Characterization of the Endocannabinoid System in Human Neuronal Cells and Proteomic Analysis of Anandamide-induced Apoptosis

Abstract

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the "endocannabinoid system." Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an ϳ3 to ϳ5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as antiapoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.Endocannabinoids bind to and activate both type 1 (CB1R) 4 and type 2 (CB2R) cannabinoid receptors and are widely recognized as important regulators of central and peripheral functions (1-3). The most studied endocannabinoids are anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) (1, 3). AEA, unlike 2-AG, binds to and activates also transient receptor potential vanilloid 1 (TRPV1), thus being considered a true "endovanilloid" (4).The "endocannabinoid system (ECS)" includes the receptors, their ligands AEA and 2-AG, and the enzymes that synthesize (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) and diacylglycerol lipase (DAGL)) or degrade (fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)) AEA and 2-AG, respectively (1-3). On the other hand, there is still controversy about the identity and even the existence of purported "endocannabinoid transporters" that have not yet been cloned, isolated, or characterized (5). A growing interest is concerned with the ability of AEA to induce apoptosis in neuronal and non-neuronal cells (for comprehensive reviews see Refs. 6 -9). Such a pro-apoptotic activity of AEA

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor

“…These data indicate that both CB subtypes were functional to a similar extent in human NHEM cells (Table 1). However, some discrepancies were observed between the activity and mRNA expression of AEA-and 2-AG-metabolizing enzymes, a finding that is not unprecedented for ECS elements (35,44). Overall, the present data demonstrate that primary human melanocytes have a complete and fully functional ECS.…”

“…2B), a typical marker of programmed cell death (Ref. 35 and references therein). Thus, p53 levels were checked to investigate the mechanism of cell death induced by mAEA, showing that either AEA or capsaicin, a selective agonist of TRPV1 receptors (4), at 5 M concentration significantly enhanced p53 mRNA, whereas ACEA, a selective CB 1 agonist (3), JWH133, a selective CB 2 agonist (3), or 2-AG, which activates both CB 1 and CB 2 receptors but not TRPV1 (4), had no effect (Fig.…”

“…NHEM cells were transfected with 600 pmol of siRNA using Lipofectamine 2000 reagent as reported (35). Briefly, 600 pmol of siRNA and 30 l of Lipofectamine 2000 were diluted with Opti-MEM to a final volume of 3 ml, mixed, and added to NHEM cells grown to 60% confluence in 100-mm-diameter plates.…”

“…The target transcripts were amplified using an ABI PRISM 7700 sequence detector system (Applied Biosystems, Foster City, CA). Human primers for CB 1 , CB 2 , TRPV1, NAPE-PLD, FAAH, DAGL, MAGL, p53, and ␤-actin were as reported (35). In addition, the following primers were used: human TYR F, 5Ј-ATGACCTCTTTGTCTGGATGC-3Ј; human TYR R, 5Ј-AA-GAGGAGAAGAATGATGC-3Ј; human MITF F, 5Ј-ATGA-CATCACGCATCTT GC-3Ј; human MITF R, 5Ј-TACTGCTT-TACC TGCTGC-3Ј.…”

“…Specific determination of mono-and oligonucleosomes in the cytoplasmic fraction of cell lysates was performed by measuring the absorbance values at 405 nm as reported (34). Apoptosis was also quantified by measuring mRNA expression of p53, a typical marker of programmed cell death (35), by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) (see below).…”