α2-Adrenergic receptors activate MAPK and Akt through a pathway involving arachidonic acid metabolism by cytochrome P450-dependent epoxygenase, matrix metalloproteinase activation and subtype-specific transactivation of EGFR

doi:10.1016/j.cellsig.2005.06.014

Cellular Signalling

Volume 18, Issue 5, May 2006, Pages 729-739

https://doi.org/10.1016/j.cellsig.2005.06.014 Get rights and content

Abstract

Previous study carried out on PC12 cells expressing each α₂-adrenergic receptor subtype individually (PC12/α_2A, /α_2B or /α_2C) have shown that epinephrine causes activation of PI3K and phosphorylation of Erk 1/2. The signal transduction mechanisms whereby each α₂-AR subtype triggers these actions were investigated in the present study. In all three clones, epinephrine-induced phosphorylation of MAPK or Akt was abolished by prior treatment with ketoconazole, but not with indomethacin or nordihydroguaiaretic acid. On the other hand, treatment of the clones with epinephrine caused a rapid increase of AA release, which was fully abolished by the PLC inhibitor U73122, but was unaffected by the PLA₂ inhibitor quinacrine. The effects of epinephrine on MAPK and Akt were mimicked by cell exposure to exogenous AA. Furthermore, whereas U73122 abolished the effects of epinephrine, quinacrine only prevented the effects of epinephrine, suggesting that AA release through PLC and its metabolites are responsible for MAPK and Akt activation by α₂-ARs. Treatment with 1,10-phenanthroline, CRM197, or tyrphostin AG1478 suppressed MAPK and Akt phosphorylation by epinephrine or AA, in a subtype-specific manner. Furthermore, conditioned culture medium from epinephrine-treated PC12/α₂ induced MAPK and Akt phosphorylation in wild-type PC12. Inhibition of NGFR tyrosine phosphorylation had no effect but the src inhibitor PP1 abolished MAPK and Akt phosphorylation in all three clones. Our results provide evidence for a putative pathway by which α₂-ARs activate MAPK and Akt in PC12 cells, involving stimulation of PLC, AA release, AA metabolism by cytochrome P450-dependent epoxygenase, stimulation of matrix metalloproteinases and subtype-specific transactivation of EGFR through src activation and heparin-binding EGF-like growth factor release.

Introduction

The α₂-adrenergic receptors (α₂-ARs) are G-protein-coupled receptors (GPCRs) which mediate the effects of the endogenous catecholamines, epinephrine and norepinephrine, by modulating the activity of a large panel of effectors including adenyl cyclase, GIRK, phospholipase Cβ, MAPKs and PI3K. Three α₂-AR subtypes (α₂A, α₂B and α₂C) differing in pharmacological properties, tissue distribution, regulatory mechanisms and signaling pathways, have been characterized in various species [1], [2], [3], [4]. Although the α₂A-AR is largely predominant, all three subtypes are expressed in the central nervous system. According to studies carried out on transgenic mice, central α₂A-ARs are responsible for the sedative, hypnotic, sympatholytic and analgesic actions of α₂-agonists [5], [6], [7]. The role of α₂B-AR is still unclear, but central α₂B-AR would play a role in salt-induced experimental hypertension [8]. On the other hand, the α₂C-AR is primarily found in the olfactory tubercles, cerebral cortex, basal ganglia and hippocampus and is believed to exert modulatory effects on several brain functions possibly by regulating dopamine and serotonin metabolism [9], [10]. Moreover, α₂C-AR subtype was recently shown to participate with α₂A-AR to presynaptic inhibition of neurotransmitter release [11], [12].

PC12 cells do not express endogenously adrenergic receptors, but using clones of these cells stably transfected with the human α₂-AR genes we have previously shown that stimulation of all three subtypes causes activation of PI3K and phosphorylation of Erk 1/2 (p42/p44 MAPK) in neuronal cells [13]. MAPK activation was found to be completely inhibited by pretreatment with the α₂-antagonist RX821002, however the signaling mechanisms whereby α₂-ARs promote these changes are still poorly characterized in this particular cell type. Like other ARs, it is probable that they are highly dependent upon the receptor subtype considered. Previous studies on Gi/Go coupled receptors including α₂-AR have shown the activation of MAPK to be pertussis toxin sensitive, to depend upon Gβγ-subunit release and to occur in a process that does not require receptor internalization [14], [15], [16]. More detailed examination of the molecular mechanisms of MAPK activation has shown that GPCRs transactivate growth factor receptors, including the EGF, IGF1 and PDGF receptors [17], [18], [19] and that these tyrosine kinase receptors may serve as a scaffolding structure or as an adaptor to which other signaling proteins are recruited [14], [20]. These activated receptor tyrosine kinase phosphorylate several cellular signaling proteins and form receptor complexes composed of Shc, Grb2 and Sos, which in turn trigger activation of mitogen-activated protein kinase (MAPK) cascade. Furthermore, PI3K which leads to the activation of the downstream signaling kinase Akt and which is critically important for cell survival, has been shown to participate to MAPK activation by Gi/Go coupled receptors [21], [22]. Other experiments carried out on LLC-PK1 transfected with the gene encoding the rat α₂B-AR have shown that in these renal proximal tubule cells the α₂B-subtype promotes MAPK activation via a mechanism involving arachidonic acid (AA) release and metabolism [23]. Release of arachidonic acid secondary to activation of specific phospholipases is a well recognized cellular response to a variety of growth factors, hormones and cytokines. Furthermore, metabolites of AA play important roles as intracellular second messengers, with well documented autocrine effects of cγ-cyclooxygenase and lipoxygenase metabolites. There is however now abundant evidence that eicosanoids derived from the cytochrome P450-dependent epoxygenase (third pathway) may also serve as second messengers and have a multitude of potent biological activities. Based on the use of several inhibitors, the present study shows that activation of Erk1/2 and PI3-K in PC12 expressing α₂-ARs occurs via a pathway involving activation of PLC, generation of AA metabolites by cytochrome-P450 epoxygenase, activation of matrix metalloproteinases, release of HB-EGF and transactivation of EGFR.

Section snippets

Reagents

Epinephrine bitartrate, arachidonic acid, indomethacin, ketoconazole, nordihydroguaiaretic acid (NDGA), 1,10-phenanthroline, tyrphostin AG1478 and [Glu⁵²]diphtheria toxin (CRM197) were purchased from Sigma (St. Louis, MO). [³H]AA (180 Ci/mmol) was from Hartmann Analytic GmbH. NGF and EGF were from Promega (Madison, WI). Dulbecco's modified Eagles medium (DMEM), fetal bovine serum (FBS) and horse serum were from Biochrom KG. Vitrogen 100 was obtained from Collagen Co (Fremont, CA). U73122, PP1

PLC-mediated AA release is involved in MAPK and Akt phosphorylation by α₂-AR

α₂-ARs have been found to increase AA release in several cell systems via mechanisms involving either cPLA₂ or the combined action of PLC and DAG lipase. Both cPLA₂ and PLC have been found activated upon stimulation of α₂-AR in several cell systems [25], [26]. As shown in Fig. 1, treatment of each PC12-α₂ clone with epinephrine caused a rapid acceleration of AA release, which was prevented by the prior addition of the selective α₂-antagonist, RX821002. The mechanism of AA release was further

Discussion

The α₂-ARs exert their regulatory effect on target cells via the modulation of the activity of several effectors, including notably adenylate cyclase, GIRK, PLCβ and MAPK. Activation of Erk1/2 by α₂-AR subtypes has been studied in a number of transfected cell types, like CHO transfected with the rat α₂A-, α₂B- and α₂C-AR subtypes [44], LLC-PK1 transfected with the RNG gene [45] and COS or HEK 293 cells transfected with the α₂C2 gene encoding the human α₂B-AR subtype [46], [47]. In addition MAPK

Acknowledgments

The authors thank Prof. Mika Scheinin, Turku University for his support.

This work was supported in part by ELPEN A.E., AstraZeneca A.E. and Novartis (HELLAS) A.E.B.E. and by a grant (PENED 2001-372) of the General Secretariat for Research and Technology of the Greek Ministry of Development.

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¹: Present address: Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, PA, USA, 19107.

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α2-Adrenergic receptors activate MAPK and Akt through a pathway involving arachidonic acid metabolism by cytochrome P450-dependent epoxygenase, matrix metalloproteinase activation and subtype-specific transactivation of EGFR

Abstract

Introduction

Section snippets

Reagents

PLC-mediated AA release is involved in MAPK and Akt phosphorylation by α2-AR

Discussion

Acknowledgments

Trends Pharmacol. Sci.

J. Biol. Chem.

Neuroscience

Neuroscience

Eur. J. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Hypertension

α₂-Adrenergic receptors activate MAPK and Akt through a pathway involving arachidonic acid metabolism by cytochrome P450-dependent epoxygenase, matrix metalloproteinase activation and subtype-specific transactivation of EGFR

PLC-mediated AA release is involved in MAPK and Akt phosphorylation by α₂-AR