title: Changes of cell-surface thiols and intracellular signaling in human monocytic cell line THP-1 treated with diphenylcyclopropenone.
abstract: Changes of cell-surface thiols induced by chemical treatment may affect the conformations of membrane proteins and intracellular signaling mechanisms. In our previous study, we found that a non-toxic dose of diphenylcyclopropene (DPCP), which is a potent skin sensitizer, induced an increase of cell-surface thiols in cells of a human monocytic cell line, THP-1. Here, we examined the influence of DPCP on intracellular signaling. First, we confirmed that DPCP induced an increase of cell-surface thiols not only in THP-1 cells, but also in primary monocytes. The intracellular reduced-form glutathione/oxidized-form glutathione ratio (GSH/GSSG ratio) was not affected by DPCP treatment. By means of labeling with a membrane-impermeable thiol-reactive compound, Alexa Fluor 488 C5 maleimide (AFM), followed by two-dimensional gel electrophoresis and analysis by liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), we identified several proteins whose thiol contents were modified in response to DPCP. These proteins included cell membrane components, such as actin and β-tubulin, molecular chaperones, such as heat shock protein 27A and 70, and endoplasmic reticulum (ER) stress-inducible proteins. Next, we confirmed the expression in DPCP-treated cells of spliced XBP1, a known marker of ER stress. We also detected the phosphorylation of SAPK/JNK and p38 MAPK, which are downstream signaling molecules in the IRE1α-ASK1 pathway, which is activated by ER stress. These data suggested that increase of cell-surface thiols might be associated with activation of ER stress-mediated signaling.
authors.affiliation: Shiseido Research Center, Kanagawa, Japan. morihiko.hirota@to.shiseido.co.jp
authors.forename: Morihiko
authors.initials: M
authors.lastname: Hirota
authors.forename: Akira
authors.initials: A
authors.lastname: Motoyama
authors.forename: Mie
authors.initials: M
authors.lastname: Suzuki
authors.forename: Masashi
authors.initials: M
authors.lastname: Yanagi
authors.forename: Masato
authors.initials: M
authors.lastname: Kitagaki
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kouzuki
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Hagino
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Itagaki
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sasa
authors.forename: Saori
authors.initials: S
authors.lastname: Kagatani
authors.forename: Setsuya
authors.initials: S
authors.lastname: Aiba
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003521:Cyclopropanes / Q000633:toxicity*; D017449:Dermatitis, Allergic Contact / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D006241:Haptens / Q000633:toxicity*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D003521:Cyclopropanes; D006241:Haptens; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; C029402:diphenylcyclopropenone