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The pathogenic ceramide metabolizing enzyme sphingomyelin deacylase in atopic dermatitis is identical to the beta-subunit of acid ceramidase
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Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
propose that self-genomic dsDNA fragments promote cell proliferation and hinder normal cell differentiation in human keratinocytes via inducing a TNF-␣-mediated immune cascade, thus functioning as a potential trigger of psoriasis. http://dx.doi.org/10.1016/j.jdermsci.2017.02.016 P01-14[C10-4] High fat diet exacerbates psoriasis-like skin lesion induced by imiquimod through inducing IL-17A and inflammasomes in mice Yuko Higashi 1,∗ , Munekazu Yamakuchi 2 , Tomoko Fukushige 1 , Teruto Hashiguchi 2 , Takuro Kanekura 1 1
The Department of Dermatology, University of Kagoshima, Kagoshima, Japan 2 Department of Laboratory and Vascular Medicine, University of Kagoshima, Kagoshima, Japan Psoriasis, a chronic inflammatory skin disease, turned out to be closely related with systemic metabolism. Recent studies revealed that an elevated body mass index (BMI) is a risk factor of psoriasis and assembly of NLRP3 inflammasome is produced by adipose tissue macrophages in obese subjects. We hypothesized that hyperlipidemia is involved in the pathogenesis of psoriasis and examined the effect of high fat diet (HFD) in the development of psoriasis in mice treated with imiquimod (IMQ). Mice fed with regular or HFD were treated with topical IMQ on their back skin. They were sacrificed at day 3 and skin and systemic conditions were evaluated. Body weight and serum lipid levels were measured routinely. Severity of skin lesions was represented by the skin score based on thickness of epidermis and degrees of erythema, induration, and scale. Skin infiltrates, cytokine levels, and activation of caspase1 and IL-1 were examined by immunohistochemical staining, quantitative PCR, and Western blotting, respectively. Body weight and serum level of cholesterol were significantly higher in mice fed with HFD (HFD-mice) compared with those fed with regular diet (RD-mice). HFD-mice showed the higher skin scores and increased number of neutrophils infiltrating into the lesional dermis. IL-17A mRNA expression was significantly increased in the skin of HFD-mice. Expression of IL-22, IL-23, and TNF-␣ mRNA was not enhanced in HFD-mice compared with RD-mice. Caspase-1 and IL-1 were activated in the skin of HFD-mice. These results strongly suggest that hyperlipidemia is involved in the development and progression of psoriasis via IL-17A and inflammasomes.
P01-15[C10-7] The pathogenic ceramide metabolizing enzyme sphingomyelin deacylase in atopic dermatitis is identical to the beta-subunit of acid ceramidase Yasuhiro Teranishi 1 , Hiroshi Kuwahara 1 , Makoto Kawashima 2 , Genji Imokawa 3 , Mari Nogami-Itoh 4,5,∗ 1 Innovative Drug Discovery Laboratories, Sumitomo Dainippon Pharma, Japan 2 The Department of Dermatology, Tokyo Women’s Medical University, Japan 3 Research Institute for Biological Functions, Chubu University, Japan 4 Itoh Immunology and Allergy Institute, Japan 5 National Institutes of Biomedical Innovation, Health and Nutrition, Japan A ceramide deficiency (CerD) in the stratum corneum is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). We previously reported that sphingomyelin (SM) deacylase (SMDase), which hydrolyzes SM to yield its lysoform sphingosylphosphorylcholine (SPC) instead of ceramide, is over-expressed in AD skin, resulting in the CerD. Although its enzymatic properties have been clarified, the enzyme itself remains unidentified. In this study, we have purified SMDase from rat skin. The enzymatic activities of SMDase and acid ceramidase (AC) were measured using SM and ceramide as substrates by monitoring the production of SPC and sphingosine, respectively, using tandem mass spectrometry. By successive column chromatography on Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SMDase was purified to homogeneity with a single band of ∼43 kDa with an enrichment of 14,067-fold. Analysis by MALDI-TOF MS/MS of the purified SMDase separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the beta-subunit of AC, which consists of alpha- and beta-subunits linked by two S-S bonds. Western blotting of samples treated with mercaptoethanol revealed that whereas recombinant(r)AC was detectable with antibodies to the alpha subunit at ∼56 kDa and ∼13 kDa and the beta subunit at ∼43 kDa, the purified SMDase was detectable only with an antibody to the beta subunit at ∼43 kDa. Consistently, breaking the two S–S bonds of rAC with dithiothreitol elicited the activity of SMDase with ∼40 kDa upon gel chromatography in contrast to AC activity with ∼50 kDa in untreated rAC. These results provide a new insight into the essential role of SMDase expressed as an AC-breaking beta-subunit in evoking CerD.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.017 http://dx.doi.org/10.1016/j.jdermsci.2017.02.018
Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
propose that self-genomic dsDNA fragments promote cell proliferation and hinder normal cell differentiation in human keratinocytes via inducing a TNF-␣-mediated immune cascade, thus functioning as a potential trigger of psoriasis. http://dx.doi.org/10.1016/j.jdermsci.2017.02.016 P01-14[C10-4] High fat diet exacerbates psoriasis-like skin lesion induced by imiquimod through inducing IL-17A and inflammasomes in mice Yuko Higashi 1,∗ , Munekazu Yamakuchi 2 , Tomoko Fukushige 1 , Teruto Hashiguchi 2 , Takuro Kanekura 1 1
The Department of Dermatology, University of Kagoshima, Kagoshima, Japan 2 Department of Laboratory and Vascular Medicine, University of Kagoshima, Kagoshima, Japan Psoriasis, a chronic inflammatory skin disease, turned out to be closely related with systemic metabolism. Recent studies revealed that an elevated body mass index (BMI) is a risk factor of psoriasis and assembly of NLRP3 inflammasome is produced by adipose tissue macrophages in obese subjects. We hypothesized that hyperlipidemia is involved in the pathogenesis of psoriasis and examined the effect of high fat diet (HFD) in the development of psoriasis in mice treated with imiquimod (IMQ). Mice fed with regular or HFD were treated with topical IMQ on their back skin. They were sacrificed at day 3 and skin and systemic conditions were evaluated. Body weight and serum lipid levels were measured routinely. Severity of skin lesions was represented by the skin score based on thickness of epidermis and degrees of erythema, induration, and scale. Skin infiltrates, cytokine levels, and activation of caspase1 and IL-1 were examined by immunohistochemical staining, quantitative PCR, and Western blotting, respectively. Body weight and serum level of cholesterol were significantly higher in mice fed with HFD (HFD-mice) compared with those fed with regular diet (RD-mice). HFD-mice showed the higher skin scores and increased number of neutrophils infiltrating into the lesional dermis. IL-17A mRNA expression was significantly increased in the skin of HFD-mice. Expression of IL-22, IL-23, and TNF-␣ mRNA was not enhanced in HFD-mice compared with RD-mice. Caspase-1 and IL-1 were activated in the skin of HFD-mice. These results strongly suggest that hyperlipidemia is involved in the development and progression of psoriasis via IL-17A and inflammasomes.
P01-15[C10-7] The pathogenic ceramide metabolizing enzyme sphingomyelin deacylase in atopic dermatitis is identical to the beta-subunit of acid ceramidase Yasuhiro Teranishi 1 , Hiroshi Kuwahara 1 , Makoto Kawashima 2 , Genji Imokawa 3 , Mari Nogami-Itoh 4,5,∗ 1 Innovative Drug Discovery Laboratories, Sumitomo Dainippon Pharma, Japan 2 The Department of Dermatology, Tokyo Women’s Medical University, Japan 3 Research Institute for Biological Functions, Chubu University, Japan 4 Itoh Immunology and Allergy Institute, Japan 5 National Institutes of Biomedical Innovation, Health and Nutrition, Japan A ceramide deficiency (CerD) in the stratum corneum is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). We previously reported that sphingomyelin (SM) deacylase (SMDase), which hydrolyzes SM to yield its lysoform sphingosylphosphorylcholine (SPC) instead of ceramide, is over-expressed in AD skin, resulting in the CerD. Although its enzymatic properties have been clarified, the enzyme itself remains unidentified. In this study, we have purified SMDase from rat skin. The enzymatic activities of SMDase and acid ceramidase (AC) were measured using SM and ceramide as substrates by monitoring the production of SPC and sphingosine, respectively, using tandem mass spectrometry. By successive column chromatography on Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SMDase was purified to homogeneity with a single band of ∼43 kDa with an enrichment of 14,067-fold. Analysis by MALDI-TOF MS/MS of the purified SMDase separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the beta-subunit of AC, which consists of alpha- and beta-subunits linked by two S-S bonds. Western blotting of samples treated with mercaptoethanol revealed that whereas recombinant(r)AC was detectable with antibodies to the alpha subunit at ∼56 kDa and ∼13 kDa and the beta subunit at ∼43 kDa, the purified SMDase was detectable only with an antibody to the beta subunit at ∼43 kDa. Consistently, breaking the two S–S bonds of rAC with dithiothreitol elicited the activity of SMDase with ∼40 kDa upon gel chromatography in contrast to AC activity with ∼50 kDa in untreated rAC. These results provide a new insight into the essential role of SMDase expressed as an AC-breaking beta-subunit in evoking CerD.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.017 http://dx.doi.org/10.1016/j.jdermsci.2017.02.018