Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 1. CP55940-induced enhanced phosphorylation of CB2 receptors, increased GRK5 expression levels, and reduced GRK2 expression levels in rat PFCx. Rats were injected with CP55940 (0.05 mg/kg, intraperitoneally) once a day for 7 days. After decapitation, the brains were collected, and PFCx was dissected. A, phosphorylated proteins were separated and detected as described under “Experimental Procedures.” 30 g of isolated phosphorylated protein was used in Western blot detection. B–E, CB2 receptor and GRK protein levels were evaluated by Western blot. Proteins (8 g) were resolved by SDS-PAGE, and antibodiesforCB2receptor(AandB),GRK5(C),GRK6(D),andGRK2(E)wereusedtodetecttheproteinsofinterest.RepresentativeWesternblotsareshown,and integratedopticaldensitywascalculatedasdescribedunder“ExperimentalProcedures.”-Actinwasusedasaloadingcontrol.F,GRK5,GRK6,andGRK2mRNA levels were evaluated by qRT-PCR as described under “Experimental Procedures.” **, p 0.01; *, p 0.05, significant effect of CP55940 treatment compared with vehicle-treated controls. The data represent mean S.E. (error bars) (n 6–8).

Article Snippet: MAY 31, 2013 • VOLUME 288 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15713 by guest on June 28, 2015 http://w w w .jbc.org/ D ow nloaded from Lentivirus and Stable Transduction of shRNAs in CLU213 Cells—GRK5 shRNA (rat), -arrestin 2 shRNA (rat), CB1 shRNA (rat), CB2 shRNA (rat), control shRNA lentiviral particles, Polybrene, and puromyocin were purchased from Santa Cruz Biotechnology, Inc. Optimal transduction conditions were determined, and transfection of cells with lentiviral particles was conducted as described previously (3).

Techniques: Phospho-proteomics, Expressing, Injection, Isolation, Western Blot, SDS Page, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 3. GP1a, a selective CB2 receptor agonist, up-regulated GRK5 via CB2 receptors, -arrestin 2, and ERK1/2 signaling in CLU213 cells. A, cells were incubated with either vehicle (ethanol 0.01% final concentration), 1 nM GP1a, or 15 nM ACEA for 72 h. qRT-PCR was used to show the effect of selective CB1 or CB2 receptor agonist treatment on GRK5 mRNA levels. **, p 0.01 significant effect of GP1a treatment compared with vehicle-treated controls. B, cells stably transfected with control, CB1, or CB2 shRNA lentiviral particles were treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to examine the effect of CB1 or CB2 receptor knockdown on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment on GRK5 mRNA levels in control or CB1 shRNA lentivirus-treated cells compared with vehicle-treated controls. ##, p 0.01, significant effect of CB2 shRNA lentivirus transfection on the GP1a-induced up-regulation of GRK5. *, p 0.05, significant effect of GP1a treatment in CB2 shRNA-transfected cells compared with vehicle-treated/CB2 shRNA-transfected cells. C, cells were pretreated with vehicle or 200 nM PD198306, potent ERK1/2 inhibitor, and then treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to show the effect of PD198306 pretreatment on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment compared with vehicle-treated controls. ##, p 0.01, significant effect of PD198306 pretreatment on GP1a-induced increases in GRK5 mRNA levels compared with vehicle-treated controls. D, cells were stably transfected with control or -arrestin 2 shRNA lentivirus particles and then treated with vehicle or 1 nM GP1a for 72 h. qRT-PCR was used to examine the effect of -arrestin 2 knockdown on GP1a-induced increases in GRK5 mRNA levels. **, p 0.01, significant effect of GP1a treatment on GRK5 mRNA levels in control shRNA lentivirus-treated cells compared with vehicle-treated controls. ##, p 0.01, significant effect of -arrestin 2 shRNA lentivirus transfection on the GP1a-induced up-regulation of GRK5. *, p 0.05, significant effect of GP1a treatment in -arrestin 2 shRNA-transfected cells compared with vehicle-treated -arrestin 2 shRNA-transfected cells. The data represent mean S.E. (error bars) (n 3).

Article Snippet: MAY 31, 2013 • VOLUME 288 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15713 by guest on June 28, 2015 http://w w w .jbc.org/ D ow nloaded from Lentivirus and Stable Transduction of shRNAs in CLU213 Cells—GRK5 shRNA (rat), -arrestin 2 shRNA (rat), CB1 shRNA (rat), CB2 shRNA (rat), control shRNA lentiviral particles, Polybrene, and puromyocin were purchased from Santa Cruz Biotechnology, Inc. Optimal transduction conditions were determined, and transfection of cells with lentiviral particles was conducted as described previously (3).

Techniques: Incubation, Concentration Assay, Quantitative RT-PCR, Stable Transfection, Transfection, Control, shRNA, Knockdown

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 4. GRK5 is necessary for the CP55940 and GP1a-induced up-reg- ulation of 5-HT2A receptors in CLU213 cells. Cells were transfected with control or GRK5 shRNA lentiviral particles as described under “Experimental Procedures.” A, qRT-PCR to show reduced GRK5 mRNA levels in cells treated with GRK5 shRNA lentiviral particles. B, Western blot to show reduced GRK5 protein levels in cells treated with GRK5 shRNA lentiviral particles. A and B, **, p 0.01, significant effect of GRK5 shRNA compared with control shRNA- treated cells. C and D, cells stably transfected with control or GRK5 shRNA lentiviral particles were treated with either vehicle, 1 nM CP55940, or 1 nM GP1a for 72 h. C, qRT-PCR to show the effect of GRK5 knockdown on CP55940- induced increases on 5-HT2A receptor mRNA levels. **, p 0.01, significant effect of CP55940 treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lentivirus transfection on the CP55940-induced up-regulation of 5-HT2A receptors. **, p 0.01, significant effect of CP55940 treatment in GRK5 shRNA-transfected cells compared with vehicle-treated GRK5 shRNA-transfected cells. D, qRT-PCR to show the effect of GRK5 knockdown on GP1a-induced increases in 5-HT2A receptor mRNA levels. **, p 0.01, significant effect of GP1a treatment on 5-HT2A receptor mRNA levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lenti- virustransfectionontheGP1a-inducedup-regulationof5-HT2Areceptors.**, p 0.01, significant effect of GP1a treatment in GRK5 shRNA-transfected cells compared with vehicle-treated GRK5 shRNA-transfected cells. The data rep- resent mean S.E. (error bars) (n 3).

Article Snippet: MAY 31, 2013 • VOLUME 288 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15713 by guest on June 28, 2015 http://w w w .jbc.org/ D ow nloaded from Lentivirus and Stable Transduction of shRNAs in CLU213 Cells—GRK5 shRNA (rat), -arrestin 2 shRNA (rat), CB1 shRNA (rat), CB2 shRNA (rat), control shRNA lentiviral particles, Polybrene, and puromyocin were purchased from Santa Cruz Biotechnology, Inc. Optimal transduction conditions were determined, and transfection of cells with lentiviral particles was conducted as described previously (3).

Techniques: Transfection, Control, shRNA, Quantitative RT-PCR, Western Blot, Stable Transfection, Knockdown

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 6. CB2 and GRK5 are necessary for the CP55940 and GP1a-in- duced increases in 5-HT2A receptor-mediated Ca2 release in CLU213 cells. Cells were stably transfected with either control, CB2, or GRK5 shRNA lentiviral particles as described under “Experimental Procedures.” A, cells sta- bly transfected with control or CB2 shRNA lentiviral particles were pretreated with either vehicle, 1 nM CP55940, 1 nM GP1a, or 15 nM ACEA for 72 h. Cells were then preincubated with 10 nM SB242084 for 20 min and then stimulated with 0.4 nM 5-HT. B, cells stably transfected with control or GRK5 shRNA lenti- viral particles were pretreated with either vehicle, 1 nM CP55940, or 15 nM GP1a. Cells were then preincubated with 10 nM SB242084 for 20 min and then stimulated with 0.4 nM 5-HT. **, p 0.01, significant effect of CP55940 pre- treatment/5-HT stimulation or GP1a pretreatment/5-HT stimulation in con- trol shRNA-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GP1a pretreatment/5-HT stimulation in control shRNA-transfected cells compared with GP1a pretreatment/5-HT stimulation in CB2 or GRK5 shRNA-transfected cells. @@, p 0.01, significant effect of CP55940 pretreatment/5-HT stimulation in control shRNA-transfected cells compared with CP55940 pretreatment/5-HT stimulation in CB2 or GRK5 shRNA-transfected cells. The data represent mean S.E. (error bars) (n 3).

Article Snippet: MAY 31, 2013 • VOLUME 288 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15713 by guest on June 28, 2015 http://w w w .jbc.org/ D ow nloaded from Lentivirus and Stable Transduction of shRNAs in CLU213 Cells—GRK5 shRNA (rat), -arrestin 2 shRNA (rat), CB1 shRNA (rat), CB2 shRNA (rat), control shRNA lentiviral particles, Polybrene, and puromyocin were purchased from Santa Cruz Biotechnology, Inc. Optimal transduction conditions were determined, and transfection of cells with lentiviral particles was conducted as described previously (3).

Techniques: Stable Transfection, Transfection, Control, shRNA

Journal: Journal of Biological Chemistry

Article Title: G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

doi: 10.1074/jbc.m113.454843

Figure Lengend Snippet: FIGURE 7. GRK5 mediates GP1a-induced increases in CB2 phosphoryla- tionandenhanced-arrestin2/ERKinteractioninCLU213cells.Toexam- ine the role of GRK5 in the GP1a-induced increases in CB2 receptor phospho- rylation and -arrestin 2/ERK interaction, cells stably transfected with control of GRK5 shRNA lentiviral particles were treated with either vehicle or 1 nM GP1a for 72 h. A, phosphorylated proteins were separated and detected as described under “Experimental Procedures.” 30 g of isolated phosphoryl- ated protein was used in Western blot detection. **, p 0.01, significant effect of GP1a treatment on CB2 phosphorylation levels in control shRNA lentivirus-transfected cells compared with vehicle-treated controls. ##, p 0.01, significant effect of GRK5 shRNA lentivirus transfection on the GP1a- induced increases in CB2 phosphorylation. The data represent mean S.E. (n 3). B, CB2 receptor protein levels in whole cell lysate were evaluated by Western blot as described under “Experimental Procedures.” Representative Western blots are shown, and -actin was used as a loading control. The data represent mean S.E. (n 3). C, co-immunoprecipitation was used to exam- inetheroleofGRK5inGP1a-inducedincreasesin-arrestin2/ERKinteraction. Co-immunoprecipitation was conducted as described under “Experimental Procedures.” Negative controls (lanes 9–12) received the same concentration of -arrestin 2 antibody except that the coupling resin was replaced with control agarose resin that is not amine-reactive. All columns were incubated with whole cell lysate (300 g) from vehicle-treated (lanes 1 and 3) or GP1a- treated (lanes 4 and 6) cells. Cell lysate (15 g of protein) was used as an input control (lanes 1–4). The data represent mean S.E. (error bars) (n 4).

Article Snippet: MAY 31, 2013 • VOLUME 288 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15713 by guest on June 28, 2015 http://w w w .jbc.org/ D ow nloaded from Lentivirus and Stable Transduction of shRNAs in CLU213 Cells—GRK5 shRNA (rat), -arrestin 2 shRNA (rat), CB1 shRNA (rat), CB2 shRNA (rat), control shRNA lentiviral particles, Polybrene, and puromyocin were purchased from Santa Cruz Biotechnology, Inc. Optimal transduction conditions were determined, and transfection of cells with lentiviral particles was conducted as described previously (3).

Techniques: Stable Transfection, Transfection, Control, shRNA, Isolation, Western Blot, Phospho-proteomics, Immunoprecipitation, Concentration Assay, Incubation

Journal: The FASEB Journal

Article Title: Inhibition of FAAH suppresses RANKL ‐induced osteoclastogenesis and attenuates ovariectomy‐induced bone loss partially through repressing the IL17 pathway

doi: 10.1096/fj.202200911R

Figure Lengend Snippet: PF inhibits osteoclast differentiation – CB1 or CB2 independence. (A–C) BMMs were infected with the lentivirus carrying FAAH‐specific shRNA or the control lentivirus and then treated with RANKL for 3 days, and the mRNA and protein expression of CB1 and CB2 were detected by qPCR and Western blot respectively. (D–F) BMMs were treated with indicated concentrations of PF (0, 0.5, 1, and 2.5 μM) with or without stimulation by 75 ng/ml RANKL for 3 days, and the mRNA and protein expression of CB1 or CB2 were determined using qPCR and Western blot respectively. # p < .05, ## p < .01, compared with the control groups; ns, non‐significant, compared with the RANKL groups. (G and H) BMMs were treated with PF (2.5 μM), AM251 (0.1 μM), and AM630 (0.1 μM) as indicated in the presence of 75 ng/ml RANKL. After 5–7 d, trap staining was conducted, and multinucleated (>3 nuclei) cells were quantified (scale bar = 400 μm). (I–K) BMMs were treated with PF (2.5 μM), AM251 (0.1 μM), and AM630 (0.1 μM) as indicated in the presence of 75 ng/ml RANKL for 3 days, and the mRNA and protein expression of osteoclast‐specific genes were determined using qPCR and Western blot respectively. # p < .05, ## p < .01, compared with the RANKL groups; ns, non‐significant, compared with the RANKL + PF groups. All data are presented as the mean ± SD. N = 3 per group.

Article Snippet: Primary antibodies targeting FAAH, CB1, and CB2 were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Infection, shRNA, Control, Expressing, Western Blot, Staining

Journal: Current opinion in anaesthesiology

Article Title: An overview of the cannabinoid type 2 (CB2) receptor system and its therapeutic potential

doi: 10.1097/ACO.0000000000000616

Figure Lengend Snippet: Components of the endogenous cannabinoid system. The cannabinoid (CB) receptors CB1 and CB2 belong to the G-protein-coupled receptor superfamily, coupled to Gi/o proteins and, under certain conditions, coupled to Gs. CB1 receptors are expressed mainly in the brain and CB2 are expressed mostly in the peripheral immune system and in the CNS in the hippocampal CA2/3 pyramidal neurons and glial cells. The endocannabinoid system also includes two arachidonic acid derivatives ligands (anandamide and 2-arachidonoylglycerol) [1], two enzymes responsible for synthesizing endogenous ligands (1,2-diacylglcerol lipase and phospholipase A), and two enzymes responsible for the metabolism of endogenous ligands (fatty acid amide hydrolase and monoglyceride lipase).

Article Snippet: [ 105 ] The authors discuss the possibility that plasma levels were suboptimal, although the plasma levels obtained were greater than those in preclinical studies demonstrating efficacy in the rat model. AstraZeneca CB2 ligand, AZD1940, is a peripherally restricted CB1/CB2 receptor agonist.

Techniques:

Journal: Current opinion in anaesthesiology

Article Title: An overview of the cannabinoid type 2 (CB2) receptor system and its therapeutic potential

doi: 10.1097/ACO.0000000000000616

Figure Lengend Snippet: Cannabinoid type 2 (CB2) receptors are expressed in reactive microglia in different neuroinflammatory and neurodegenerative conditions such as neuropathic pain and Alzheimer’s disease. The figure depicts 3D immunofluorescence confocal images of the microglial marker ionized calcium binding adaptor molecule (1 Iba1) (red) and the immunosignal of cannabinoid type 2 (CB2) receptor (green) in microglia; the colocalization of CB2 and microglia is shown in yellow. No substantial CB2 expression is seen in the healthy microglia (a), but increased expression of CB2 is seen in reactive microglia (arrow, b). Note the change from a highly branched and ramified morphology under normal physiological conditions to an amoeboid form in the presence of neuroinflammation. Treatment with a selective CB2 agonist, 1-(3-benzyl-3-methyl-2,3-dihydro-1-benzofuran-6-yl)carbonyl) piperidine (MDA7) restored microglial function and normal morphology (c). Scale bar = 10 μm.

Article Snippet: [ 105 ] The authors discuss the possibility that plasma levels were suboptimal, although the plasma levels obtained were greater than those in preclinical studies demonstrating efficacy in the rat model. AstraZeneca CB2 ligand, AZD1940, is a peripherally restricted CB1/CB2 receptor agonist.

Techniques: Immunofluorescence, Marker, Binding Assay, Expressing

Journal: Current opinion in anaesthesiology

Article Title: An overview of the cannabinoid type 2 (CB2) receptor system and its therapeutic potential

doi: 10.1097/ACO.0000000000000616

Figure Lengend Snippet: (a) Modulation of interleukein-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) pathways by a selective CB2 agonist, 1-(3-benzyl-3-methyl-2,3-dihydro-1-benzofuran-6-yl)carbonyl) piperidine (MDA7) (generated by Ingenuity pathway analysis using comparison analysis between the 6 arrays). The brightness of node colors is proportional to the fold changes of gene expression levels. Color indicates up-regulated (red) and down-regulated (green) genes. In addition fold change expression bar charts of the six arrays have been included for each gene. In the IL-1 pathway, a complex is formed including IL-1R-associated kinases (IRAK) and the adapter protein MyD88. IRAK is rapidly phosphorylated and associates with TNF receptor-associated factor 6 (TRAF6); this association is necessary for downstream IL-1-induced translocation of signaling molecules to the nucleus, which ultimately leads to expression of genes that mediate inflammation and, frequently, tissue destruction. MDA7 acts by inhibiting IL-1 and TNF-α decreased expression of MyD88 and TNF receptor-associated factor 2 (TRAF2). (b) Signaling cascades initiated via toll-like receptor 2 (TLR2)- and TLR4-dependent activation and its modulation by MDA7. In addition fold change expression bar charts of the six arrays have been included for each gene. Engagement of TLR2 on the cell surface as a heterodimer with either TLR1 or TLR6 leads to recruitment of the adaptor protein MyD88 and interaction with TIR-domain-containing adaptor protein (TIRAP) via death-domain interactions. Phosphorylated IL-1 receptor-associated kinase (IRAK), together with TNF receptor-associated factor 6 (TRAF6), dissociates from the receptor, and then TRAF6 interacts with TAK1 binding protein 1 (TAB1)—induces autophosphorylation of the transforming growth factor β (TGFβ)-activating kinase (TAK1), and TAB2. This leads to phosphorylation of the IκBα-kinase (IKK) complex (IKKα, IKKβ, and IKKγ) and mitogen-activated protein kinases (MAPK), such as c-Jun NH2-terminal kinase (JNK), inducing the nuclear translocation of NF-κB and subsequent induction of target genes such as TNFα and ILs. The transcriptional activity of NF-κB is tightly regulated by its association with the inhibitory IκB that sequesters NF-κB in the cytosol. Beneficial effects of MDA7 include modulation of general genes that will ultimately inhibit phosphorylation of IκB proteins by the IKKs and prevention of IκB degradation. TLR4-MyD88-independent pathway activation involves signaling through the Toll-interleukin-1 receptor (TIR) adaptor TRIF (also known as TICAM1), TRIF-related adaptor molecule (TRAM; also known as TICAM2), TRAF3, receptor-interacting protein (RIP) and the transcription factor interferon regulatory factor 3 (IRF3). The green and red bar charts and shading represent the respective differential gene modulation (repression or induction, respectively) by MDA7. The numbers represent fold change. (Reproduced from Xu et al.[73] with permission).

Article Snippet: [ 105 ] The authors discuss the possibility that plasma levels were suboptimal, although the plasma levels obtained were greater than those in preclinical studies demonstrating efficacy in the rat model. AstraZeneca CB2 ligand, AZD1940, is a peripherally restricted CB1/CB2 receptor agonist.

Techniques: Generated, Expressing, Translocation Assay, Activation Assay, Binding Assay, Activity Assay