title: Actin filament-associated protein 1 (AFAP-1) is a key mediator in inflammatory signaling-induced rapid attenuation of intrinsic P-gp function in human brain capillary endothelial cells.
abstract: The purpose of this study was to identify regulatory molecule(s) involved in the inflammatory signaling-induced decrease in P-glycoprotein (P-gp) efflux function at the blood-brain barrier (BBB) that may occur in brain diseases. We confirmed that in vivo P-gp efflux activity at the BBB was decreased without any change in P-gp protein expression level in a mouse model of acute inflammation induced by 3 mg/kg lipopolysaccharide. In a human BBB model cell line (human brain capillary endothelial cells; hCMEC/D3), 1-h treatment with 10 ng/mL tumor necrosis factor-α (TNF-α; an inflammatory mediator) rapidly reduced P-gp efflux activity, but had no effect on P-gp protein expression level. To clarify the non-transcriptional mechanism that causes the decrease in intrinsic efflux activity of P-gp in acute inflammation, we applied comprehensive quantitative phosphoproteomics to compare hCMEC/D3 cells treated with TNF-α and vehicle (control). Actin filament-associated protein-1 (AFAP-1), MAPK1, and transcription factor AP-1 (AP-1) were significantly phosphorylated in TNF-α-treated cells, and were selected as candidate proteins. In validation experiments, knockdown of AFAP-1 expression blocked the reduction in P-gp efflux activity by TNF-α treatment, whereas inhibition of MAPK function or knockdown of AP-1 expression did not. Quantitative targeted absolute proteomics revealed that the reduction in P-gp activity by TNF-α did not require any change in P-gp protein expression levels in the plasma membrane. Our results demonstrate that AFAP-1 is a key mediator in the inflammatory signaling-induced, translocation-independent rapid attenuation of P-gp efflux activity in human brain capillary endothelial cells.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Hoshi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
authors.forename: Yasuo
authors.identifier: 0000-0001-9860-1979
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Tachikawa
authors.affiliation: Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.|Japan Agency for Medical Research and Development (AMED) CREST, Tokyo, Japan.
authors.forename: Sumio
authors.initials: S
authors.lastname: Ohtsuki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018836:Inflammation Mediators / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C497360:AFAP1 protein, human; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018836:Inflammation Mediators; D008840:Microfilament Proteins; D014409:Tumor Necrosis Factor-alpha
keywords: P-glycoprotein; actin filament-associated protein-1; blood-brain barrier; inflammation; phosphorylation; proteomics