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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1738.)
© 2001 American Heart Association, Inc.

Vascular Biology

Induction of Nuclear Orphan Receptor NGFI-B Gene and Apoptosis in Rat Vascular Smooth Muscle Cells Treated With Pyrrolidinedithiocarbamate

Tokumitsu Watanabe; Masao Yoshizumi; Masahiro Akishita; Masato Eto; Kenji Toba; Masayoshi Hashimoto; Koichiro Nagano; Yi-Qiang Liang; Yumiko Ohike; Katsuya Iijima; Noriko Sudoh; Seungbum Kim; Takashi Nakaoka; Naohide Yamashita; Junya Ako; Yasuyoshi Ouchi

From the Department of Geriatric Medicine (T.W., M.Y., M.E., M.H., K.N., Y.-Q.L., Y. Ohike, K.I., N.S., S.K., J.A., Y. Ouchi), Graduate School of Medicine, University of Tokyo; the Department of Geriatric Medicine (M.A., K.T.), Kyorin University School of Medicine; and the Department of Advanced Medicine (T.N., N.Y.), Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Correspondence to Yasuyoshi Ouchi, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail youchi-tky{at}umin.ac.jp

Abstract—— NGFI-B is one of the orphan nuclear receptors, and its gene is implicated in the apoptosis of T cells. The aim of this study was to investigate the expression and the role of NGFI-B in vascular smooth muscle cells (VSMCs). Pyrrolidinedithiocarbamate (PDTC) is a modulator of an oxidative state and is reported to induce apoptosis only when the density of VSMCs is low. Under low VSMC density (10 000 cells/cm²), addition of PDTC (0.1 to 10 µmol/L) caused apoptosis of VSMCs, which was confirmed by Hoechst 33258 staining under fluorescence microscopy. At low VSMC density, expression of NGFI-B mRNA was induced 1 hour after the addition of PDTC, peaking at 6 hours, and persisted for up to 12 hours. The protein level of NGFI-B was increased 4 hours after PDTC addition and persisted for up to 12 hours. Under low VSMC density, PDTC-induced expression of NGFI-B mRNA was correlated with the magnitude of apoptosis, which was quantified by enzyme immunoassay for histone-associated DNA fragments. In contrast, when the density of VSMCs was high (50 000 cells/cm²), PDTC did not induce apoptosis, and the expression of NGFI-B was only transient. This transient expression pattern was also seen when VSMCs were treated with phorbol ester, calcium ionophore, hydrogen peroxide, or angiotensin II, even at low cell density. We next investigated whether the NGFI-B gene may act as a transcription factor under treatment with PDTC by measuring the promoter activity of luciferase reporter plasmids that contained typical NGFI-B–responsive elements. The PDTC-induced transcriptional activity of NGFI-B was 2-fold higher at low cell density than at high cell density. These data demonstrate that NGFI-B can be induced in VSMCs and suggest that NGFI-B may play a role in PDTC-induced VSMC apoptosis.

Key Words: vascular smooth muscle cells • apoptosis • nuclear receptor • cell density

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