Induction of Neurite Outgrowth in PC12 Cells by α-Phenyl-N-tert-butylnitron through Activation of Protein Kinase C and the Ras-Extracellular Signal-regulated Kinase Pathway

Abstract

The spin trap ␣-phenyl-N-tert-butylnitron (PBN) is widely used for studies of the biological effects of free radicals. We previously reported the protective effects of PBN against ischemia-reperfusion injury in gerbil hippocampus by its activation of extracellular signalregulated kinase (ERK) and suppression of both stressactivated protein kinase and p38 mitogen-activated protein kinase. In the present study, we found that PBN induced neurite outgrowth accompanied by ERK activation in PC12 cells in a dose-dependent manner. The induction of neurite outgrowth was inhibited significantly not only by transient transfection of PC12 cells with dominant negative Ras, but also by treatment with mitogen-activated protein kinase/ERK kinase inhibitor PD98059. The activation of receptor tyrosine kinase TrkA was not involved in PBN-induced neurite outgrowth. A protein kinase C (PKC) inhibitor, GF109203X, was found to inhibit neurite outgrowth. The activation of PKC⑀ was observed after PBN stimulation. PBN-induced neurite outgrowth and ERK activation were counteracted by the thiol-based antioxidant N-acetylcysteine. From these results, it was concluded that PBN induced neurite outgrowth in PC12 cells through activation of the Ras-ERK pathway and PKC.

Phenyl N-tert-Butylnitrone Down-Regulates Interleukin-1β-Stimulated Matrix Metalloproteinase-13 Gene Expression in Human Chondrocytes: Suppression of c-Jun NH2-Terminal Kinase, p38-Mitogen-Activated Protein Kinase and Activating Protein-1

“…8) indicates that PBN is not an inhibitor of the activity of ERKp44/p42. In other studies, PBN has been shown to stimulate the phosphorylation of ERKp44/p42 in PC 12 cells (Tsuji et al, 2001), and IL-1 is known to stimulate the phosphorylation of ERKp44/p42 in OA chondrocytes (Geng et al, 1996), we did not find a synergistic stimulatory effect of IL-1 and PBN on ERKp44/p42 in OA chondrocytes (Fig. 5A).…”

“…PBN has also been shown to decrease basal protein phosphorylation with concomitantly increased phosphatase activity and also inhibited the IL-1␤-induced activation of p38 MAPK and H 2 O 2 biosynthesis in rat glial cells (Robinson et al, 1999). Other studies have shown that PBN inhibits stress-sensitive signaling pathways in gerbil hippocampus and the induction of neurite outgrowth in PC12 cells via activation of Ras-ERK1/2 pathway and activation of protein kinase C (Tsuji et al, 2000(Tsuji et al, , 2001. However, studies on the modulation of IL-1␤-induced stimuli in OA chondrocytes by PBN have not yet been reported.…”

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells

“…In addition, the transitional metal manganese has been shown to increase neuritogenesis (53). Furthermore, the free radical-trapping agent ␣-phenyl-N-tert-butylnitron has been reported to induce neurite outgrowth by a redox mechanism (54), and NGF-induced neuritogenesis has been reported to require the generation of ROS (13)(14)(15). Therefore, it is possible that ROS are common signals for neurite outgrowth in PC12 cells.…”

Nerve Growth Factor Regulates Adrenergic Expression

“…To isolate nuclear protein and total RNA, cells were grown in 100-mm culture dishes to a density of 5 ϫ 10 5 to 1 ϫ 10 7 cells/dish before drug treatment. NGF (0-100 ng/ml), 10 nM PACAP, and the signaling pathway drugs H89 (30 M), a selective protein kinase A (PKA) inhibitor (Chijiwa et al, 1990;; forskolin (10 M), an adenylate cyclase activator; GF109203X (100 nM), a protein kinase C (PKC) inhibitor (Tsuji et al, 2001;; phorbol 12-myristate 13-acetate (PMA; 80 nM), a PKC activator at low concentrations (Morita et al, 1995); U0126 (10 M), an ERK1/2 MAPK inhibitor (Harada et al, 2001;Hamelink et al, 2002;Hou et al, 2003); SB203580 (50 M), a p38 MEK inhibitor (Cheng et al, 2000); wortmannin and LY294002 (10-50 M), phosphatidylinositol 3-kinase inhibitors (Tsuji et al, 2001;Chang et al, 2003;Ha et al, 2003); and U-73122 (50 M), a phospholipase C inhibitor (Hamelink et al, 2002) were obtained from Sigma-Aldrich. Initial treatment concentrations were based on literature values, and doseresponse curves and time courses were executed to optimize treatment conditions (data not shown).…”