Nerve growth factor-induced up-regulation of cytosolic phospholipase A2α level in rat PC12 cells

doi:10.1016/j.neulet.2004.05.001

Neuroscience Letters

Volume 365, Issue 3, 29 July 2004, Pages 218-222

https://doi.org/10.1016/j.neulet.2004.05.001 Get rights and content

Abstract

Nerve growth factor (NGF) regulates various types of gene transcription in neurons. One of the cytosolic phospholipase A₂s, cPLA₂α, which preferentially cleaves phospholipids at the sn-2 position to arachidonic acid (AA), is involved in neuronal responses including survival. We investigated the effect of NGF on cPLA₂α expression and its signaling pathways in PC12 cells, which differentiate into neuronal-like cells with neurites by NGF treatment. Treatment with NGF increased cPLA₂α mRNA level after 4 h and its protein level 24 h after NGF addition. The NGF-induced increase in cPLA₂α mRNA was inhibited by actinomycin D. NGF caused phosphorylation of mitogen-activated protein kinases (MAPKs); sustained phosphorylation of extracellular-regulated kinases (ERK1/2) and transient phosphorylation of p38 MAPK. NGF responses (cPLA₂α mRNA and its protein) were inhibited by selective inhibitors for the ERK1/2 pathway, p38 MAPK and c-Jun NH₂-terminal kinase. Epidermal growth factor, which transiently activates ERK1/2, did not modify cPLA₂α expression. Although phorbol 12-myristate 13-acetate, an activator of protein kinase C (PKC), alone showed no effect, NGF-induced cPLA₂α mRNA expression decreased due to the inhibition of PKC. These findings suggest that NGF-induced cPLA₂α expression is regulated by gene transcription via the ERK1/2, p38 MAPK and PKC pathways in PC12 cells.

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Citation Excerpt :
Because NGF is required for neurite growth of sensory neurons (22) and NGF-treated neuronal cells showed elevated TRPV4 expression during neurite growth (Fig. 2), we hypothesized that NGF utilizes TRPV4 to control neurite growth. Furthermore, because NGF would stimulate cytosolic phospholipase A2 (PLA2, an enzyme that releases AA from phosphoglycerides) (37), we considered that AA and its metabolite, 5,6-EET, were found to stimulate neuritogenesis. Therefore, we examined whether NGF regulates the expression levels of PLA2 and TRPV4.
Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca²⁺ overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A₂ and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A₂-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.
Release of arachidonic acid by 2-arachidonoyl glycerol and HU210 in PC12 cells; roles of Src, phospholipase C and cytosolic phospholipase A<inf>2</inf>α
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The phospholipase A₂ (PLA₂)-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (α type cytosolic PLA₂ (cPLA₂α), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA₂α, the activities of Src family kinases, Ca²⁺-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 μM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB₁ receptor) and by pyrrophenone (a selective inhibitor of cPLA₂α) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA₂α and an increase in the intracellular Ca²⁺ level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca²⁺ response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA₂α in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB₁ receptor and/or CB₁-like receptor in neuronal cells.
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Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B₂ BK receptor antagonist Hoe140, suggesting the involvement of B₂ BK receptors. BK-induced COX-2 expression and translocation of PKC-δ from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-δ might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-δ significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E₂ (PGE₂) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-δ might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-κB into nucleus which was blocked by helenalin, rottlerin and PD98059, implying the involvement of NF-κB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE₂ release was sequentially mediated through PKC-δ-dependent activation of p42/p44 MAPK and NF-κB. Understanding the regulation of COX-2 expression and PGE₂ release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.
The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells
2006, Neuroscience Letters
The nervous system is highly sensitive to various environmental stresses, such as ischemia. Stress response mechanisms that result in neuroprotection, including the induction of heat shock proteins (HSP), are not well understood. We examined the effect of KNK437, a compound that inhibits the synthesis of inducible heat shock proteins, on neuronal differentiation in rat pheochromocytoma PC12 cells. KNK437 decreased the expression of HSP70, and induced the neurite outgrowth of PC12 cells in the absence of stress stimulation, although with lower efficacy than nerve growth factor (NGF). Neurite outgrowth stimulated by KNK437 and NGF was blocked by inhibitors of ERK mitogen-activated protein (MAP) kinase, p38 MAP kinase, and glycogen synthase kinase 3β signaling pathways. NGF, and not KNK437, induced acetylcholine esterase (AChE) activity, a functional differentiation marker, indicating that KNK437 utilizes a mechanism distinct from that of NGF. KNK437 enhanced the activity of low dose NGF treatment on neurite outgrowth induction and ERK phosphorylation in PC12 cells, a finding that identifies KNK437 as a possible nerve regeneration agent. This compound may be a useful tool for the investigation of neuronal differentiation and neuroprotection against environmental stress.
Up-regulation of cytosolic phospholipase A<inf>2</inf>α expression by N,N-diethyldithiocarbamate in PC12 cells; involvement of reactive oxygen species and nitric oxide
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Disulfiram (an alcohol-aversive drug) and related compounds are known to provoke several side effects involving behavioral and neurological complications. N,N-diethyldithiocarbamate (DDC) is considered as one of the main toxic species of disulfiram and acts as an inhibitor of superoxide dismutase. Since arachidonic acid (AA) formation is regulated by reactive oxygen species (ROS) and related to toxicity in neuronal cells, we investigated the effects of DDC on AA release and expression of the α type of cytosolic phospholipase A₂ (cPLA₂α) in PC12 cells. Treatment with 80–120 μM DDC that causes a moderate increase in ROS levels without cell toxicity stimulated cPLA₂α mRNA and its protein expression. The expression was mediated by extracellular-signal-regulated kinase (ERK1/2), one of the mitogen-activated protein kinases. Treatment with N^G nitro-l-arginine methyl ester (an inhibitor of nitric oxide synthase, 1 mM) and oxy-hemoglobin (a scavenger of nitric oxide, 2 mg/mL) abolished the DDC-induced responses (ERK1/2 phosphorylation and cPLA₂α expression). We also showed DDC-induced up-regulation of the mRNA expression of lipocortin 1, an inhibitor of PLA₂. Furthermore, DDC treatment of the cells enhanced Ca²⁺-ionophore-induced AA release in 30 min, although the effect was limited. Changes in AA metabolism in DDC-treated cells may have a potential role in mediating neurotoxic actions of disulfiram. In this study, we show the first to demonstrate the up-regulation of cPLA₂α expression by DDC treatment in neuronal cells.
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Excess production of reactive oxygen species (ROS), including H₂O₂, leads to neuronal death in pathological conditions. Although ROS stimulates α-type cytosolic phospholipase A₂ (cPLA₂α) activity, their role in cPLA₂α expression has not been elucidated. We investigated the effect of ROS on cPLA₂α mRNA levels and signaling pathways in rat pheochromocytoma PC12 cells. Treatment with H₂O₂ and xanthine–xanthine oxidase (X/XO) for 4 h decreased cPLA₂α mRNA levels without changing the mRNA levels of other tested proteins. H₂O₂ and X/XO caused cell toxicity not after 4 h but 24 h after their addition. The H₂O₂-induced decrease in cPLA₂α mRNA levels was inhibited in cells treated with N-acetyl-cysteine and selective inhibitors of mitogen-activated protein kinase (MAPK) pathways (extracellular signal-regulated kinase and p38 MAPK). Treatment with dopaminergic neurotoxins, including 1,2,3,4-tetrahydroisoquinoline (TIQ)-inducing ROS formation, decreased cPLA₂α mRNA levels. These findings suggest that ROS decreases cPLA₂α mRNA levels via MAPK pathways in PC12 cells.

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¹: These authors equally contributed to this work.

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Nerve growth factor-induced up-regulation of cytosolic phospholipase A2α level in rat PC12 cells

Abstract

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Brain Res.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2

Nature

Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fla-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells

J. Neurosci.

Transcriptional regulation and structural organization of the human cytosolic phospholipase A2 gene

Am. J. Physiol.

Nerve growth factor-induced up-regulation of cytosolic phospholipase A₂α level in rat PC12 cells

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A₂

Transcriptional regulation and structural organization of the human cytosolic phospholipase A₂ gene