Expression and function of cannabinoid receptors CB1 and CB2 and their cognate cannabinoid ligands in murine embryonic stem cells

Abstract

Background: Characterization of intrinsic and extrinsic factors regulating the self-renewal/division and differentiation of stem cells is crucial in determining embryonic stem (ES) cell fate. ES cells differentiate into multiple hematopoietic lineages during embryoid body (EB) formation in vitro, which provides an experimental platform to define the molecular mechanisms controlling germ layer fate determination and tissue formation.

Methods and findings: The cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) are members of the G-protein coupled receptor (GPCR) family, that are activated by endogenous ligands, the endocannabinoids. CB1 receptor expression is abundant in brain while CB2 receptors are mostly expressed in hematopoietic cells. However, the expression and the precise roles of CB1 and CB2 and their cognate ligands in ES cells are not known. We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES)-derived embryoid bodies. Furthermore, mES cells as well as ES-derived embryoid bodies at days 7 and 14, expressed endocannabinoids, the ligands for both CB1 and CB2. The CB1 and CB2 antagonists (AM251 and AM630, respectively) induced mES cell death, strongly suggesting that endocannabinoids are involved in the survival of mES cells. Treatment of mES cells with the exogenous cannabinoid ligand Delta(9)-THC resulted in the increased hematopoietic differentiation of mES cells, while addition of AM251 or AM630 blocked embryoid body formation derived from the mES cells. In addition, cannabinoid agonists induced the chemotaxis of ES-derived embryoid bodies, which was specifically inhibited by the CB1 and CB2 antagonists.

Conclusions: This work has not been addressed previously and yields new information on the function of cannabinoid receptors, CB1 and CB2, as components of a novel pathway regulating murine ES cell differentiation. This study provides insights into cannabinoid system involvement in ES cell survival and hematopoietic differentiation.

Figure 1. Expression of CB1 and CB2 in Rosa26.6 (Panel A) and E14 (Panel B) ES cells.

Cells were washed with PBS, and then RNA was isolated and analyzed by RT-PCR using specific primers for CB1, CB2, GAPDH and CXCR4. Panel C: RT-PCR analysis of the in vitro differentiation of Rosa26.6 ES cells, using specific primers for GAPDH, Flk-1, PECAM-1 and Sca-1. EBs: Embryoid bodies. ES cells: undifferentiated control ES cells. The following primers were used: GAPDH: 292 bp S 5′-CTCACTGGCATGGCCTTCCG-3′ AS 5′-ACCACCCTGTTGCTGTAGCC-3′ CB1: 430 bp S 5′-CGTGGGCAGCCTGTTCCTCA-3′ AS 5′-CATGCGGGCTTGGTCTGG-3′ CB2: 479 bp S 5′-CCGGAAAAGAGGATGGCAATGAAT-3′ AS 5′CTGCTGAGCGCCCTGGAGAAC-3′ PECAM-1: 260 bp S 5′-GTCATGGCCATGGTCGAGTA-3′ AS 5′-CTCCTCGGCATCTTGCTGAA-3′ Flk-1: 239 bp S 5′-CACCTGGCACTCTCCACCTTC-3′ AS 5′-GATTTCATCCCACTACCGAAAG-3′

Figure 2. The expression of CB1 and CB2 receptors in Rosa26.6 and E14 ES cells as analyzed by Western blot analysis.

Cells were lysed in RIPA buffer and 100 mg of total cell lysates were analyzed by SDS-PAGE, followed by Western blotting with CB1 or CB2 specific antibodies (at a dilution of 1:500). The cell lines 293T and SH-SY5Y were used as negative and positive controls, respectively, for CB1 expression. Actin was used as a control for loading. ES cells: undifferentiated ES cells; EBs: Embryoid bodies at different time points as indicated.

Figure 3. Comparison of endocannabinoid levels in mES cells and EBs at days 7 and 14, when the number of cells in each group is normalized to 10⁷ ( = 1e⁷).

The groups depict the logarithms of each value. AEA, DHEA and EEA endocannabinoid levels were detected but were lower than the limit of quantitation (<0.05 ng/1e⁷ cells) for the number of cells analyzed.

Figure 4. Effects of cannabinoid ligands on the chemotaxis of ES cells and hematopoietic differentiated ES-derived EB cells (EBs-day 10).

Cells were placed in the upper well of the transwell in the presence or absence of specific inhibitors, as indicated. The ligands: 2-AG, Δ⁹-THC, JWH-015 and SDF-1α were placed in the lower chambers. Data show the mean value of 3 independent experiments (mean±SD). Error bars indicate SD. * P values with asterisk (*, P<0.05) show significant differences from control with media alone.

Figure 5. Effects of Δ⁹-THC and cannabinoid inhibitors (AM251 and AM630) on Rosa ES cell survival.

Rosa ES cells were either untreated (as control) or treated with Δ⁹-THC, control DMSO (0.01%), control methanol (0.01%), or with the inhibitors AM251 (for the CB1 receptor) or AM630 (for the CB2 receptor), as indicated. After 48 hours, the cells were analyzed for their viability by light microscopy. This is a representative experiment out of three experiments.

Figure 6. Effects of Δ⁹-THC on the differentiation of ES cells. Rosa ES cells were either untreated or treated with Δ⁹-THC in the presence or absence of cannabinoid inhibitors (AM630 and AM251), as indicated.

After 14 days, the number of EBs was counted. Data represent the mean value of 3 independent experiments (mean±SD). * P values with asterisk (*, P<0.05) show significant differences from ES cells.