CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

研究成果: ジャーナルへの寄稿記事

53 引用 (Scopus)

抄録

P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury.

元の言語英語
ページ(範囲)301-310
ページ数10
ジャーナルPurinergic Signalling
8
発行部数2
DOI
出版物ステータス出版済み - 6 1 2012

Fingerprint

Purinergic P2X4 Receptors
Microglia
Lysosomes
Exocytosis
Green Fluorescent Proteins
Fluorescent Antibody Technique
Hypersensitivity
Membrane Proteins
CCR Receptors
Time-Lapse Imaging
Biotinylation
Purinergic P2 Receptors
Hexosaminidases
Pain
Peripheral Nerve Injuries
Wounds and Injuries
Neuroglia
Nervous System
Cultured Cells
Adenosine Triphosphate

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cellular and Molecular Neuroscience
  • Cell Biology

これを引用

@article{0f8a630d7a4b44b0b6e5f7a5273e7006,
title = "CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia",
abstract = "P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury.",
author = "Emika Toyomitsu and Makoto Tsuda and Tomohiro Yamashita and Hidetoshi Tozaki-Saitoh and Yoshitaka Tanaka and Kazuhide Inoue",
year = "2012",
month = "6",
day = "1",
doi = "10.1007/s11302-011-9288-x",
language = "English",
volume = "8",
pages = "301--310",
journal = "Purinergic Signalling",
issn = "1573-9538",
publisher = "Springer Netherlands",
number = "2",

}

TY - JOUR

T1 - CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

AU - Toyomitsu, Emika

AU - Tsuda, Makoto

AU - Yamashita, Tomohiro

AU - Tozaki-Saitoh, Hidetoshi

AU - Tanaka, Yoshitaka

AU - Inoue, Kazuhide

PY - 2012/6/1

Y1 - 2012/6/1

N2 - P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury.

AB - P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury.

UR - http://www.scopus.com/inward/record.url?scp=84861009038&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84861009038&partnerID=8YFLogxK

U2 - 10.1007/s11302-011-9288-x

DO - 10.1007/s11302-011-9288-x

M3 - Article

C2 - 22222817

AN - SCOPUS:84861009038

VL - 8

SP - 301

EP - 310

JO - Purinergic Signalling

JF - Purinergic Signalling

SN - 1573-9538

IS - 2

ER -