Effects of Gαi2 and Gαz protein knockdown on alpha2A-adrenergic and cannabinoid CB1 receptor regulation of MEK-ERK and FADD pathways in mouse cerebral cortex

Abstract

Background Alpha 2A -adrenergic (α 2A -AR) and cannabinoid CB 1 (CB 1 -R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α 2A -AR-and CB 1 -R in vivo are unclear. This study investigated the roles of Gαi 2 and Gαz proteins in mediating α 2A -AR-and CB 1 -R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex. Methods Gαi 2 or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3 nmol/day, 5 days) prior to UK-14,304 (UK or brimonidine; 1 mg/kg) or WIN55212-2 (WIN; 8 mg/kg) acute treatments. Inactivated (p-T 286 ) MEK1, activated (p-S 217/221 ) MEK1/2, activated (p-T 202 /Y 204 ) ERK1/2, p-S 191 FADD, and the corresponding total forms of these proteins were quantified by immunoblotting. Results Increased (+ 88%) p-T 286 MEK1 cortical density, with a concomitant reduction (-43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi 2 or Gαz antisense ODNs. Contrastingly, WIN induced Gαi 2 -and Gαz-independent upregulations of p-T 286 MEK1 (+ 63%), p-S 217/221 MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (− 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S 191 FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi 2 or Gαz protein integrity. ConclusionThe results indicate that α 2A -AR (UK), but not CB 1 -R (WIN), agonists use Gαi 2 and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex.

Cannabinoid CB2 receptor ligands disrupt the sequential regulation of p-MEK1/2 to p-ERK1/2 in mouse brain cortex

“…The endocannabinoid system consists of two major types of G-protein (Gi/o)-coupled receptors, the cannabinoid 2 (CB 2 ) and CB 1 receptors that are stimulated by the endogenous ligands anandamide and/or 2-arachidonoylglycerol [6]. In the context of MEK-ERK signaling, the CB 1 receptor agonist WIN55212-2 was shown to induce, as expected, the parallel upregulation of p-MEK1/2 and p-ERK1/2 in rat and mouse cerebral cortex [7,8]. Moreover, the non-selective CB 1/2 receptor agonist CP55940 was also reported to stimulate p-ERK1/2 in transfected Chinese hamster ovary (CHO) cells expressing the human CB 2 receptor (CHO-CB 2 ), through pertussis toxin (Gi/o protein-dependent) mechanisms [9].…”

“…The sequential in vivo activation (p) of cortical MEK1/2 (p-MEK/total-MEK ratio) to p-ERK (p-ERK/total-ERK ratio) kinases was disrupted in mouse brain cortex after the acute regulation with various, and markedly different, cannabinoid CB 2 receptor ligands (i.e., direct agonist JWH133: modest decrease of MEK with ERK upregulation; endogenous agonist acting on overexpressed receptors: MEK downregulation with ERK upregulation; and direct inverse agonist/antagonist AM630: MEK upregulation with ERK unaltered) (see Figure 2). In the endocannabinoid system, these paradoxical findings for the CB 2 receptor contrast with the well-known canonical activation from MEK to ERK signaling after the stimulation of CB 1 receptors with the agonist WIN55212-2 (both kinases were concomitantly upregulated in rodent brain cortex [7,8]). In the paradoxical context of MEK-ERK physiological regulation, it is important to remark that the p-MEK1/2 kinases (i.e., MAP-ERK1 and 2 kinases) are equally competent activators of their unique substrates p-ERK1 and 2, and consequently, the cellular functions of p-ERK1/2 are under the tight control of their upstream activators p-MEK1/2 [1,5].…”

“…In fact, the acute treatment with the selective MEK1/2 inhibitor SL-327 blocked the concomitant p-ERK activity in the mouse brain [7] (Figure 2). In the endocannabinoid system, and as mentioned above, the canonical activation of p-MEK to p-ERK signaling has been well demonstrated after stimulation of CB 1 receptors with selective full agonists (e.g., WIN55212-2) in rat and mouse brain cortex [7,8]. Therefore, it was shocking to observe that treatment of mice with a selective CB 2 receptor agonist (JWH133) resulted in disruption of p-MEK (decreased) to p-ERK (increased) signaling in the brain cortex (Figure 1A and Figure 2).…”

“…It is known that the stimulation of cannabinoids CB 2 receptors can engage multiple waves of different signaling pathways [12,13], but at present the molecular signals underpinning the reported in vivo MEK-ERK dysregulation in the brain remain unknown. Moreover, some in vitro studies have reported that p-ERK1/2 signaling in rat-1 fibroblasts can be activated (with very high concentrations of peroxynitrites) via some MEK-independent pathway [18], but such a possibility contradicts the accepted postulates (mainly for in vivo experiments; e.g., see [7,8] for specific details) of the canonical activation of MEK-ERK signaling (i.e., p-ERK1/2 are the sole cytoplasmic substrates of p-MEK1/2 kinases [3,4], which have been validated by quantitative phosphoproteomic analysis [19]).…”

“…In the paradoxical context of MEK-ERK physiological regulation, it is important to remark that the p-MEK1/2 kinases (i.e., MAP-ERK1 and 2 kinases) are equally competent activators of their unique substrates p-ERK1 and 2, and consequently, the cellular functions of p-ERK1/2 are under the tight control of their upstream activators p-MEK1/2 [1,5]. In fact, the acute treatment with the selective MEK1/2 inhibitor SL-327 blocked the concomitant p-ERK activity in the mouse brain [7] (Figure 2). In the endocannabinoid system, and as mentioned above, the canonical activation of p-MEK to p-ERK signaling has been well demonstrated after stimulation of CB 1 receptors with selective full agonists (e.g., WIN55212-2) in rat and mouse brain cortex [7,8].…”