Hypoxia-induced production of peptidylarginine deiminases and citrullinated proteins in malignant glioma cells.
BACKGROUND
Recently, it has been reported that hypoxia highly enhances expression of peptidylarginine deiminase (PAD) 4 and production of citrullinated proteins in some tumor cells. However, little is known about malignant gliomas on this issue. Therefore, we here investigated whether expression of PADs was induced by hypoxia and whether PADs citrullinated intracellular proteins if induced using U-251 MG cells of a human malignant glioma cell line.


METHODS
Expression of PADs in U-251 MG cells, cultured under hypoxia or normoxia for 24 h, was investigated by quantitative polymerase chain reaction (qPCR). Citrullination of proteins in the cells and the cell lysates incubated for 48 h with or without Ca2+was detected by western blotting. Citrullinated proteins were identified by mass spectrometry.


RESULTS
The mRNA levels of PAD1, 2, 3, and 4 were up-regulated by hypoxia in a hypoxia-inducible factor-1-dependent manner in U-251 MG cells. In spite of the increased expression, intracellular proteins were not citrullinated. However, the induced PADs citrullinated U-251 MG cell-derived proteins when the cells were lysed. Multiple proteins citrullinated by hypoxia-induced PADs were identified. In addition, the extracellular domain of vascular endothelial growth factor receptor 2 was citrullinated by human PAD2 in vitro.


CONCLUSIONS
Our data may contribute to understanding of pathophysiology of malignant gliomas from the aspects of protein citrullination.
Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan; Department of Neurosurgery, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan. Electronic address: sasetaigen@marianna-u.ac.jp.
Taigen
T
Sase
Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan. Electronic address: m-ari@marianna-u.ac.jp.
Mitsumi
M
Arito
Department of Neurosurgery, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan. Electronic address: dera@marianna-u.ac.jp.
Hidetaka
H
Onodera
Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan. Electronic address: omoteyama@marianna-u.ac.jp.
Kazuki
K
Omoteyama
Disease Biomarker Analysis and Molecular Regulation, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan. Electronic address: manae@marianna-u.ac.jp.
Manae S
MS
Kurokawa
Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. Electronic address: yakagami@tmig.or.jp.
Yayoi
Y
Kagami
Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. Electronic address: ishigami@tmig.or.jp.
Akihito
A
Ishigami
Department of Neurosurgery, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan. Electronic address: tanaka@marianna-u.ac.jp.
Yuichiro
Y
Tanaka
Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa 216-8511, Japan. Electronic address: t3kato@marianna-u.ac.jp.
Tomohiro
T
Kato
D045744:Cell Line, Tumor; D002956:Citrulline / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D000072258:Tumor Hypoxia
D002956:Citrulline; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D010100:Oxygen
Citrullinated proteins; Hypoxia; Malignant gliomas; Peptidylarginine deiminase