Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats

Rei Matsubara, Toshio Kukita, Yuka Ichigi, Ippei Takigawa, Peng Fei Qu, Noboru Funakubo, Hiroshi Miyamoto, Kazuaki Nonaka, Akiko Kukita

研究成果: ジャーナルへの寄稿記事

16 引用 (Scopus)

抄録

Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1+c-fms+ and Kat1+c-fms- cells but not Kat1-c-fms+ cells. Kat1-c-fms+ cells appeared in early stages of culture, but Kat1+c-fms+ and Kat1+c-fms- cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1+CD11b+ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1+c-fms+ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.

元の言語英語
記事番号e47930
ジャーナルPloS one
7
発行部数10
DOI
出版物ステータス出版済み - 10 24 2012

Fingerprint

Rats
osteoclasts
Osteoclasts
Macrophage Colony-Stimulating Factor
Macrophages
Chemokine Receptors
rats
Bone
cells
Fusion reactions
bone marrow
Macrophage-1 Antigen
Bone Marrow
Nucleic Acid Synthesis Inhibitors
macrophages
Gene encoding
Hydroxyurea
Membrane Proteins
Experimental Arthritis
hydroxyurea

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

これを引用

Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats. / Matsubara, Rei; Kukita, Toshio; Ichigi, Yuka; Takigawa, Ippei; Qu, Peng Fei; Funakubo, Noboru; Miyamoto, Hiroshi; Nonaka, Kazuaki; Kukita, Akiko.

:: PloS one, 巻 7, 番号 10, e47930, 24.10.2012.

研究成果: ジャーナルへの寄稿記事

Matsubara, R, Kukita, T, Ichigi, Y, Takigawa, I, Qu, PF, Funakubo, N, Miyamoto, H, Nonaka, K & Kukita, A 2012, 'Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats', PloS one, 巻. 7, 番号 10, e47930. https://doi.org/10.1371/journal.pone.0047930
Matsubara, Rei ; Kukita, Toshio ; Ichigi, Yuka ; Takigawa, Ippei ; Qu, Peng Fei ; Funakubo, Noboru ; Miyamoto, Hiroshi ; Nonaka, Kazuaki ; Kukita, Akiko. / Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats. :: PloS one. 2012 ; 巻 7, 番号 10.
@article{e9a794cc121940ee8bdca61ec7bb169e,
title = "Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats",
abstract = "Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1+c-fms+ and Kat1+c-fms- cells but not Kat1-c-fms+ cells. Kat1-c-fms+ cells appeared in early stages of culture, but Kat1+c-fms+ and Kat1+c-fms- cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1+CD11b+ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1+c-fms+ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.",
author = "Rei Matsubara and Toshio Kukita and Yuka Ichigi and Ippei Takigawa and Qu, {Peng Fei} and Noboru Funakubo and Hiroshi Miyamoto and Kazuaki Nonaka and Akiko Kukita",
year = "2012",
month = "10",
day = "24",
doi = "10.1371/journal.pone.0047930",
language = "English",
volume = "7",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "10",

}

TY - JOUR

T1 - Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats

AU - Matsubara, Rei

AU - Kukita, Toshio

AU - Ichigi, Yuka

AU - Takigawa, Ippei

AU - Qu, Peng Fei

AU - Funakubo, Noboru

AU - Miyamoto, Hiroshi

AU - Nonaka, Kazuaki

AU - Kukita, Akiko

PY - 2012/10/24

Y1 - 2012/10/24

N2 - Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1+c-fms+ and Kat1+c-fms- cells but not Kat1-c-fms+ cells. Kat1-c-fms+ cells appeared in early stages of culture, but Kat1+c-fms+ and Kat1+c-fms- cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1+CD11b+ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1+c-fms+ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.

AB - Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1+c-fms+ and Kat1+c-fms- cells but not Kat1-c-fms+ cells. Kat1-c-fms+ cells appeared in early stages of culture, but Kat1+c-fms+ and Kat1+c-fms- cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1+CD11b+ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1+c-fms+ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.

UR - http://www.scopus.com/inward/record.url?scp=84868117017&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84868117017&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0047930

DO - 10.1371/journal.pone.0047930

M3 - Article

C2 - 23110133

AN - SCOPUS:84868117017

VL - 7

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 10

M1 - e47930

ER -