title: Analysis of amphotericin B-induced cell signaling with chemical inhibitors of signaling molecules.
abstract: Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor-kappaB (NF-kappaB), a well-known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte-like cell line. Among chemical inhibitors of signaling molecules, U-73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA-AM (calcium chelator), LFM-A13 (Bruton's tyrosine kinase (Btk)-specific inhibitor), and PP2 (c-Src kinase inhibitor) suppressed AmB-induced phosphorylation of p65 and translocation of p65 into the nucleus. U-73122 and Gö6976 reduced AmB-mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)-alpha and interleukin (IL)-6) in RAW264.7 cells. Furthermore, AmB-induced activation of NF-kappaB was observed in toll-like receptor (TLR) 2-expressed cells, and the activation of NF-kappaB was inhibited by U-73122, whereas peptidoglycan-induced NF-kappaB activation, which was also dependent on TLR2, was not inhibited by U-73122. Finally, U-73122 partially suppressed in vivo production of TNF-alpha and IL-6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c-Src and NF-kappaB. These signaling molecules may become a target for chemotherapy suppressing AmB-induced proinflammatory cytokine production.
authors.affiliation: Microbiology and Oral Infection, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Matsuo
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hotokezaka
authors.forename: Naoya
authors.initials: N
authors.lastname: Ohara
authors.forename: Yuji
authors.initials: Y
authors.lastname: Fujimura
authors.forename: Atsutoshi
authors.initials: A
authors.lastname: Yoshimura
authors.forename: Yukio
authors.initials: Y
authors.lastname: Okada
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hara
authors.forename: Noriaki
authors.initials: N
authors.lastname: Yoshida
authors.forename: Koji
authors.initials: K
authors.lastname: Nakayama
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000077329:Agammaglobulinaemia Tyrosine Kinase; D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D000081247:CSK Tyrosine-Protein Kinase; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D016207:Cytokines / Q000096:biosynthesis; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051193:Toll-Like Receptors / Q000378:metabolism; D051996:Transcription Factor RelA / Q000276:immunology / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019061:src-Family Kinases
chemical_list: D002121:Calcium Channel Blockers; D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D051996:Transcription Factor RelA; D000666:Amphotericin B; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625951:Btk protein, mouse; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002118:Calcium