title: Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.
abstract: Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan ; Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
authors.forename: Ami
authors.initials: A
authors.lastname: Oizumi
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan ; Laboratory of Biochemistry, Juntendo University School of Health Care and Nursing, Urayasu, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakayama
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Iwahara
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
authors.forename: Katsunari
authors.initials: K
authors.lastname: Kina
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Matsumoto
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan.
authors.forename: Hideoki
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan ; Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Takamori
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
authors.affiliation: Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Suga
authors.affiliation: Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan ; Laboratory of Biochemistry, Juntendo University School of Health Care and Nursing, Urayasu, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Iwabuchi
mesh_terms: D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism*; D008246:Lysophospholipids / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D055576:Neutral Ceramidase / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011549:Pseudomonas / Q000378:metabolism*; D049349:Receptors, Lysosphingolipid / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099157:Sphingosine Kinase
chemical_list: D019332:Endothelin-1; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D016209:Interleukin-8; D008246:Lysophospholipids; D000072000:NF-KappaB Inhibitor alpha; D055576:Neutral Ceramidase; D017853:Phosphotransferases (Alcohol Group Acceptor); D049349:Receptors, Lysosphingolipid; D013110:Sphingosine; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000099157:Sphingosine Kinase; C000606204:NFKBIA protein, human; C060506:sphingosine 1-phosphate