Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via Gi/o proteins

Abstract

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.

15d-Prostaglandin J2 Enhancement of Nerve Growth Factor–Induced Neurite Outgrowth Is Blocked by the Chemoattractant Receptor– Homologous Molecule Expressed on T-Helper Type 2 Cells (CRTH2) Antagonist CAY10471 in PC12 Cells

“…In accordance with previous reports (8,9), NGF increased the phosphorylation of all 3 kinases (Fig. 3A).…”

“…Since activation of ERK, p38 MAP kinase, and Akt are all known to be implicated in neuritogenesis in PC12 cells (8,9), we examined the involvement of CRTH2 on the activation of these kinases. In accordance with previous reports (8,9), NGF increased the phosphorylation of all 3 kinases (Fig.…”

“…15d-PGJ 2 interacts with CRTH2 with a much higher affinity than it does with DP (5). Therefore, we examined the possible involvement of CRTH2 in the promoting effects of 15d-PGJ 2 on NGFinduced neurite outgrowth in PC12 cells as well as on the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein (MAP) kinase, and Akt, all of which are known to be implicated in neuritogenesis in PC12 cells (8,9). NGF (Alomone Labs, Jerusalem, Israel); 15d-PGJ 2 , 13,14-dihydro-15-keto-PGD 2 (DK-PGD 2 ), CAY10471, BWA868C, rosiglitazone (Cayman Chemical Co., Ann Arbor, MI, USA); dibutyryl-cyclic AMP (dbcAMP; Sigma Chemical Co., St. Louis, MO, USA); and SB 203580 (Calbiochem, San Diego, CA, USA) were diluted with test medium (indicated below) before use.…”

G protein signaling controls the differentiation of multiple cell lineages

“…The induction of PI3K/Akt pathway by NGF is in part mediated through the Gβγ subunit released from activated G i/o , which promotes the phosphorylation of translation regulator tuberlin to enhance neuronal survival [14,15]. In a similar manner, G i/o mediates part of the NGF-induced p38 and JNK phosphorylations, which are required for neuronal differentiation [17,18]. These examples suggest bidirectional crosstalk between GPCR and NGF signaling in the regulation of neuronal survival and differentiation.…”

“…These include the findings that demonstrate the localization of GPCRs and their signaling components in lipid rafts and caveolae [63], dimerization of GPCRs [64], and trans-activation of receptor tyrosine kinases by GPCRs [65]. In the case of cell differentiation, examples of novel GPCR signaling mechanism include transactivation of TrkA by purinergic P2Y2 receptor [12], and mediation of partial NGF signaling by G i/o proteins [14,17,18]. With new discoveries in the signaling mechanisms of GPCRs and novel ligands for orphan receptors, we will undoubtedly appreciate the significance of G protein-linked signaling in the differentiation of various cell lineages and development of tissues and organs.…”