Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol

Abstract

Cb2 is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that 2 distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to proteincoding exon-2. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion in Cb2, exon-1B/exon-2 Cb2 messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-2 Cb2 transcripts were present and receptors could not be detected. To elucidate the function of Cb2 in myeloid leukemia cells, a set of in vitro experiments was carried out using 32D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-2 Cb2 complementary DNA and a myeloid cell line carrying a virus insertion in Cb2 (ie, NFS 78). We demonstrate that a major function of the Cb2 receptor is stimulation of migration as determined in a transwell assay. The cannabinoid receptors belong to the superfamily of 7-transmembrane G protein-coupled receptors (GPCRs). Several GPCRs have been shown to be involved in cell growth and oncogenesis as the result of aberrant expression. [5][6][7] Examples of GPCRs with transforming ability are the ␣1B-adrenergic, 8 thrombin, 9 and serotonin 1c receptors 10 and the receptor encoded by MAS oncogene. 11,12 Our previous observation that Cb2 is a common virus integration site suggests that aberrant expression of this 7-transmembrane receptor may be a critical event in transformation in certain cases of leukemia. 3 The protein-coding region of Cb2 is located on a single exon (exon-2) of approximately 4 kilobases. Recently we identified 2 distinct 5Ј noncoding exons (ie, exon-1A and exon-1B) previously designated exon-1 and exon-1Ј, respectively. 3 In the study presented here we first carried out experiments to investigate Cb2 messenger RNA (mRNA) transcripts and protein expression in leukemic cells with or without retroviral insertion in the Cb2 locus. Secondly, we performed studies to determine the function of the peripheral cannabinoid receptor when overexpressed in myeloid cells.GPCRs have been related to many functions, including cell proliferation, maturation, survival, apoptosis, or migration. 6,13,14 In the present study, we investigated the function of the peripheral cannabinoid receptor when overexpressed on myeloid cells (ie, 32D/G-CSF-R [granulocyte colony-stimulating factor receptor]) in which we overexpressed exon-1B/exon-2 Cb2 splice variant and a myeloid leukemia cell line containing a virus insertion in the Cb2 locus, NFS 78. We also wished to determine which of the large panel of Cb2 ligands that have been identified previously is the true agonist of the receptor. We investigated the effects of natural (␦ 8 24 ) cannabinoids. We show that 2-AG is the most potent agonist for the Cb2 receptor and that a major function of 2-AG is stimulation of migration. We further studied whether 2-AG acts as a chemotactic or chemokinetic age...

Expression and Function of Cannabinoid Receptors CB1 and CB2 and Their Cognate Cannabinoid Ligands in Murine Embryonic Stem Cells

“…We observed induction of Sca-1 expression, as well as induction of PECAM-1 and Flk-1 expression during ES cell differentiation (Fig. 1C), which is in agreement with other published reports [30].…”

“…The chemotaxis assays were performed using 5 µm-pore size and 6.5 mm-diameter Costar Transwells (Corning-Costar, Cambridge, MA), as previously described [30]. Cells were washed twice with Hank's balanced salt solution (HBSS) medium, resuspended in 100 µl medium [Iscove's Modified Dulbecco's Medium (IMDM) plus 0.5% BSA] and placed in the upper chamber of the Transwells.…”

Functional selectivity at G-protein coupled receptors: Advancing cannabinoid receptors as drug targets

“…This view is supported by demonstration that β-arrestin2-null mice evidence enhanced sensitivity to the principal psychoactive phytocannabinoid in marijuana, THC [94]. In contrast, CB2R-selective antagonists/inverse agonists that preferentially activate the β-arrestin2 pathway incite cytoskeletal rearrangements in immune cells [95,96] and in lung airway [97] to modulate chemotaxis and invasion of immune and cancerous cells. Although these studies illustrate the important role of β-arrestin in normal, G-protein-dependent GPCR desensitization mechanisms and do not exemplify β-arrestin-biased signaling per se , the results suggest that the degree of β-arrestin bias in vivo is an important characteristic of potential, CBR-targeted therapeutics.…”

Endogenous lipid activated G protein-coupled receptors: Emerging structural features from crystallography and molecular dynamics simulations

“…The quite specific localization of CB 2 , as well as the fact that CB 2 knock-out mice fail to respond to the immunomodulatory effects of classical cannabinoids (Buckley et al, 2000), suggest that CB 2 receptor ligands would have potential therapeutic applications as immunomodulators for the treatment of inflammation and allergy. Several papers report the role of the CB 2 receptor in modulating leukocyte migration (Franklin and Stella, 2003; Jorda et al, 2002; Kishimoto et al, 2003; Massi et al, 2000), activation (Kishimoto et al, 2004), and antigen processing (McCoy et al, 1999). Additional applications could arise from studies on bone physiology, as blockage of CB 2 has been reported to protect from bone loss in ovariectomized mice (Idris et al, 2005).…”