QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

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

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Waldeck-Weiermair, M. Articles by Graier, W. F. Search for Related Content PubMed PubMed Citation Articles by Waldeck-Weiermair, M. Articles by Graier, W. F.

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

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.

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.

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.

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.

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.

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.