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First published online 29 April 2008
doi: 10.1242/jcs.020958

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Integrin clustering enables anandamide-induced Ca²⁺ signaling in endothelial cells via GPR55 by protection against CB₁-receptor-triggered repression

Markus Waldeck-Weiermair^1,*, Cristina Zoratti^1,*, Karin Osibow¹, Nariman Balenga², Edith Goessnitzer³, Maria Waldhoer², Roland Malli¹ and Wolfgang F. Graier^1,*,

¹ Institute of Molecular Biology and Biochemistry, Medical University Graz, Graz, A8010, Austria
² Institute of Experimental and Clinical Pharmacology, Medical University Graz, Graz, A8010, Austria
³ Institute of Pharmaceutical Chemistry, University Graz, Graz Austria

Author for correspondence (e-mail: wolfgang.graier{at}meduni-graz.at)

Accepted 17 February 2008

	Summary

Top Summary Introduction Results Discussion Materials and Methods References

 
Although the endocannabinoid anandamide is frequently described to act predominantly in the cardiovascular system, the molecular mechanisms of its signaling remained unclear. In human endothelial cells, two receptors for anandamide were found, which were characterized as cannabinoid 1 receptor (CB₁R; CNR1) and G-protein-coupled receptor 55 (GPR55). Both receptors trigger distinct signaling pathways. It crucially depends on the activation status of integrins which signaling cascade becomes promoted upon anandamide stimulation. Under conditions of inactive integrins, anandamide initiates CB₁R-derived signaling, including G_i-protein-mediated activation of spleen tyrosine kinase (Syk), resulting in NFB translocation. Furthermore, Syk inhibits phosphoinositide 3-kinase (PI3K) that represents a key protein in the transduction of GPR55-originated signaling. However, once integrins are clustered, CB₁R splits from integrins and, thus, Syk cannot further inhibit GPR55-triggered signaling resulting in intracellular Ca²⁺ mobilization from the endoplasmic reticulum (ER) via a PI3K-Bmx-phospholipase C (PLC) pathway and activation of nuclear factor of activated T-cells. Altogether, these data demonstrate that the physiological effects of anandamide on endothelial cells depend on the status of integrin clustering.

Key words: Anandamide, Bmx/Etk, Cannabinoid signaling, CB₁ receptor, GPR55, Ca²⁺ signaling, Integrins, Syk

	Introduction

Top Summary Introduction Results Discussion Materials and Methods References

 
The endogenous agonists of cannabinoid receptors exhibit multiple biological functions in various tissues, such as neurons, the immune system and the cardiovasculature (Hillard, 2000; Randall et al., 2002). Among these endocannabinoids, arachidonoylethanolamide (anandamide) recognizes several receptors (Di Marzo et al., 2002) and acts predominantly in the cardiovascular system (Randall and Kendall, 1997; Randall and Kendall, 1998). Anandamide is produced by vascular endothelial cells (Deutsch et al., 1997; Sugiura et al., 1998) that express either the cannabinoid 1 receptor [CB₁R; CNR1 (Maccarrone et al., 2000)] or the CB₂ receptor [CB₂R; CNR2 (Zoratti et al., 2003)], depending on the source and species of the cells. Notably, most of our recent knowledge on the CB receptor type underlying anandamide-induced physiological effects is based on the use of two selective inhibitors of CB₁R and CB₂R: rimonabant (SR141716A; N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) (Rinaldi-Carmona et al., 1994; Showalter et al., 1996) and SR144528 [N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le-3-carboxamide (Griffin et al., 2000; Rinaldi-Carmona et al., 1998; Showalter et al., 1996)], respectively. Endothelium-dependent relaxation was found to be sensitive to rimonabant (Chaytor et al., 1999; Mukhopadhyay et al., 2002; Wagner et al., 1999; White and Hiley, 1997) but not to gap junction inhibitors (Chaytor et al., 1999) and blockade of the vanilloid receptor 1 (Grainger and Boachie-Ansah, 2001; Mukhopadhyay et al., 2002), another putative receptor of anandamide on endothelial cells. However, in mice that lack CB₁R and CB₂R, anandamide-induced mesenteric vasodilation was still prevented by rimonabant (Offertaler et al., 2003), thus, leading to the concept of an `atypical' endothelial anandamide receptor (e-aR) that is also sensitive to rimonabant. Hence, this receptor was found to be activated by the cannabinoid analogs abnormal cannabidiol (–)-4-(3-3,4-trans-p-menthadien-[1,8]-yl)-olivetol) and O1602 [5-methyl-4-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-1,3-benzenediol; an agonist of `atypical' non-CB₁/CB₂ endothelial anandamide receptor (Offertaler et al., 2003), and to be inhibited by O1918 (Offertaler et al., 2003). The e-aR was shown to be coupled to G_i/o protein and to trigger Ca²⁺-activated formation of nitric oxide in endothelial cells (Mukhopadhyay et al., 2002).

Considering the large amount of recent reports using rimonabant and the variety of vascular effects of anandamide, which include inhibition of endothelin 1 expression (Ronco et al., 2006), relaxation of mesenteric arteries (Hoi and Hiley, 2006), or the inhibition of CB₁R-mediated tumor growth and metastatic spreading (Portella et al., 2003), endocannabinoid-triggered signaling in endothelial cells needs to be explored in more detail.

Despite the intriguing reports on the existence of e-aR in human endothelial cells, most reports on endothelial cells did not differentiate between the signal transduction triggered by the two receptor types present. Thus, the underlying signal transduction cascades beyond each individual receptor type are still unclear. Moreover, as these receptors are presumably activated by anandamide at the same time, interplay between the two signaling cascades activated by each of these receptors may occur and needs further investigation.

Consequently, in this study we intended to explore the signaling cascades downstream of CB₁R and e-aR in human endothelial cells. Furthermore, the interrelation and integration of the two signaling pathways on the initiation of cytosolic Ca²⁺ signaling as a highly specific readout of activation of e-aR were investigated.

	Results

Top Summary Introduction Results Discussion Materials and Methods References

 
The effect of anandamide on endothelial Ca²⁺ signaling inversely depended on external Ca²⁺ and was due to atypical endothelial anandamide receptors
In the presence of 2 mM external Ca²⁺, addition of 10 µM anandamide yielded very small cytosolic Ca²⁺ elevation in only 10% of the cells, which was far less than that obtained with 100 µM histamine under identical conditions (Fig. 1A). Similarly, the hydrolytically more stable analog methanandamide failed to initiate Ca²⁺ elevation in the presence of external Ca²⁺, thus excluding Ca²⁺-dependent hydrolysis as being responsible for the lack of Ca²⁺ signaling in response to anandamide in the presence of external Ca²⁺.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Endocannabinoids trigger Ca2+ signaling and ER depletion in human endothelial cells. (A) Comparison of the effects of 100 µM histamine (n=18) and 10 µM anandamide (n=31) on cytosolic free-Ca2+ concentration in the presence of 2 mM extracellular Ca2+. (B) Cytoplasmic free-Ca2+ concentration upon stimulation with 100 µM histamine (n=19) and 10 µM anandamide (n=21) in the absence of extracellular Ca2+, followed by re-addition of 2 mM extracellular Ca2+ to reveal store-operated Ca2+ entry due to previous ER depletion. (C) Comparison of the effects of 100 µM histamine (n=4) and 10 µM anandamide (n=3) on free ER Ca2+ concentration in the absence of extracellular Ca2+, followed by re-addition of 2 mM extracellular Ca2+ to visualize ER Ca2+ refilling efficiency. (D) Concentration-response curves for anandamide (n=10) and metanandamide (n=10) on peak responses of cytosolic Ca2+ elevation in the nominal absence of extracellular Ca2+. Cytosolic (A,B,D) and ER (C) Ca2+ concentrations were recorded using fura-2 and YC4er, respectively. *P<0.01 versus histamine.

To define the receptor being involved in anandamide-triggered endothelial Ca²⁺ signaling, the expression of CB₁R but not CB₂R was confirmed by RT-PCR in the human endothelial cells used in this study (Fig. 2A). Involvement of CB₂R was further excluded by the lack of the CB₂R antagonist SR144528 [1 µM (Rinaldi-Carmona et al., 1998)] (Table 1) to inhibit anandamide-induced Ca²⁺ signaling in the nominal absence of extracellular Ca²⁺ (supplementary material Fig. S2A). Similarly, inhibition of TRPV1 (VR1) receptors by SB366791 [4'-chloro-3-methoxycinnamanilide (Fowler et al., 2003)] (10 µM) failed to affect anandamide-induced Ca²⁺ signaling under the same conditions (supplementary material Fig. S2B). By contrast, rimonabant (1 µM), an inhibitor of CB₁R and e-aR (Jarai et al., 1999; Wagner et al., 1999), inhibited anandamide-induced Ca²⁺ signaling in nominally Ca²⁺-free solution (Fig. 2B).

View larger version (32K): [in this window] [in a new window]   Fig. 2. `Atypical' endothelial anandamide receptors are identified as GPR55 that mediates anandamide-initiated Ca2+ signaling, while activation of CB1 receptors prevent anandamide-induced Ca2+ signaling. (A) Representative RT-PCR on CB1R in a human endothelial cell line (lane 2) in comparison with human brain as positive control (lane 3). (B) In Ca2+-free solution, the effect of 10 µM anandamide on cytosolic free-Ca2+ concentration was analyzed in the absence (n=19) or presence of the CB1R antagonist rimonabant (1 µM) (n=28). (C) In the presence of the CB1R antagonist AM251 (10 µM), 10 µM anandamide triggered Ca2+ signaling even in the presence of extracellular Ca2+ (n=59). (D) In the presence of 2 mM extracellular Ca2+, the effect of the e-aR agonist O1602 (10 µM) on cytosolic free-Ca2+ concentration was tested (n=17). (E) In the absence of extracellular Ca2+, the effect of O1918 (10 µM), an inhibitor of the e-aR, on anandamide (10 µM, AEA) -induced Ca2+ signaling was assessed (n=8). (F) The effect of the CB1R agonist HU-210 (10 µM) on O1602 (e-aR agonist, 10 µM) -induced Ca2+ signaling was tested in the presence of 2 mM extracellular Ca2+ (n=10). (G) Representative RT-PCR on GPR55 in the human endothelial cell line using various annealing temperatures. (H) Pretreatment with siRNA against GPR55 (n=49; control, n=48) on anandamide (10 µM) -induced cytosolic Ca2+ signaling was monitored in nominally Ca2+-free solution. (I) Pretreatment with siRNA against GPR55 (n=62; control, n=18) on O1602 (10 µM) -induced cytosolic Ca2+ signaling in Ca2+-containing solution. (J) Consequences of GPR55 overexpression on anandamide (AEA, 10 µM) -induced Ca2+ signaling in the nominal absence of extracellular Ca2+ (n=43; control, n=42). (K) Consequences of GPR55 overexpression on O1602 (10 µM) -induced Ca2+ signaling in the nominal absence of extracellular Ca2+ (n=21; control, n=25). Cytosolic free-Ca2+ concentrations were recorded using fura-2. *P<0.005 versus the absence of the respective receptor blocker/activator.

To further differentiate whether CB₁R or e-aR is involved in anandamide-induced Ca²⁺ signaling, the effects of specific receptor agonists and antagonists were tested. Inhibition of CB₁R by 10 µM of the potent CB₁R antagonist AM251 [N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (Table 1) (Pertwee, 2005) unmasked anandamide (10 µM)-induced Ca²⁺ signaling even in the presence of extracellular Ca²⁺ (Fig. 2C). In addition, the synthetic CB₁R agonist HU-210 [10 µM (Felder et al., 1995)], which has been shown to couple with intracellular Ca²⁺ signaling in HEK 293 cells, cultured hippocampal neurons (Lauckner et al., 2005) and rat insulinoma beta cells (De Petrocellis et al., 2007), failed to initiate endothelial Ca²⁺ signaling in the presence and nominal absence of external Ca²⁺ (supplementary material Fig. S2C). By contract, 10 µM of O1602, an agonist of the e-aR (Table 1) (Bukoski et al., 2002; Jarai et al., 1999) evoked a very strong cytosolic Ca²⁺ elevation even in the presence of extracellular Ca²⁺ (Fig. 2D), which was not affected by the TRPV1 receptor inhibitor SB366791 (10 µM) (supplementary material Fig. S2D). In line with these findings, 10 µM of O1918 [1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-benzene] (Table 1), a cannabidiol analog that acts as a selective antagonist of `atypical' cannabidiol at the non-CB₁/CB₂ endothelial anandamide receptor (Jarai et al., 1999; Mo et al., 2004; Offertaler et al., 2003), prevented anandamide-induced Ca²⁺ signaling in the nominal absence of extracellular Ca²⁺ (Fig. 2E). Moreover, in the presence of the CB₁R-specific agonist HU-210 (10 µM), activation of the e-aR with 10 µM O1602 failed to evoke Ca²⁺ signaling, while, after washout of HU-210, O1602 initiated Ca²⁺ signaling in the same cells (Fig. 2F). These data point to involvement of both anandamide receptors in endothelial cells in which CB₁R inhibits the signal cascade beyond e-aR, which yields intracellular Ca²⁺ mobilization and becomes overt in the nominal absence of external Ca²⁺.

The `atypical' non-CB₁ and CB₂ endothelial anandamide receptor (e-aR) was identified as G-protein-coupled receptor 55 (GPR55)
To identify the e-aR molecularly, the endothelial cells used for this study were tested for GPR55, a putative anandamide-binding protein (Baker et al., 2006; Ryberg et al., 2007). Using the respective primers, mRNA of GPR55 was found in endothelial cells (Fig. 2G). Hence, treatment with siRNA against GPR55, which was proved to reduce the expression of GPR55 by 56% (supplementary material Fig. S2E), strongly diminished anandamide-triggered Ca²⁺ signaling in Ca²⁺-free solution, as well as the effect of O1602 in the presence of extracellular Ca²⁺ (Fig. 2H,I, respectively), thus pointing to the involvement of GPR55 in anandamide-induced Ca²⁺ signaling in endothelial cells. Overexpression of GPR55 (supplementary material Fig. S2F) strongly elevated anandamide-triggered Ca²⁺ signaling in the nominal absence of extracellular Ca²⁺ (Fig. 2J). Consistent with this, the effect of O1602 (10 µM) on endothelial cytosolic free-Ca²⁺ was largely elevated in GPR55-overexpressing cells (Fig. 2K, Table 1).

Recently, GPR55 was described as a receptor of lysophosphatidylinositol [LPI (Oka et al., 2007)]. These data could be confirmed in endothelial cells in which the effect of 10 µM LPI was strongly reduced in cells treated with siRNA against GPR55 (supplementary material Fig. S2G), whereas overexpression of GPR55 yield enhanced Ca²⁺ signaling by LPI (supplementary material Fig. S2H).

The effect of anandamide on endothelial Ca²⁺ signaling was sensitive to divalent cations acting on extracellular bindings sites
Based on the unusual sensitivity of anandamide-triggered Ca²⁺ signaling to external Ca²⁺, we tested whether or not the inhibitory effect of extracellular Ca²⁺ was due to Ca²⁺-dependent chelating of anandamide in the medium. Using NMR spectroscopy, no evidence for a Ca²⁺-anadamide complex was found (supplementary material Fig. S3A,B). Moreover, anandamide uptake and binding did not differ in the nominal absence or presence of extracellular Ca²⁺ (supplementary material Fig. S3C). The concentration-response curve of the inhibitory action of extracellular Ca²⁺ on anandamide-triggered intracellular Ca²⁺ signaling revealed an IC₅₀ of 7.7 (6.7-8.8) µM (Fig. 3A). Similarly, Sr²⁺ and Ba²⁺ inhibited anandamide-triggered Ca²⁺ signaling in endothelial cells in a concentration-dependent manner (Fig. 3B). These results point to an extracellular Ca²⁺-binding site being responsible for its inhibitory effect on anandamide-induced Ca²⁺ signaling.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Anandamide/GPR55-induced Ca2+ signaling is linked to integrins. (A,B) The sensitivity of anandamide (10 µM) -triggered cytosolic Ca2+ signaling to various concentrations of extracellular Ca2+ (n=10), Ba2+ (n=24) and Sr2+ (n=31) was tested. (C) Comparison between the effect of 10 µM anandamide in Ca2+-containing buffer in the absence (n=20) or presence of 70 µM Mn2+ (n=40). (D) The effect of 10 µM anandamide on cytosolic free-Ca2+ concentration was monitored in the nominal Ca2+-free solution in the absence (n=25) or presence of the ROCK inhibitor Y27632 (10 µM, n=29). (E-G) The effects of antibodies (2 µM) against vβ3 integrin (LM609) (control, n=34; LM609, n=30) and the β1 (JB1A) (control, n=28; JB1A, n=33) and β3 subunits (B3A) (control, n=38; B3A, n=30) on cytosolic Ca2+ signaling induced by 10 µM anandamide was assessed in nominally Ca2+-free solution. (H) The effect of function-blocking β3-integrin antibody B3A on O1602 (10 µM) -initiated Ca2+ signaling in the presence of extracellular Ca2+ (control, n=65; B3A, n=84). Cytosolic free-Ca2+ concentrations were recorded using fura-2. *P<0.0001 versus the absence of the respective compound.

Clustered vβ3 and 5β1 integrins were involved in anandamide-induced Ca²⁺ signaling in human endothelial cells

To further challenge the concept that integrins are involved in the process of anandamide-triggered Ca²⁺ signaling, the effects of the crosslinking antibody against vβ3 integrin [LM609, 2 µM (Charo et al., 1990; Cheresh, 1987)] and two function-blocking antibodies against the β1 [JB1A, 2 µM (Mohri et al., 1996)] and the β3 subunits [B3A, 2 µM (Vignali et al., 1990)]. During preincubation for 15 minutes in Ca²⁺-containing medium, none of the integrin-targeting antibodies affected basal endothelial Ca²⁺ levels, while all prevented the effect of anandamide in the absence of extracellular Ca²⁺ (Fig. 3E-G). In line with these findings, B3A also prevented O1602-induced Ca²⁺ signaling in Ca²⁺-containing buffer (Fig. 3H). These data point to the involvement of vβ3 and 5β1 integrins in GPR55-mediated Ca²⁺ signaling by anandamide and O1602 in endothelial cells.

Anandamide induced diverse tyrosine phosphorylation patterns depending on the presence of extracellular Ca²⁺
As tyrosine kinases are known to represent early downstream targets of integrin-mediated signaling, the effect of anandamide on the activity of endothelial tyrosine kinases [using the FRET-based sensor Picchu 936X (Kurokawa et al., 2001; Schaeffer et al., 2003)] and the patterns of tyrosine phosphorylation were assessed in the presence and absence of extracellular Ca²⁺. Notably, anandamide yielded fast activation of PP1-sensitive tyrosine kinases under any conditions indicated by increase in the FRET signal of Picchu 936X (Fig. 4A,B) but not the inactive mutant Picchu 938X. Although this is a rather general sensor for several tyrosine kinases and, thus, no conclusion can be drawn on the actual kinase(s) involved, it uniquely reveals the kinetics of tyrosine phosphorylation upon anandamide. However, corresponding western blot analyses revealed markedly different effects of anandamide on protein tyrosine phosphorylation, depending on extracellular Ca²⁺. In the presence of extracellular Ca²⁺ a fast, strong and long lasting phosphorylation of a 60 kDa band occurred in response to anandamide stimulation, while phosphorylation of a 70-80 kDa band reached its maximum 5 minutes after agonist stimulation (supplementary material Fig. S4A). By contrast, in the absence of external Ca²⁺ (i.e. under conditions of anandamide-induced Ca²⁺ signaling) the 70-80 kDa band appeared much earlier, while the 60 kDa band was delayed compared with the presence of extracellular Ca²⁺ (supplementary material Fig. S4A). Identical findings were obtained in the presence of 70 µM Mn²⁺ in Ca²⁺-containing buffer.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Anandamide initiates tyrosine phosphorylation. (A,B) The effect of anandamide (10 µM) -induced activation of tyrosine kinases was visualized in single endothelial cells in the presence (A) and absence (B) of extracellular Ca2+. As indicated, 10 µM PP1 was added to the media (n=4 under each condition). Tyrosine kinase activity was monitored by measuring the excitation ratio of F535/F480 nm (FRET) at 440 nm excitation. (C) The effect of 10 µM anandamide on cytosolic free-Ca2+ concentration was tested in nominally Ca2+-free solution in the absence (n=24) or presence of 10 µM PP1 (n=25). (D) In Ca2+-containing solution, Ca2+ intracellular Ca2+ signaling was initiated by 10 µM O1602 in the presence of 10 µM PP2, followed by an additional stimulation with 10 µM O1602 after washout of PP2 as indicated (n=14). For visualization of tyrosine kinase activity in single endothelial cells, cells were transiently transfected with Picchu 936X. Cytosolic free-Ca2+ concentration was recorded using fura-2. *P<0.001 versus the absence of PP1.

View larger version (22K): [in this window] [in a new window]   Fig. 5. PI3K is involved in anandamide/GPR55-evoked Ca2+ signaling. (A) The effect of 10 µM anandamide on cytosolic free-Ca2+ concentration was tested in the nominal Ca2+-free solution in the absence (n=33) or presence (n=38) of 0.1 µM wortmannin. (B) In the presence of extracellular Ca2+, intracellular Ca2+ signaling was initiated by 10 µM O1602 in the presence of 0.1 µM wortmannin followed by an additional stimulation with 10 µM O1602 after washout of the PI3K inhibitor as indicated (n=14). (C) The effect of the PLC inhibitor U73122 (2 µM, n=26; control, n=30) on anandamide (10 µM) -trigged cytosolic Ca2+ signaling was tested in Ca2+-free buffer. (D) In Ca2+-containing buffer, intracellular Ca2+ signaling was initiated by 10 µM O1602 in the presence of 1 µM U73122 followed by an additional stimulation with 10 µM O1602 after washout of the PLC inhibitor as indicated (n=14). (E) Concentration-response relationship of 2APB on cytosolic Ca2+ signaling induced by either 100 µM histamine (n=4) or 10 µM anandamide (n=6) in nominally Ca2+-free solution. Cytosolic free-Ca2+ concentrations were recorded in single cells using fura-2. *P<0.001 versus the absence of the inhibitor.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Bmx/Etk is the downstream target for PI3K upon GPR55 stimulation by anandamide. (A) Phosphorylation of Bmx/Etk upon stimulation with 10 µM anandamide in the nominal absence of extracellular Ca2+ was tested by immunoprecipitation using anti-Bmx/Etk antibody (upper blot) and subsequent staining with the anti-phosphotyrosine antibody 4G10 (lower blot). (B,C) The effects of the Tec-family (Bmx/Etk) kinase inhibitor LFM-A13 (10 µM, n=46; control, n=30) or pretreatment with siRNA against Bmx/Etk (n=6; control, n=27) on anandamide (10 µM) -induced cytosolic Ca2+ signaling were monitored in nominally Ca2+-free solution. (D) The effect of the Tec-family (Bmx/Etk) kinase inhibitor LFM-A13 (10 µM, n=75; control, n=71) on O1602 (10 µM) -trigged cytosolic Ca2+ signaling in Ca2+-containing buffer was tested. Standard immunoprecipitation was applied. In experiments with siRNA, cells were transiently transfected with a vector encoding approved siRNA against Bmx/Etk. Experiments were performed 48 hours after transfection. Cytosolic free-Ca2+ concentrations were recorded using fura-2. *P<0.0001 versus the absence of the inhibitor.

View larger version (40K): [in this window] [in a new window]   Fig. 7. Syk is the mediator of CB1R-originated repression of anandamide-induced Ca2+ signaling. (A,B) The inhibition of Syk by either its inhibitor piceatannol (5 µM, n=24) or siRNA against Syk (n=7; control, n=4) allowed strong cytosolic Ca2+ elevation in response to 10 µM anandamide in the presence of 2 mM extracellular Ca2+. (C) The effect of the Gi protein inhibitor pertussis toxin (400 ng/ml for 3 hours, n=8; control, n=8) on anandamide (10 µM) -induced Ca2+ signaling in the presence of 2 mM extracellular Ca2+ was verified in human endothelial cells. (D) Immunoprecipitation using anti-β1-integrin antibody and subsequent staining with either anti-β1-integrin (upper blot) or anti-CB1R (lower blot). Endothelial extracts were harvested from cells under basal conditions (-) and after stimulation with 10 µM anandamide (+) in the presence of 2 mM extracellular Ca2+ (Ca2+) or 2 mM extracellular Ca2+ plus 70 µM Mn2+ (Mn2+). Right panels shows precipitation with beads that were not preloaded with anti-β1-integrin were used. (E) Immunoprecipitation using anti-β1-integrin antibody and subsequent staining with either anti-β1-integrin (upper blot), anti-V-integrin (middle blot) or anti-CB1R (lower blot). Endothelial extracts were harvested from cells under basal conditions (-) and after stimulation with 10 µM anandamide (+) in the and absence (EGTA) of extracellular Ca2+. Right panels shows precipitation with beads that were not preloaded with anti-β1-integrin were used. Standard immunoprecipitation was applied. In experiments with siRNA, cells were transiently transfected with a vector encoding approved siRNA against Syk 48 hours prior to the experiments. Cytosolic free-Ca2+ concentrations were recorded using fura-2. *P<0.0001 versus the absence of the inhibitor.

PI3K, Bmx/Etk and PLC represented downstream targets of integrin-dependent anandamide/GPR55-induced signal transduction in endothelial cells
At 0.1 µM, the selective inhibitor of phosphoinositide 3 kinase (PI3K) wortmannin (Table 2) diminished tyrosine phosphorylation of the 70-80 kDa band (supplementary material Fig. S5) and abolished anandamide-triggered Ca²⁺ signaling in nominally Ca²⁺-free solution (Fig. 5A) and in the presence of 70 µM Mn²⁺ in Ca²⁺ containing buffer (data not shown). Notably, wortmannin was less active in reducing the 70-80 kDa band in the presence of extracellular Ca²⁺, suggesting two different proteins between 70-80 kDa: a wortmannin-sensitive one becoming phosphorylated in the absence of extracellular Ca²⁺ (or the presence of 70 µM Mn²⁺); and a wortmannin-insensitive protein being phosphorylated in the presence of extracellular Ca²⁺. In line with the inhibitory potential of wortmannin on anandamide-induced Ca²⁺ signaling in nominal Ca²⁺-free/Mn²⁺-containing solutions, this PI3K inhibitor prevented Ca²⁺ signaling in response to O1602 in the presence of extracellular Ca²⁺ (Fig. 5B, Table 2).

To test the involvement of phospholipase C (PLC) activation in the integrin/PI3K-dependent Ca²⁺ recruitment by anandamide, U73122 [(1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5dione (Bleasdale et al., 1990), Table 2], an inhibitor of PLC (Aschner et al., 1997) was used. Cytosolic Ca²⁺ elevations induced by anandamide in the absence of external Ca²⁺ (Fig. 5C) or O1602 in Ca²⁺-containing buffer (Fig. 5D) were prevented by 2 µM U73122. Moreover, the rather unspecific and membrane permeable Ins(1,4,5)P₃-receptor antagonist 2APB [2-aminoethoxydiphenylborate (Maruyama et al., 1997; Koganezawa and Shimada, 2002)] prevented endothelial Ca²⁺ elevation in response to 10 µM anandamide and 10 µM histamine at comparable potency with IC₅₀ values of 43 (30-60) and 87 (50-152) µM, respectively (Fig. 5E). These data suggest that anandamide-triggered Ca²⁺ signaling is due to PLC-mediated formation of Ins(1,4,5)P₃ that, in turn, elevates cytosolic Ca²⁺ concentration by intracellular mobilization from the ER.

One potential candidate for bridging PI3K activation to PLC stimulation is bone marrow kinase, X-linked/epithelial and endothelial tyrosine kinase (Bmx/Etk) (Qiu and Kung, 2000), a member of the Tec family (Takesono et al., 2002). Immunoprecipitation revealed a 3.2±0.5-fold (n=4) increase in phosphorylation of Bmx/Etk upon anandamide in the absence but not presence of extracellular Ca²⁺ (Fig. 6A). Accordingly, siRNA against Bmx/Etk (efficiency shown in supplementary material Fig. S6A) diminished anandamide-induced Ca²⁺ signaling in the absence of external Ca²⁺ or in the presence of 70 µM Mn²⁺ in Ca²⁺-containing solution (Fig. 6B). Moreover, 10 µM of the selective Tec family inhibitor LFM-A13 [2-cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, Table 2] (Chau et al., 2002) prevented anandamide-triggered Ca²⁺ signaling in the absence of external Ca²⁺ or (Fig. 6C) in the presence of Mn²⁺ in Ca²⁺-containing solution. These data are in line with phosphorylation of the 70-80 kDa band upon anandamide stimulation (supplementary material Fig. S5) representing Bmx/Etk that, in turn, activates PLC leading to intracellular Ca²⁺ mobilization via the generation of Ins(1,4,5)P₃. Consistent with this, inhibition of Bmx/Etk by 10 µM LFM-A13 also prevented O1602 (10 µM)-triggered Ca²⁺ signaling in Ca²⁺ containing solution (Fig. 6D).

In the presence of external Ca²⁺, activation of Syk via G_i/o exhibited inhibitory action on PI3K-Bmx-PLC pathway and prevented anandamide/GPR55-induced Ca²⁺ signaling
Based on the western blot (supplementary material Fig. S5) that revealed a wortmannin-insensitive tyrosine phosphorylation at 70-80 kDa in the presence but not absence of extracellular Ca²⁺, a negative-feedback mechanism against the PI3K-Bmx-PLC signaling via tyrosine phosphorylation [possibly via spleen tyrosine kinase (Syk)] was hypothesized. RT-PCR of the endothelial cell line used proved the expression of Syk in its long form (Wang et al., 2003) (supplementary material Fig. 6B), which matches the 70-80 kDa band in western blots (supplementary material Fig. S5). To further challenge the concept of inhibitory action of Syk on PI3K-Bmx-PLC signaling, the effect of the Syk inhibitor piceatannol [3,4,3',5'-tetrahydroxy-trans-stilbene (Geahlen and McLaughlin, 1989; Ashikawa et al., 2002)] was investigated. In cells that only weakly responded to anandamide in the presence of external Ca²⁺, this agonist yielded strong Ca²⁺ signaling in the presence of 5 µM piceatannol (Fig. 7A, Table 2). These findings were further supported by the induction of Ca²⁺ signaling by anandamide despite the presence of extracellular Ca²⁺ (Fig. 7B) in cells, which were transiently transfected with siRNA against Syk (supplementary material Fig. S6A). To verify the signaling mechanisms by which endothelial CB₁R triggers activation of Syk, G_i/o protein, which has been reported to be coupled to this cannabinoid receptor (Mukhopadhyay et al., 2002), was inhibited by ADP ribosylation with pertussis toxin. Inhibition of G_i/o protein by pertussis toxin (Table 2) (400 ng/ml, 3 hours) yielded anandamide-induced Ca²⁺ signaling even in the presence of extracellular Ca²⁺ (Fig. 7C), thus pointing to G_i/o-mediated activation of Syk that, in turn, inhibits PI3K-Bmx-PLC-Ca²⁺ signaling (see Fig. 10).

View larger version (80K): [in this window] [in a new window]   Fig. 10. Schemes of the two signaling cascades activated by anandamide in endothelial cells. (A) Anandamide-induced Ca2+ signaling under conditions of unclustered integrins originates from CB1R that is linked to β1 integrin and stimulates Syk via Gi/o, subsequently resulting in the activation of NFB, Erk1 and Erk2. Additionally, Syk inhibits PI3K and, thus, the downstream signaling of the GPR55 is prevented. (B) Once integrins are clustered, CB1R uncouples from β1 integrin and, thus, no negative feedback on PI3K via Syk occurs. Consequently, GPR55-initiated signaling becomes promoted, resulting in the activation of a PI3K-Bmx-PLC cascade that triggers in the production of IP3 and subsequent intracellular Ca2+ mobilization, yielding activation of NFAT, Erk1 and Erk2. Based on the data presented, involvement of Gq/s in GPR55-triggered signaling cannot be not confirmed or excluded at this stage. Abbreviations: 1β3, 1β3 integrin; v5, v5 integrin; Bmx/Etk, bone marrow kinase, X-linked/epithelial and endothelial tyrosine kinase; CB1R, cannabinoid 1 receptor; ER, endoplasmic reticulum; Erk1/2, extracellular signal regulated kinases 1 and 2; GPR55, G-protein-coupled receptor 55 (e-aR, `atypical' endothelial anandamide receptor); IP3, Ins(1,4,5)P3; Gi, Gi protein; Gq, Gq (G13) protein; NFAT, nuclear factor of activated T-cells; NFB, nuclear factor B; p50, nuclear factor B p50; p65, nuclear factor B, subunit 3; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; Syk, spleen tyrosine kinase; Tk, tyrosine kinase.

Clustered β1 integrin released binding to CB₁ receptor upon anandamide stimulation
To explore how integrin-clustering shields anandamide-induced PI3K-Bmx-PLC-Ca²⁺ signaling from the inhibitory effect of CB₁R-G_i/o-Syk signaling, interaction of integrins with CB₁R was investigated by immunoprecipitation. Under conditions of unclustered integrins (i.e. in the presence of external Ca²⁺), the binding of CB₁R to β1 integrin was not affected by anandamide (normalized CB₁R ratio without versus with anandamide 1: 1.03±0.04, n=4, n.s.; Fig. 7D). By contrast, if integrin clustering was facilitated by Mn²⁺, anandamide yielded detachment of the CB₁R from β1 integrin (CB1R was normalized to β1 integrin; CB₁R ratio without anandamide versus with anandamide 1: 0.31±0.13, n=4, P<0.05; Fig. 7D). Identical results were obtained in Ca²⁺-free solution (CB1R was normalized to β1 integrin; CB₁R ratio without anandamide versus with anandamide 1: 0.30±0.21, n=3, P<0.05; Fig. 7E). Although under conditions of clustered integrins the binding of the CB₁R to β1 integrin was affected by anandamide, it did not alter the localization/distribution of CB₁R (supplementary material Fig. S6C).

Thus, under conditions of clustered integrins, anandamide yielded detachment of the CB₁R from β1 integrin, which, in turn, may preserve anandamide-induced signaling pathways via e-aR leading to cytosolic Ca²⁺ elevation.

Depending on integrin clustering, anandamide triggered either nuclear accumulation of NFB or NFAT while MAPK activation occurred under all conditions
To investigate transcriptional consequences of the two distinct signaling pathways initiated by anandamide via either CB₁R or e-aR, the nuclear recruitment of nuclear factor B (NFB) and nuclear factor of activated T-cells (NFAT) upon anandamide stimulation was investigated under conditions of clustered and unclustered integrins. In the presence of extracellular Ca²⁺ (i.e. unclustered integrins), anandamide evoked nuclear accumulation of the p65 subunit of NFB (Fig. 8A) but not NFAT (Fig. 9A), whereas if integrins were clustered by Mn²⁺ in the presence of external Ca²⁺, anandamide failed to stimulate p65 translocation (Fig. 8B) but evoked strong nuclear accumulation of NFAT (Fig. 9B).

View larger version (56K): [in this window] [in a new window]   Fig. 8. NFB is activated independently of anandamide-triggered Ca2+ signals. The effect of 10 µM anandamide on the (trans)location of GFP-tagged p65 subunit of NFB was visualized in endothelial cells in buffer containing 2 mM Ca2+ in the absence (A) or presence (B) of 70 µM Mn2+. Images of endothelial cells that were transiently transfected with p65-GFP were taken 48 hours after transfection using a conventional fluorescence microscope. Representative images of at least 10 cells out of three experiments on alternate days are shown. Scale bars: 10 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 9. Anandamide-induced Ca2+ signal subsequently activates NFAT, Erk1 and Erk2. (A,B) The effect of 10 µM anandamide on the (trans)location of GFP-tagged NFAT was visualized in endothelial cells in the presence (A) or absence (B) of 2 mM extracellular Ca2+. (C,D) Time dependency of the effect of 10 µM anandamide on Erk1 and Erk2 phosphorylation in endothelial cells in 2 mM Ca2+-containing buffer in the absence (C) or presence (D) of 70 µM Mn2+. Images of endothelial cells that were transiently transfected with NFAT-GFP were taken 48 hours after transfection using a conventional fluorescence microscope. Data presented are representative for at least eight cells out of three experiments on alternate days. Scale bars: 10 µm. For assessment of Erk1 and Erk2 activation, standard western blotting was applied.

	Discussion

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Our data demonstrate that two distinct receptors for anandamide exist in human endothelial cells, which were characterized as CB₁R and GPR55. Both receptors trigger distinct signaling pathways, whereby one exhibits negative feedback on the other, depending on the status of integrin configuration. As illustrated in Fig. 10A, the presence of extracellular Ca²⁺ anandamide binding to CB₁R results in G_i protein-mediated activation of Syk and its subsequent signal transduction pathway, resulting in activation of NFB. Additionally, Syk inhibits PI3K that represents a key signaling protein in the transduction of GPR55-originated signaling. However, once integrins are clustered (Fig. 10B), either by removal of extracellular Ca²⁺ or addition of Mn²⁺, Syk does not further inhibit GPR55-triggered signaling. Subsequently, the latter causes intracellular Ca²⁺ mobilization from the ER via a PI3K-Bmx-PLC pathway and, in turn, activation of Ca²⁺-activated NFAT (Fig. 10B). Notably, although the activation of transcription factors varies depending on the signaling pathway, Erk1 and Erk2 are activated under all circumstances (Fig. 10). Although the physiological consequences of these concomitant and interrelated signaling pathways in response to anandamide in endothelial cells are unclear and need to be further explored, the crucial role of integrin-clustering may point to a circumstance-dependent bivalent contribution of this endocannabinoid to cell stimulation, proliferation, angiogenesis and/or migration under conditions such like inflammation, permeability or hypoxia.

Receptor(s) involved in anandamide-induced endothelial cell activation
Endocannabinoids interact with two cannabinoid receptors identified by molecular cloning: CB₁R, which is expressed predominantly in the brain (Matsuda et al., 1990) but is also present in peripheral tissues (Shire et al., 1995), and CB₂R, expressed mainly by cells of the immune system (Munro et al., 1993) but also calf pulmonary aortic endothelial cells (Zoratti et al., 2003). In human and murine vasculature, the actions of endogenous cannabinoids have been widely interpreted as being mediated by CB₁R, although there is a growing amount of evidence, particularly in isolated heart and blood vessel preparations, that another cannabinoid receptor, referred as e-aR may exist (Offertaler et al., 2003).

Our findings that, despite the presence of CB₁R, its very selective synthetic agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol] (Felder et al., 1995) failed to stimulate intracellular Ca²⁺ signaling while the e-aR agonist O1602 (Bukoski et al., 2002; Jarai et al., 1999) evoked a strong Ca²⁺ signal in Ca²⁺ containing solution, indicate that anandamide-triggered Ca²⁺ signaling is mediated by e-aR in the human umbilical vein-derived endothelial cell line used in this study. However, anandamide failed to induce a Ca²⁺ signal in the presence of extracellular Ca²⁺. Accordingly, this points to a repressive effect of CB₁R-originated signal transduction on signaling pathways beyond e-aR. This hypothesis was further confirmed by our findings that activation of CB₁R with HU210 strongly diminished the O1602-initiated Ca²⁺ signal. Notably, caution is advised on the specificity of CBR antagonist/agonists, and, thus, additional efforts to characterize involved receptors were undertaken.

However, in the absence of external Ca²⁺, anandamide initiated strong cytosolic Ca²⁺ signaling. Notably, this signal was insensitive to the CB₂R antagonist SR144528 but was prevented by rimonabant, an inhibitor of CB₁R as well as of e-aR (Offertaler et al., 2003). Importantly, as O1918, the antagonist of the e-aR (Jarai et al., 1999; Mo et al., 2004; Offertaler et al., 2003), prevented anandamide-induced Ca²⁺ signaling in the absence of external Ca²⁺, these data indicate that in the nominal absence of extracellular Ca²⁺ the CB₁R-originated suppressive effect is uncoupled from the e-aR pathway, and, thus, allows the generation of a Ca²⁺ signal. Hence, using RT-PCR and siRNA, the putative anandamide receptor GPR55 (Brown, 2007; Hiley and Kaup, 2007; Ryberg et al., 2007) was identified as the e-aR. Hence, overexpression of GPR55 strongly increased Ca²⁺ signaling upon anandamide and O1602 treatment in Ca²⁺-free and Ca²⁺-containing buffer, respectively. However, the potency of anandamide was less in this study than previously reported (Ryberg et al., 2007), which may be due to the different cell models (i.e. transfected HEK 293 cells versus endothelial cells). Hence, lysophosphatidylinositol, a recently described agonist of the GPR55 (Oka et al., 2007), was found to trigger Ca²⁺ signaling via constitutive GPR55 in endothelial cells. Accordingly, there are several questions arising: first, how does the e-aR/GPR55 trigger Ca²⁺ signaling; second, what is the molecular mechanism of the CB₁R-mediated suppressive effect on anandamide-induced Ca²⁺ signaling; third, why and how is the Ca²⁺ signal via e-aR/GPR55 shielded from CB₁R-mediated suppression in the absence of extracellular Ca²⁺; and, fourth, what are the physiological consequences of both signaling pathways for transcriptional control in endothelial cells?

GPR55 interacts with integrins and triggers Ca²⁺ signaling via PI3K and Bmx
In the absence of extracellular Ca²⁺, integrins might be involved in the transition of GPR55-originated signaling that yields intracellular Ca²⁺ mobilization from the ER (Fig. 10B). In particular, the inhibitory properties of the crosslinking antibody against vβ3 integrin [LM609 (Charo et al., 1990; Cheresh, 1987)] and the functional-blocking antibodies against the β1 [JB1A (Mohri et al., 1996)] and β3 subunits [B3A (Vignali et al., 1990)] on anandamide/O1602-induced Ca²⁺ signaling fuel the hypothesis that, upon anandamide/O1602 stimulation, GPR55 interacts with vβ3 and 5β1 integrins, which is prerequisite to transmitting outside-in signaling towards intracellular downstream targets (Fig. 10A). This conclusion is further supported by the inhibitory effects of fibronectin (Rupp and Little, 2001) or its integrin-binding motif RGD (Bar-Shavit et al., 1991; Ruoslahti, 2003; Ruoslahti and Pierschbacher, 1987) on anandamide-/O1602-induced Ca²⁺ signaling. The importance of integrin clustering was additionally confirmed by our results that the inhibition of ROCK, which is required for clustering of the integrins (Rodriguez-Fernandez et al., 2001), by Y27632 (Uehata et al., 1997) strongly diminished anandamide-induced Ca²⁺ signaling.

Anandamide-induced Ca²⁺ signaling in Ca²⁺-free medium was sensitive to the inhibition of PI3K, PLC and the Ins(1,4,5)P₃ receptor. Thus, the involvement of PI3K upstream of PLC activation and Ins(1,4,5)P₃-mediated intracellular Ca²⁺ mobilization can be assumed (Fig. 10B). The 70-80 kDa protein Bmx/Etk, a unique tyrosine kinase of the Tec family in endothelial and epithelial cells (Takesono et al., 2002), was identified as mediator between PI3K and PLC. PI3K-induced activation of Bmx/Etk has been described in prostate cancer cells and in breast cancer cells (Bagheri-Yarmand et al., 2001; Qiu et al., 1998). In support of this, the Tec family inhibitor LFM-A13 (Chau et al., 2002; Mahajan et al., 1999), as well as Bmx/Etk-targeted siRNA inhibited anandamide- and O1602-induced Ca²⁺ signaling. Our data that the wide-range tyrosine kinase inhibitors PP1 and PP2 (Liu et al., 1999) abolished anandamide- and O1602-induced Ca²⁺ signaling, and the enhanced tyrosine phosphorylation lead us to suggest that Bmx/Etk undergoes tyrosine phosphorylation by Src family kinases.

Overall, our data point to anandamide-triggered Ca²⁺ signaling as the apparent result of a signaling cascade starting with anandamide binding to GPR55 that clusters with vβ3 and 5β1 integrins to transmit outside-in signaling, leading to PI3K-mediated activation/translocation of Bmx/Etk. After Bmx/Etk phosphorylation by a member of the Src family (Stephens et al., 2001), it activates PLC (Qin and Chock, 2001) that, in turn, instigates Ins(1,4,5)P₃-triggered intracellular Ca²⁺ mobilization from the ER (Fig. 10B).

CB₁-receptor mediated suppression on anandamide-triggered Ca²⁺ signaling is mediated via G_i/o and Syk
Notably, inhibition of G_i/o by pertussis toxin unmasked intracellular Ca²⁺ signaling upon anandamide in the presence of extracellular Ca²⁺, pointing to the involvement of G_i/o downstream of CB₁R (Howlett et al., 2002) in a negative-feedback loop that prevents intracellular Ca²⁺ signaling upon anandamide under these conditions (Fig. 10A).

A potential executor of the inhibitory effects on GPR55-PI3K-Bmx signaling downstream of CB₁R and G_i/o is Syk. This tyrosine kinase with splicing variants of 72 kDa and 68 kDa (Wang et al., 2003) inhibits activation of PI3K in breast cancer cells (Mahabeleshwar and Kundu, 2003) and is expressed in endothelial cells (Inatome et al., 2001). Accordingly, the long form of Syk could be found in the human endothelial cell line used for this study. Moreover, selective inhibition of Syk by piceatannol (Ashikawa et al., 2002) and the respective siRNA unmasked anandamide-triggered Ca²⁺ signaling in the presence of extracellular Ca²⁺. Additionally, wortmannin-resistant tyrosine phosphorylation of a 70-80 kDa protein was found in response to anandamide in the presence but not absence of extracellular Ca²⁺. These data indicate that endothelial Syk indeed executes repression of anandamide-triggered Ca²⁺ signaling downstream to CB₁R and G_i/o (Fig. 10A).

Integrins shield GPR55-triggered signaling from CB₁R-controlled suppression
Neither NMR spectroscopy nor anandamide uptake experiments provided evidence for a Ca²⁺-anandamide chelate to prevent binding/uptake of free anandamide and, thus, Ca²⁺ signaling in the presence of extracellular Ca²⁺. Therefore, we speculated that the interaction of extracellular Ca²⁺ with cell membrane constituents is responsible for the observed inhibitory effect of extracellular Ca²⁺ on anandamide-induced Ca²⁺ signaling in endothelial cells. Hence, the existence of a rather specific Ca²⁺-binding site was further supported by the findings that the Ca²⁺ surrogates Sr²⁺ and Ba²⁺ also prevented anandamide-induced Ca²⁺ signaling in a concentration-dependent manner. Notably, the IC₅₀ value of Ca²⁺ (8 µM) to prevent anandamide-triggered Ca²⁺ signaling is strikingly similar to that found for Ca²⁺-induced inhibition of integrin clustering (Leitinger et al., 2000; Leitinger et al., 1999). Consequently, the involvement of these dynamically regulated transmembrane heteromultimers in unmasking GPR55-triggered Ca²⁺ signaling in the absence of extracellular Ca²⁺ was postulated. Clustered integrins (i.e. in the absence of extracellular Ca²⁺) may protect GPR55-initiated signaling against the repressive effects of CB₁R-originated pathways. In line with this assumption, Mn²⁺, a potent activator of integrin clustering (Mould et al., 1998; Smith and Cheresh, 1990; Thamilselvan et al., 2003), allowed anandamide-induced Ca²⁺ signaling even in the presence of extracellular Ca²⁺, while Mn²⁺ did not induce any Ca²⁺ signal by itself.

In addition, binding of CB₁R to the β1-integrin subunit was released upon anandamide only if integrins were clustered, which further supports the concept that clustering of integrins shields anandamide-induced PI3K-Bmx-PLC-Ca²⁺ signaling from the inhibitory effect of CB₁R activation (Fig. 10). Besides CB₁R, phospho-Syk was also found to interact directly with the cytoplasmic domain of β1-, β2- and β3-integrin subunits (Woodside et al., 2002).

Accordingly, the interaction of CB₁R and Syk with inactive (i.e. unclustered) β1 integrin might either disturb the cytoplasmic integrin rearrangement necessary to transmit the GPR55-initiated signal to its downstream target or allow Syk-mediated inhibition of PI3K owing to the proximity of both proteins (Fig. 10A). However, once integrins cluster either by activation of ROCK, extracellular Mn²⁺ or removal of extracellular Ca²⁺, CB₁R and Syk are released from β1 integrin and no further suppression of PI3K occurs, thus allowing GPR55-originated Ca²⁺ signaling to be transmitted (Fig. 10B). Though the involvement of a G₁₃ in GPR55-triggered Ca²⁺ signaling (Ryberg et al., 2007) seem unlikely in the cells used, owing to the involvement of integrins, it cannot be excluded.

In contrast to anandamide that binds to CB₁R and GPR55, activation of GPR55 only by either O1602 or LPI yielded Ca²⁺ signaling independently of extracellular Ca²⁺, thus indicating that the negative feedback by CB₁R needs to be activated in order to achieve inhibition of the GPR55-originated pathway.

Transcription factor activation upon anandamide depends on the signaling pathway activated
Syk induces tyrosine phosphorylation of IB, causing dissociation, phosphorylation and nuclear translocation of the p65 subunit of NFB (Takada et al., 2003). This is in line with the data indicating that, in the presence of extracellular Ca²⁺, anandamide triggered nuclear translocation of p65 (Fig. 10A). However, under conditions of clustered integrins, anandamide failed to evoke the translocation of p65. The lack of NFB activation under these conditions is consistent with reports pointing to the importance of Ca²⁺ oscillation for the activation of this transcription factor (Hu et al., 1999). By contrast, nuclear translocation of Ca²⁺/calcineurin activated NFAT (Clipstone and Crabtree, 1992; Loh et al., 1996) upon stimulation with anandamide was exclusively obtained under conditions of clustered integrins (Fig. 10B). These findings are in agreement with our previous report that intracellular Ca²⁺ mobilization activates NFAT in human endothelial cells (Bochkov et al., 2002). Notably, although the contribution of NFB and NFAT to gene expression and regulation of cell function often overlaps, there are considerable differences as NFB is frequently referred to as being involved in endothelial cell apoptosis (von Albertini et al., 1998) and the initiation of inflammatory processes (Badrichani et al., 1999), while NFAT has been implicated in angiogenesis (Qin et al., 2006) and cell growth (Li et al., 2005).

Interestingly, anandamide-activated mitogenic signaling results in phosphorylation of Erk1 and Erk2 under all conditions, which is consistent with frequent reports that integrins and cannabinoid receptors induce mitogen-activated protein kinase (MAPK) signaling pathways (Liu et al., 2000). The phenomenon of Erk1 and Erk2 phosphorylation under the various conditions (i.e. with and without Ca²⁺ signaling) might be related to the activation of different pathways, resulting in phosphorylation of Erk1 and Erk2. Consistently, Syk stimulates Erk1 and Erk2 via the Ras-MAPK pathway (Kawakami et al., 2003) but also Ca²⁺ that is known to influence cell proliferation and differentiation (Berridge et al., 2000a; Berridge et al., 2000b) stimulates the Ras-MAPK pathway (Cullen and Lockyer, 2002).

In addition to such alternative activation of distinct transcription factors, the observed GPR55-triggered Ca²⁺ signaling may also mediate Ca²⁺-sensitive cell functions such as activation of nitric oxide synthase, formation of prostaglandins or endothelium-derived hyperpolarizing factor (Graier et al., 1994), proliferation (Munaron, 2002), or permeability (Tiruppathi et al., 2006). Accordingly, depending on the status of integrins, anandamide initiates alternate processes in endothelial cells that may even lead to opposite cell functions (e.g. apoptosis versus cell proliferation).

Although both signaling cascades and their mutual interrelation could be described in herein, important questions still remain to be answered. What are the events that favor one of the two alternative signaling pathways? What are the physiological and/or pathological consequences of the respective signaling cascades? Particularly in view of the known initiators of integrin clustering such as inflammation (Staunton et al., 2006), adhesion (Shattil and Newman, 2004) and migration (Lindbom and Werr, 2002), the physiological implication of the dual signaling induced by anandamide in endothelial cells urges its investigation.

	Materials and Methods

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Chemicals, supplies and materials
Fura-2/AM was from Molecular Probes Europe (Leiden, Netherlands). ³H-anandamide was obtained from APB (Vienna, Austria). Anandamide (arachidonylethanolamide), R-(+)-methandamide, U73122, 2APB, LMF-A13, HU210 and piceatannol were purchased from Tocris Cookson (Northpoint, Avonmouth, Bristol, UK). O1602, O1918 and AM251 were from Cayman Europe (Tallinn, Estonia). SR144528 and rimonabant were kind gifts from Sanofi-Recherche (Monpellier, France). Bradykinin, arachidonic acid, ATP and lysophosphatidylinositol were from Sigma Chemicals (St Louis, MO, USA). RNeasy Mini kit and the EndoFree Plasmid Maxi kit were purchased from Qiagen (VWR, Vienna, Austria). RQ1 RNase-free DNase I and TransFas were from Promega (Mannheim, Germany). RNAguard and random hexamer primers were from APB. First-strand buffer, moloney murine leukemia virus reverse transcriptase, cell culture media and media substitutes were obtained from Life Technologies (Gibco, Invitrogen, Vienna, Austria). Fetal calf serum was from PAA (Linz, Austria). DyNAzyme II DNA polymerase was from Finnzymes Oy (Vienna, Austria). Primers were synthesized at MWG Biotech (Ebersberg, Germany). Restriction enzymes and T4 DNA ligase were purchased at New England BioLabs (Frankfurt, Germany) or Promega. The expression vectors pcDNA 3 and pBudCE4.1 were obtained from Invitrogen (Vienna, Austria). The dNTPs and all other chemicals were from Roth (Karlsruhe, Germany). The CB1R antibody (H-150) was from Santa Cruz Biotechnology (Heidelberg, Germany). Antibodies for β1 integrin (JB1A) and V integrin (SAM-1) were obtained from Biomedica/Chemicon (Millipore, Vienna, Austria) and Protein G Sepharose 4 Fast Flow for immunoprecipitation was from GE Healthcare (Vienna, Austria).

Cell culture and transfection
The human umbilical vein derived endothelial cell line, EA.hy926 (Edgell et al., 1983) at over 45 passages was grown in DMEM containing 10% FCS and 1% HAT. In addition, we have performed experiments in short-term cultured human umbilical vein endothelial cells and human uterine artery endothelial cells (Trenker et al., 2007). If necessary, dishes were coated with 20 µg/ml fibronectin (Short et al., 1998). Transient transfection was performed using Transfast according to the manufacturer's protocol.

Reverse transcriptase-PCR
Total RNA was isolated using the RNeasy Mini Kit according to the manufacturer's protocol and subjected to RT-PCR as described previously (Zoratti et al., 2003). The identity of all PCR products was verified on TAE agarose gels and was further confirmed by automated sequencing of the isolated PCR.

Reduction of gene expression by siRNA
The siRNA against human Bmx/Etk was selected at position 1482 bp of the human mRNA and ligated into pSuppressor using the SalI/XbaI sites. For identification of siRNA-expressing cells, the U6 promotor and Bmx/Etk or Syk siRNA were cloned into pBudCE4.1 encoding mtDsRed (Malli et al., 2003) via KpnI/XhoI at the second multicloning site. The siRNA against human Syk in pSuppressor was purchased by Imgenex. siRNA against GPR55 was from Quiagen (Hs_GPR55_5_HP). The efficiency of siRNAs and of an appropriate negative control was approved by real-time PCR (supplementary material Fig. S2E; see Fig. 4A).

Western blot analysis
EA.hy926 cells were scraped in lysis buffer and disrupted by three cycles of freezing/thawing. Equal amounts of total protein were subjected to western blot and detected by enhanced chemiluminescence as previously described (Schaeffer et al., 2003). If required, membranes were stripped with 0.1 M glycine (pH 2.5), and re-probed.

Immunoprecipitation
Cell lysates were incubated with 2-5 µg (200 µg/ml) of specific antibody overnight. The obtained immune complexes were precipitated with Protein G Sepharose 4 Fast Flow, according to the manufacturer's protocol and subjected to western blot analyses.

Immunohistochemistry
Fixed and permeabilized cells grown on glass cover were incubated overnight at 4°C with anti-NFAT antibody. After incubation with Alexa Fluor 488 secondary antibody (2 µg/ml), the glass cover slips were sealed and fluorescence was visualized using deconvolution microscopy.

Measurement of tyrosine kinase activity
Tyrosine kinase activity was monitored in single endothelial cells using a probe for tyrosine phosphorylation of the CrkII adaptor protein [Picchu-936X (Kurokawa et al., 2001)], as previously described (Schaeffer et al., 2003).

Cytosolic free Ca²⁺ measurements
Cytosolic free-Ca²⁺ was measured using Fura2/AM as previously described (Frieden et al., 2002; Malli et al., 2005; Trenker et al., 2007). [Ca²⁺]_cyto was expressed as (F₃₄₀/F₃₈₀)/F₀.

FRET measurement of endoplasmic free-Ca²⁺ concentration and kinase activity
Tyrosine kinase activity was monitored in single endothelial cells using a probe for tyrosine phosphorylation of the CrkII adaptor protein [Picchu-936X (Kurokawa et al., 2001)] as previously described (Schaeffer et al., 2003). For the assessment of the intraluminal free-Ca²⁺ concentration of the ER, cells were transiently transfected with Cam4_er and measurements were performed as described previously (Frieden et al., 2002; Malli et al., 2003; Malli et al., 2005). FRET-based sensors were excited at 440 nm (440AF21, Omega Optical, Brattleboro, VT, USA) and emission was collected simultaneously at 535 and 480 nm using an optical beam splitter (Dual-View MicroImager, Optical Insights, Visitron Systems, Puchheim, Germany). Kinase activity or alterations in ER Ca²⁺ were expressed as (F₅₃₅/F₄₈₀)/F₀.

	Acknowledgments

 
We thank Beatrix Petschar and Anna Schreilechner for their excellent technical assistance, R. Tsien (University of California, San Diego, CA) for Cam4_er, M. Matsuda and A. Miyawaki (Riken, Wako, Saitama, Japan) for providing the Picchu-936X constructs, Tullio Pozzan (University Padova, Padova, Italy) for mtDsRed, and C. J. S. Edgell (University of North Carolina, Chapel Hill, NC) for the EA.hy926 cells. The expression vector for p65-GFP was a gift from J. Schmid (Medical University of Vienna, Vienna, Austria). We also thank Andrew Irving (University of Dundee, UK) for the 3xHA-GPR55 cDNA. This work was supported by the Austrian Funds (P16860-B09, P20181-B05 and SFB F3010-B05) and by the Franz Lanyar Foundation at the Medical University of Graz. The Institute of Medical Molecular Biology and Biochemistry was supported by the infrastructure program (UGP4) of the Austrian ministry of education, science and culture. Nariman Balenga was supported by a PhD Fellowship in PhD Program Molecular Medicine at the Medical University of Graz.

	Footnotes

 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/10/1704/DC1

^* These authors contributed equally to this work

	References

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