Induction of nuclear orphan receptor NGFI-B gene and apoptosis in rat vascular smooth muscle cells treated with pyrrolidinedithiocarbamate

NGFI-B is one of the orphan nuclear receptors, and its gene is implicated i n the apoptosis of T cells. The aim of this study was to investigate the ex pression and the role of NGFI-B in vascular smooth muscle cells (VSMCs). Py rrolidinedithiocarbamate (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 (10000 cells/cm(2)), addition of PDTC (0.1 to 10 mu mol/L) caused apoptosis of VSMCs, which was confirmed by Hoechst 33258 staining u nder fluorescence microscopy. At low VSMC density, expression of NGFI-B mRN A was induced 1 hour after the addition of PDTC, pealing at 6 hours, and pe rsisted for up to 12 hours. The protein level of NGFI-B was increased 4 hou rs after PDTC addition and persisted for up to 12 hours. Under low VSMC den sity, PDTC-induced expression of NGFI-B mRNA was correlated with the magnit ude of apoptosis, which was quantified by enzyme immunoassay for histone-as sociated DNA fragments. In contrast, when the density of VSMCs was high (50 000 cells/cm(2)), 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 peroxid e, or angiotensin II, even at low cell density. We next investigated whethe r the NGFI-B gene may act as a transcription factor under treatment with PD TC by measuring the promoter activity of luciferase reporter plasmids that contained typical NGFI-B-responsive elements. The PDTC-induced transcriptio nal activity of NGFI-B was 2-fold higher at low cell density than at high c ell 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.