: total 13.09 J/cm2) therapy, these were not effective. Indometacin and minocycline had to be stopped by adverse effects, and systemic corticosteroid was believed unavailable because of his complications. Etretinate, 40mg per day, was started two years after the initial visit. After the administration, cutaneous lesions were improved, and recurrences were prevented. We concluded that etretinate, a vitamin A derivative, has been used as an adjunctive agent for malignant lymphoma. It may have a potential as a therapeutic option for pseudolymphoma.
Anacardic acid reduces lipogenesis in human differentiated adipocytes via inhibition of histone acetylation M Kim, E Kim, K Cho, D Lee and J Chung Department of Dermatology, Seoul National University College of Medicine, SNUH Biomedical Research Institute, Seoul, Korea (the Republic of) Here we investigated effects of anacardic acid (AA) on the regulation of lipogenesis in differentiated human adipocytes, and elucidated possible epigenetic mechanisms via p300 histone acetyltransferase activity. To investigate the role of histone acetylation in lipogenesis regulation, we evaluated triglyceride (TG) contents, expression of key lipogenic enzyme acetyl-CoA carboxylase (ACC) and sterol regulatory element binding protein 1c (SREBP-1c) in primary cultured adipocytes isolated from subcutaneous adipose tissues. Treatment of AA or knockdown of p300 by using transient transfection of p300 siRNA led to significant reduction of TG contents, SREBP-1c and ACC expression, indicating that p300 mediates SREBP-1, ACC expression, and corollary lipid production. While p300 overexpression by p300WT was associated with significantly enhanced activity of SREBP1-908luc promoter, the SREBP1908luc promoter activity was significantly reduced in the presence of p300DHAT. In addition, we performed a promoter assay using HEK293T cells treated with AA or TSA. While the SREBP1-908luc promoter activity was significantly decreased by AA, but significantly increased by TSA treatment. These findings suggest that histone acetyltransferase activity of p300, not a p300 expression per se, is critical for the transcriptional regulation of SREBP-1 and p300HAT inhibitors such as AA could be employed as anti-obesity modalities.
452
453
Housekeeping gene selection and validation for qPCR analysis of psoriasis biopsies A Zolotarenko, EV Chekalin, A Prelovskaya and S Bruskin Laboratory of functional genomics, Vavilov Institute of General Genetics RAS, Moscow, Russian Federation Real-time PCR is the “gold standard” of the existing methods for assessing the gene expression. However, despite the accuracy of the methodology as a whole, the results obtained by this method can vary greatly depending on the selected normalization method. Despite the general stability, the expression profiles of the housekeeping genes can vary greatly depending on the type of sample being analyzed. Thus, for each specific task - for example, different types of cells and tissues - it is necessary to select the optimal housekeeping genes that could be used as the reference ones. In this study, based on the data obtained from the RNA-seq of 14 pairs of biopsies taken from lesional and non-lesional skin of psoriasis patients, 14 genes were selected as the candidate reference genes in the analysis of expression profiles. This were BABAM1, GAPDH, ANAPC5, UIMC1, TAX1BP1, POLS3C, OS9, NRD1, XRCC1, USP16, SAP18, NMT1, GGNBP2, CES. Expression profiles of the selected genes were evaluated by the quantitative real-time PCR at an additional panel of 8 pairs of biopsies taken from lesional and non-lesional skin of psoriasis patients. The resulting expression data was analyzed using Bestkeeper, GeNorm, Normfinder services in order to identify the most stable reference genes for psoriatic tissues. Based on the results of the analysis, the genes NMT1, BABAM1, XRCC1 proved to be the most stable, and the most variable ones were TAX1BP1, NRD1 and GAPDH -the gene widely used in the analysis of qPCR data. The data obtained highlights the importance of choosing the right housekeeping genes for each specific cell or tissue type prior to the performance of the expression studies.
The role of purinergic signaling in development of irritant dermatitis of acrodermatitis enteropathica T Okamoto, Y Ogawa, S Shimada and T Kawamura Department of Dermatology, University of Yamanashi, Yamanashi, Japan Purinergic signaling is crucial to maintain homeostasis of inflammation. We previously elucidated that disruption of the homeostasis of skin inflammation by Zn deficiency-mediated ablation of Langerhans cells (LCs) that expressed CD39 (E-NTPDase1) was causative for development of dermatitis in acrodermatitis enteropathica. ATP was hydrolyzed into AMP by E-NTPDases (Entpd), E-NPPs (Enpp), and tissue non-specific alkaline phosphatases (TNAP). It has been well known that LCs express Entpd1 (CD39) and keratinocytes (KCs) do not express it in both mice and humans. However, the expression of other ATP-hydrolyzing molecules such as Entpd 2-3, Enpp 1-3, and TNAP in LCs and KCs is not addressed so far. The purpose of this study is 1. to determine the expression of these ATP-hydrolyzing molecules in LCs and KCs of both mice and humans by qPCR, and 2. to examine the alteration of expression of these ATP-hydrolyzing molecules under Zn deficient condition by qPCR. Normal murine KCs expressed Entpd2, Enpp1, and low levels of Enpp2 and TNAP, whereas normal human epidermal KCs (NHEKs) expressed Entpd2, Entpd3, and Enpp1. Normal murine LCs expressed Entpd1 (CD39), Entpd2, and low level of Enpp1, whereas normal human LCs expressed Entpd1 (CD39), and low levels of Enpp2 and Enpp3. KCs from Zn-deficient diet mice showed increased expression of Entpd2 and Enpp1, and loss of expression of Enpp2 and TNAP. Finally, we examined the impact of LCs in ATP hydrolysis. ATP hydrolysis activity was determined by measuring released phosphate. ATP hydrolysis activity was impaired up to 82% in LC-depleted murine epidermis compared with LC-existing murine epidermis. These data suggested that the expression of ATP-hydrolyzing molecules in LCs and KCs were different between mice and humans. Additionally, Zn deficiency altered the expression of ATP-hydrolyzing molecules in KCs. Lastly, Entpd1 (CD39)- and Entpd2-expressing LCs assumed about 80% of ATP hydrolysis, whereas Entpd2- and Enpp1-expressing KCs assumed the remaining 20% ATP hydrolysis in murine epidermis.
www.jidonline.org S269