Inhibition of PLC-γ1 activity converts nerve growth factor from an anti-mitogenic to a mitogenic signal in CHO cells

Abstract

Nerve growth factor (NGF) treatment of Chinese hamster ovary ®broblast (CHO) cells exogenously expressing 2.5610 5 TrkA receptors (CHO/TrkA) results in inhibition of serum and insulin-like growth factor-I (IGF-I) stimulated cell proliferation in a dosedependent manner. Furthermore, NGF does not stimulate [ 3 H]thymidine incorporation and inhibits IGF-I mediated DNA synthesis in CHO/TrkA cells. NGF and IGF-I induce extracellular-signal regulated kinase 1 (ERK1) and ERK2 activation, but NGF is able to stimulate a higher and more sustained activation of these enzymes compared with IGF-I. Cotreatment with NGF and IGF-I yields an ERK1/2 activity pro®le similar to that of NGF treatment alone. While pretreatment with mitogen activated protein kinase kinase (MKK) inhibitor PD98059 (30 mM) results in 100% inhibition of IGF-I stimulated MAPK phosphorylation (IC 50 51 mM), NGF mediated MAPK phosphorylation is only decreased by 50% (IC 50 =3 mM). NGF, but not IGF-I, stimulates tyrosine phosphorylation and activation of PLC-g1 which can be inhibited in a dose-dependent manner by phosphoinositide-speci®c phospholipase C (PI-PLC) inhibitor U73122 (IC 50 =4 mM). Pretreatment with U73122 (IC 50 =7 mM) results in an 87% inhibition of NGF mediated MAPK phosphorylation, while cotreatment with PD98059 and U73122 results in 97% inhibition. U73122 pretreatment has no eect on NGF stimulated Akt activation. NGF, but not IGF-I, stimulates the tyrosine phosphorylation of Suc1-associated neurotrophic factor-induced tyrosine phosphorylation target (SNT-1)/®broblast growth factor receptor substrate 2 (FRS2) which can be completely prevented by pretreatment with 10 mM U73122. Finally, inhibition of PI-PLC results in NGF's ability to stimulate DNA synthesis in the absence and presence of IGF-I.

Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKMζ

“…Zhu and Oxford (2007) To examine the role of PLC in the NGF-induced increase in AP firing, sensory neurons were exposed to U-73122, a selective inhibitor of PLC (IC 50 1-2 M; Bleasdale et al 1990;). U-73122 also blocks activation of PLC␥-1, including that produced by NGF (Jones et al 2005;Sato et al 2000;Zapf-Colby et al 1999). As shown in Fig.…”

Dimethyphenylpiperazinium, a nicotinic receptor agonist, downregulates inflammation in monocytes/macrophages through PI3K and PLC chronic activation

“…We are aware that PLC phosphorylation is not a direct proof of protein activation. However, because it is widely established that PLC phosphorylation correlates its activity (18), and that the antibody used in our study was previously used to describe PLC activity (30,31), we believe that, because DMPP provokes PLC phosphorylation, it should promote its activation.…”

Phospholipase Activity of Phospholipase C-γ1 Is Required for Nerve Growth Factor-regulated MAP Kinase Signaling Cascade in PC12 Cells

“…Moreover, pretreatment with U73122 results in 87% inhibition of NGF-mediated MAPK phosphorylation in TrkA-transfected Chinese hamster ovary cells, whereas it has no effect on NGF-stimulated Akt activation (34). NGF activates a DAG-regulated protein kinase, protein kinase C-␦, which is required for activation of the MAPK cascade and for neurite outgrowth.…”