The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB₁- and FAAH-independent mechanisms

Abstract

Background: Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between 'on demand' generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis.

Experimental approach: We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids.

Key results: Anandamide decreased TH promoter activity in N1E115 cells through CB₁ receptor activation. Unexpectedly, URB597 reduced TH expression (pEC₅₀ = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB₁ receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain.

Conclusions: While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB₁ and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when interpreting the data.

Figure 1

Endocannabinoids and URB597-mediated regulation of TH promoter activity. Luciferase activity was measured in N1E115 cells transiently transfected with pTH250-Luc and treated for 5 h with AEA, 2-AG, PEA or vehicle, each at 1 μM (A). The responses to these endocannabinoids were also measured in cells concomitantly treated with URB597 (0.1 μM). (B) Concentration–response modulation of luciferase activity with URB597; pEC₅₀ value derived from non-linear analysis of concentration–response curves is indicated in the text. Results are given as the percentages of relative luciferase activity (firefly luciferase relative to Renilla luciferase) relative to control values. Data shown are means with SEM values of three to six experiments performed in triplicate. Two-way ANOVA indicates a general effect of URB597 (***P = 0.0002, f = 20.60, residual d.f. = 22). ^#P < 0.05 using one-way ANOVA performed inside the URB597-treated group, relative to control cells treated with URB597 alone.

Figure 2

In vitro and in vivo URB597-mediated modification of TH expression. Quantifications of TH mRNA (A) and protein (B) expression were performed on neuroblastoma exposed for 5 or 24 h to 1 μM URB597. Quantitative PCR of TH mRNA was performed on total RNA extracts. The expression of TH mRNA (A) was normalized against GAPDH expression and results are given as the relative expression of treated versus control cells/animals. The densitometric analysis of the TH protein signals (60 kDa) shown in (B) was normalized against the measured signals corresponding to actin (42 kDa). A typical immunoblot is shown at the bottom right corner. Results are given as the percentages of expression relative to control cells. Data shown are means with SEM values of three experiments performed in triplicate. Two-tailed paired Student's t-test, *P < 0.05, **P < 0.01, relative to control at corresponding time (P = 0.0314, t = 5.507, residual d.f. = 2) and (P = 0.0087, t = 10.64, residual d.f. = 2) for mRNA and protein dosages respectively. In mice, TH mRNA contents were evaluated in hippocampus, striatum, cerebellum, cortex and hypothalamus tissues (C) 24 h after a single injection of URB597 (3 mg·kg⁻¹, i.p.). Results are given as the percentages relative to control animals injected with vehicle only. Values are means with SEM of seven animals in each group. Two-tailed unpaired Student's t-test, *P < 0.05, relative to control (P = 0.0305, t = 2.452, residual d.f. = 12) (P = 0.0153, t = 2.824, residual d.f. = 12) (P = 0.0705, t = 1.985, residual d.f. = 2) in the hippocampus, the striatum and the cerebellum respectively.

Figure 3

URB597-mediated regulation of TH promoter activity is CB₁ cannabinoid receptor- and FAAH-independent. (A) Luciferase activity determined on cells transfected with pTH250-Luc and treated with AEA at 10 μM in the presence or the absence of SR 141716A (1 μM) and/or URB597 (0.1 μM). One-way ANOVA followed by Tukey's post-test, ***P < 0.001, **P < 0.01, *P < 0.05, relative to control; and ^##P < 0.01, ^#P < 0.05, as indicated between treated cells. (B) illustrates the influence of SR 141716A (1 μM) on the regulation of TH promoter activity mediated by URB597 or HU 210 (both at 0.1 μM). Two-way ANOVA indicates a general effect of SR 141716A (**P = 0.024, f = 10.26, residual d.f. = 48) with a positive interaction on the treatment (P = 0.0001, f = 11.16). ^###P < 0.001 as indicated between treated cells, determined with Bonferroni post-test. The responses induced by URB597 (0.1 μM) and other FAAH inhibitors (all at 10 μM) were compared to investigate the involvement of FAAH (C). One-way ANOVA indicated a global treatment effect (P = 0.0246, f = 3.396, residual d.f. = 18) with **P < 0.01 relative to control, given by Dunnett's post-test. All the results are given as the percentages of relative luciferase activity (firefly luciferase relative to Renilla liciferase) relative to control values. Data shown are means with SEM values of three to six experiments performed in triplicate.

Figure 4

Increase in endocannabinoid and N-acylethanolamine contents in N1E115 cells induced by different FAAH inhibitors. N1E115 cell contents in AEA (A), 2-AG (B), PEA (C), OEA (D) and SEA (E) were measured by HPLC-MS using an isotope dilution method after exposure to URB597 (0.1 μM), MAFP (10 μM), CAY10499 (10 μM), CAY10402 (10 μM), PF750 (10 μM) or vehicle alone. Results are expressed as the percentages of endocannabinoid or N-acylethanolamine contents relative to control except for (A), for which the basal level of AEA was below the detection limit (N.D.). For this panel only, results ere expressed as the ratio between AEA and its internal standard d₄-AEA. Data are means with SEM values of three experiments performed in triplicate. One-way ANOVA followed by Dunnett's post-test ***P < 0.001, **P < 0.01, relative to control.

Figure 5

Involvement of non-CB₁/non-CB₂ cannabinoid receptors and PPARs in the URB597-mediated regulation of TH promoter activity. (A) RT-PCR was performed on N1E115 cell mRNA extracts with specific primers targeting the different endocannabinoid molecular targets. RT-PCR yielded the predicted amplification products for CB₁, GPR55 and GPR119 receptors as well as for PPARα and PPARγ. No bands were detected for CB₂ cannabinoid receptor and TRPV1 despite a correct amplification in spleen and brain tissues respectively. Negative controls were performed without any DNA template (not shown). The influence of SR 144528 at 1 μM (B), O-1918 at 30 μM (B), CBD at 10 μM (B) or PPAR antagonists (C) on luciferase activity was examined using transfected N1E115 cells carrying pTH250-Luc. Cells were concomitantly treated with the indicated antagonists and 0.1 μM URB597. The tested PPAR antagonists were T 0070907 (PPARγ antagonist, 10 μM) or GW 9662 (PPARγ/PPARα antagonist, 20 μM). Results are given as the percentages of relative luciferase activity (firefly luciferase relative to Renilla luciferase) relative to control values. Data are means with SEM values of at least three experiments performed in triplicate. Two-way ANOVA indicated a general effect of some of the antagonists (***P < 0.0001, f = 18.76, residual d.f. = 18) with a positive interaction (***P < 0.0001, f = 12.13) for (B). ^##P < 0.01, as indicated for treated cells and determined by Bonferroni post-test. In (C), Bartlett's statistic revealed that variances significantly differ between these groups. Despite a strong trend (P = 0.062 for a general effect of URB597 in two-way ANOVA), statistical significance was not reached using the non-parametric Kruskal–Wallis test effect.

Figure 6

Regulation of TH promoter activity by direct activation of the non-CB₁/non-CB₂ cannabinoid receptors and PPARs. To confirm the involvement of the non-CB₁/non-CB₂ cannabinoid receptors in the URB597-mediated effect, the influence of Abn-CBD (10 μM, 5 h) was investigated on N1E115 cells transfected with pTH250-Luc. The involved signalling cascades were examined by concomitantly treating the cells either with non-CB₁/non-CB₂ cannabinoid receptors antagonists (CBD, 10 μM) (A) or PPAR antagonists (T 0070907 at 10 μM or GW 9662 at 20 μM) (B). (A) Two-way ANOVA indicated a general effect of CBD as antagonist (***P < 0.0001, f = 52.37, residual d.f. = 10) with a positive interaction (***P < 0.0001, f = 65.28). ^#P < 0.05 and ^###P < 0.001, as indicated for treated cells and determined by Bonferroni post-test. (B) Two-way ANOVA indicated a general effect of PPAR antagonists (***P < 0.0001, f = 19.49, residual d.f. = 18) with a positive interaction (*P = 0.0193, f = 5.026). ^#P < 0.05 as indicated for treated cells and determined by Bonferroni post-test. (C) The involvement of PPARs in the regulation of TH expression was confirmed by examining the influence of troglitazone (TGZ, 20 μM, 5 h) alone and in the presence of the PPAR antagonists (T 0070907 at 10 μM or GW 9662 at 20 μM). One-way ANOVA indicated a global treatment effect (P = 0.0128, f = 6.949, residual d.f. = 8) with *P < 0.05 relative to control, given by Dunnett's post-test. All the results are given as the percentages of relative luciferase activity (firefly luciferase relative to Renilla luciferase) relative to control values. Data shown are means with SEM values of at least three experiments performed in triplicate.

Figure 7

Role of MAPK in URB597 control of TH promoter activity. The influence of MAPK signalling was investigated in N1E115 cells transfected with pTH250-Luc. To examine the role of this signalling cascade, cells were treated for 1 h with MEK1/2 inhibitor U0126 (5 μM) before adding URB597 (0.1 μM) for an additional 5 h (A). Results are given as the percentages of relative luciferase activity (firefly luciferase relative to Renilla luciferase) relative to control values. Data shown are means with SEM values of three experiments performed in triplicate. One-way ANOVA indicated a general effect of the MEK1/2 inhibitor (***P = 0.0003, f = 36.33, residual d.f. = 8) with a positive interaction (*P = 0.0139, f = 9.831). ^##P < 0.01 as indicated for treated cells and determined by Bonferroni post-test. (B) ERK1/2 activation was determined using immunoblot detection of phosphorylated ERK1/2 (P-ERK1/2 – 42 and 44 kDA). Cells were treated with URB597 (1 μM, 15 min) in the presence or the absence of SR 141716A (1 μM). Phosphorylated ERK1/2 values were normalized to total ERK1/2 (Tot ERK1/2). Results are expressed as percentages of relative density. A representative immunoblot is shown in (C). Data shown are means with SEM values of three experiments. One-way ANOVA indicated a general effect of URB597 (**P < 0.0016, f = 21.80, residual d.f. = 8). Neither significant effect for SR 141716A nor an interaction were observed, indicating the absence of antagonism by SR 141716A.

Figure 8

Schematic representation of the potential targets involved in the URB597-metiated regulation of TH. Besides the well-documented FAAH inhibition and endocannabinoid/NAE elevations, URB597 could either directly (striped arrow) or indirectly, putatively through inhibition of an alternative (non-FAAH) enzymatic activity (open arrow), interact with some off-targets. This results in a regulation of the Abn-CBD-sensitive receptor, which, in turn, could decrease TH gene expression by interacting with PPARs. However, our investigations also support the involvement of ERK1/2 signalling pathway. Therefore, we may hypothesize that the URB597-mediated reduction in ERK1/2 phosphorylation (through direct or indirect Abn-CBD-sensitive receptor-dependent mechanisms) also contributes to the regulation of PPARs (dotted lines). The resulting effect is a decrease in TH gene expression. Contrasting with URB597 effect, other tested FAAH inhibitors failed to regulate these targets and to modulate TH gene expression.