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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
Author(s) -
Hoshi Yutaro,
Uchida Yasuo,
Tachikawa Masanori,
Ohtsuki Sumio,
Terasaki Tetsuya
Publication year - 2017
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13960
Subject(s) - mediator , actin , function (biology) , protein filament , microbiology and biotechnology , attenuation , inflammation , neuroscience , chemistry , biophysics , biology , physics , immunology , biochemistry , optics
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), MAPK 1, 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.