Cellular and Molecular Neurobiology

, Volume 31, Issue 6, pp 921–930

The Non-Psychoactive Plant Cannabinoid, Cannabidiol Affects Cholesterol Metabolism-Related Genes in Microglial Cells

  • Neta Rimmerman
  • Ana Juknat
  • Ewa Kozela
  • Rivka Levy
  • Heather B. Bradshaw
  • Zvi Vogel
Original Paper

Abstract

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that is clinically used in a 1:1 mixture with the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) for the treatment of neuropathic pain and spasticity in multiple sclerosis. Our group previously reported that CBD exerts anti-inflammatory effects on microglial cells. In addition, we found that CBD treatment increases the accumulation of the endocannabinoid N-arachidonoyl ethanolamine (AEA), thus enhancing endocannabinoid signaling. Here we proceeded to investigate the effects of CBD on the modulation of lipid-related genes in microglial cells. Cell viability was tested using FACS analysis, AEA levels were measured using LC/MS/MS, gene array analysis was validated with real-time qPCR, and cytokine release was measured using ELISA. We report that CBD significantly upregulated the mRNAs of the enzymes sterol-O-acyl transferase (Soat2), which synthesizes cholesteryl esters, and of sterol 27-hydroxylase (Cyp27a1). In addition, CBD increased the mRNA of the lipid droplet-associated protein, perilipin2 (Plin2). Moreover, we found that pretreatment of the cells with the cholesterol chelating agent, methyl-β-cyclodextrin (MBCD), reversed the CBD-induced increase in Soat2 mRNA but not in Plin2 mRNA. Incubation with AEA increased the level of Plin2, but not of Soat2 mRNA. Furthermore, MBCD treatment did not affect the reduction by CBD of the LPS-induced release of the proinflammatory cytokine IL-1β. CBD treatment modulates cholesterol homeostasis in microglial cells, and pretreatment with MBCD reverses this effect without interfering with CBD’s anti-inflammatory effects. The effects of the CBD-induced increase in AEA accumulation on lipid-gene expression are discussed.

Keywords

Cholesteryl ester Cannabidiol Cholesterol Diacylglycerol-O-acyl transferase Lipid droplet N-arachidonoyl ethanolamine Protein tyrosine phosphatase non receptor 22 Sterol-O-acyl transferase Sterol 27-hydroxylase Microglia 

Supplementary material

10571_2011_9692_MOESM1_ESM.eps (2.1 mb)
Supplementary material 1 (EPS 2127 kb)

References

  1. Bisogno T, Hanus L, De Petrocellis L, Tchilibon S, Ponde DE, Brandi I, Moriello AS, Davis JB, Mechoulam R, Di Marzo V (2001) Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Br J Pharmacol 134:845–852PubMedCrossRefGoogle Scholar
  2. Biswas KK, Sarker KP, Abeyama K, Kawahara K, Iino S, Otsubo Y, Saigo K, Izumi H, Hashiguchi T, Yamakuchi M, Yamaji K, Endo R, Suzuki K, Imaizumi H, Maruyama I (2003) Membrane cholesterol but not putative receptors mediates anandamide-induced hepatocyte apoptosis. Hepatology 38:1167–1177PubMedCrossRefGoogle Scholar
  3. Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F (1990) Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 27:229–237PubMedCrossRefGoogle Scholar
  4. Bradshaw HB, Rimmerman N, Krey JF, Walker JM (2006) Sex and hormonal cycle differences in rat brain levels of pain-related cannabimimetic lipid mediators. Am J Physiol Regul Integr Comp Physiol 291:R349–R358PubMedCrossRefGoogle Scholar
  5. Butovsky E, Juknat A, Elbaz J, Shabat-Simon M, Eilam R, Zangen A, Altstein M, Vogel Z (2006) Chronic exposure to Delta9-tetrahydrocannabinol downregulates oxytocin and oxytocin-associated neurophysin in specific brain areas. Mol Cell Neurosci 31:795–804PubMedCrossRefGoogle Scholar
  6. Carrier EJ, Auchampach JA, Hillard CJ (2006) Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. Proc Natl Acad Sci USA 103:7895–7900PubMedCrossRefGoogle Scholar
  7. Cornicelli JA, Gilman SR, Krom BA, Kottke BA (1981) Cannabinoids impair the formation of cholesteryl ester in cultured human cells. Arteriosclerosis 1:449–454PubMedCrossRefGoogle Scholar
  8. De Petrocellis L, Di Marzo V (2010) Non-CB1, non-CB2 receptors for endocannabinoids, plant cannabinoids and synthetic cannabimimetics: focus on G-protein coupled receptors and transient receptor potential channels. J Neuroimmune Pharmacol 5:103–121PubMedCrossRefGoogle Scholar
  9. De Petrocellis L, Ligresti A, Moriello AS, Allara M, Bisogno T, Petrosino S et al (2011) Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes. Br J Pharmacol. doi:10.1111/j.1476-5381.2010.01166.x
  10. Fujimoto T, Ohsaki Y, Cheng J, Suzuki M, Shinohara Y (2008) Lipid droplets: a classic organelle with new outfits. Histochem Cell Biol 130:263–279PubMedCrossRefGoogle Scholar
  11. Gajate C, Mollinedo F (2001) The antitumor ether lipid ET-18-OCH(3) induces apoptosis through translocation and capping of Fas/CD95 into membrane rafts in human leukemic cells. Blood 98:3860–3863PubMedCrossRefGoogle Scholar
  12. Gallily R, Even-Chena T, Katzavian G, Lehmann D, Dagan A, Mechoulam R (2003) Gamma-irradiation enhances apoptosis induced by cannabidiol, a non-psychotropic cannabinoid, in cultured HL-60 myeloblastic leukemia cells. Leuk Lymphoma 44:1767–1773PubMedCrossRefGoogle Scholar
  13. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80PubMedCrossRefGoogle Scholar
  14. Heid HW, Moll R, Schwetlick I, Rackwitz HR, Keenan TW (1998) Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases. Cell Tissue Res 294:309–321PubMedCrossRefGoogle Scholar
  15. Ignatowska-Jankowska B, Jankowski M, Glac W, Swiergel AH (2009) Cannabidiol-induced lymphopenia does not involve NKT and NK cells. J Physiol Pharmacol 60(Suppl 3):99–103PubMedGoogle Scholar
  16. Jan TR, Su ST, Wu HY, Liao MH (2007) Suppressive effects of cannabidiol on antigen-specific antibody production and functional activity of splenocytes in ovalbumin-sensitized BALB/c mice. Int Immunopharmacol 7:773–780PubMedCrossRefGoogle Scholar
  17. Juknat A, Pietr M, Kozela E, Rimmerman N, Levy R, Coppola G, Geschwind D, Vogel Z (2011) Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and Δ9-tetrahydrocannabinol in BV-2 microglial cells. Br J Pharmacol (accepted)Google Scholar
  18. Kaczocha M, Glaser ST, Deutsch DG (2009) Identification of intracellular carriers for the endocannabinoid anandamide. Proc Natl Acad Sci USA 106:6375–6380PubMedCrossRefGoogle Scholar
  19. Kaczocha M, Glaser ST, Chae J, Brown DA, Deutsch DG (2010) Lipid droplets are novel sites of N-acylethanolamine inactivation by fatty acid amide hydrolase-2. J Biol Chem 285:2796–2806PubMedCrossRefGoogle Scholar
  20. Kaplan BL, Springs AE, Kaminski NE (2008) The profile of immune modulation by cannabidiol (CBD) involves deregulation of nuclear factor of activated T cells (NFAT). Biochem Pharmacol 76:726–737PubMedCrossRefGoogle Scholar
  21. Kimmel AR, Brasaemle DL, McAndrews-Hill M, Sztalryd C, Londos C (2010) Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins. J Lipid Res 51:468–471PubMedCrossRefGoogle Scholar
  22. Kozela E, Pietr M, Juknat A, Rimmerman N, Levy R, Vogel Z (2010) Cannabinoids Delta(9)-tetrahydrocannabinol and cannabidiol differentially inhibit the lipopolysaccharide-activated NF-kappaB and interferon-beta/STAT proinflammatory pathways in BV-2 microglial cells. J Biol Chem 285:1616–1626PubMedCrossRefGoogle Scholar
  23. Kozela E, Lev N, Kaushansky N, Eilam R, Rimmerman N, Levy R, Ben-Nun A, Juknat A Vogel Z (2011) Cannabidiol inhibits pathogenic T-cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice. Br J Pharmacol. doi:10.1111/j.1476-5381.2011.01379.x
  24. Lee CY, Wey SP, Liao MH, Hsu WL, Wu HY, Jan TR (2008) A comparative study on cannabidiol-induced apoptosis in murine thymocytes and EL-4 thymoma cells. Int Immunopharmacol 8:732–740PubMedCrossRefGoogle Scholar
  25. Ligresti A, Cascio MG, Pryce G, Kulasegram S, Beletskaya I, De Petrocellis L, Saha B, Mahadevan A, Visintin C, Wiley JL, Baker D, Martin BR, Razdan RK, Di Marzo V (2006) New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis. Br J Pharmacol 147:83–91PubMedCrossRefGoogle Scholar
  26. Liu J, Wang L, Harvey-White J, Osei-Hyiaman D, Razdan R, Gong Q, Chan AC, Zhou Z, Huang BX, Kim HY, Kunos G (2006) A biosynthetic pathway for anandamide. Proc Natl Acad Sci USA 103:13345–13350PubMedCrossRefGoogle Scholar
  27. Liu DZ, Hu CM, Huang CH, Wey SP, Jan TR (2011) Cannabidiol attenuates delayed-type hypersensitivity reactions via suppressing T-cell and macrophage reactivity. Acta Pharmacol Sin 31:1611–1617CrossRefGoogle Scholar
  28. Maccarrone M, Dainese E, Oddi S (2010) Intracellular trafficking of anandamide: new concepts for signaling. Trends Biochem Sci 35:601–608PubMedCrossRefGoogle Scholar
  29. Malfait AM, Gallily R, Sumariwalla PF, Malik AS, Andreakos E, Mechoulam R, Feldmann M (2000) The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc Natl Acad Sci USA 97:9561–9566PubMedCrossRefGoogle Scholar
  30. McHugh D, Hu SS, Rimmerman N, Juknat A, Vogel Z, Walker JM et al (2010) N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor. BMC Neurosci 11:44PubMedCrossRefGoogle Scholar
  31. McKallip RJ, Lombard C, Fisher M, Martin BR, Ryu S, Grant S, Nagarkatti PS, Nagarkatti M (2002) Targeting CB2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease. Blood 100:627–634PubMedCrossRefGoogle Scholar
  32. McKallip RJ, Jia W, Schlomer J, Warren JW, Nagarkatti PS, Nagarkatti M (2006) Cannabidiol-induced apoptosis in human leukemia cells: a novel role of cannabidiol in the regulation of p22phox and Nox4 expression. Mol Pharmacol 70:897–908PubMedCrossRefGoogle Scholar
  33. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279PubMedCrossRefGoogle Scholar
  34. Oddi S, Fezza F, Pasquariello N, De Simone C, Rapino C, Dainese E, Finazzi-Agro A, Maccarrone M (2008) Evidence for the intracellular accumulation of anandamide in adiposomes. Cell Mol Life Sci 65:840–850PubMedCrossRefGoogle Scholar
  35. Pietr M, Kozela E, Levy R, Rimmerman N, Lin YH, Stella N et al (2009) Differential changes in GPR55 during microglial activation. FEBS Lett 583:2071–2076PubMedCrossRefGoogle Scholar
  36. Pikuleva IA (2006) Cholesterol-metabolizing cytochromes P450. Drug Metab Dispos 34:513–520PubMedCrossRefGoogle Scholar
  37. Rakhshan F, Day TA, Blakely RD, Barker EL (2000) Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells. J Pharmacol Exp Ther 292:960–967PubMedGoogle Scholar
  38. Raz A, Goldman R (1976) Effect of hashish compounds on mouse peritoneal macrophages. Lab Invest 34:69–76PubMedGoogle Scholar
  39. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461PubMedCrossRefGoogle Scholar
  40. Rimmerman N, Hughes HV, Bradshaw HB, Pazos MX, Mackie K, Prieto AL, Walker JM (2008) Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line. Br J Pharmacol 153:380–389PubMedCrossRefGoogle Scholar
  41. Rimmerman N, Bradshaw HB, Kozela E, Levy R, Juknat A, Vogel Z (2011) Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line which is devoid of caveolin-1. Br J Pharmacol. doi:10.1111/j.14765381.2011.01380.x
  42. Ruiz-Valdepenas L, Martinez-Orgado JA, Benito C, Millan A, Tolon RM, Romero J (2011) Cannabidiol reduces lipopolysaccharide-induced vascular changes and inflammation in the mouse brain: an intravital microscopy study. J Neuroinflammation 8:5PubMedCrossRefGoogle Scholar
  43. Russo E, Guy GW (2006) A tale of two cannabinoids: the therapeutic rationale for combining tetrahydrocannabinol and cannabidiol. Med Hypotheses 66:234–246PubMedCrossRefGoogle Scholar
  44. Sarker KP, Maruyama I (2003) Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts. Cell Mol Life Sci 60:1200–1208PubMedGoogle Scholar
  45. Sarker KP, Biswas KK, Yamakuchi M, Lee KY, Hahiguchi T, Kracht M, Kitajima I, Maruyama I (2003) ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death. J Neurochem 85:50–61PubMedCrossRefGoogle Scholar
  46. Smyth GK (2005) Limma: linear models for microarray data. In: Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W (eds) Bioinformatics and computational biology solutions using R and bioconductor. Springer, New York, pp 397–420CrossRefGoogle Scholar
  47. Stella N (2010) Cannabinoid and cannabinoid-like receptors in microglia, astrocytes and astrocytomas. Glia 58:1017–1030PubMedCrossRefGoogle Scholar
  48. Tambuyzer BR, Ponsaerts P, Nouwen EJ (2009) Microglia: gatekeepers of central nervous system immunology. J Leukoc Biol 85:352–370PubMedCrossRefGoogle Scholar
  49. Ueda N, Tsuboi K, Uyama T (2010) Enzymological studies on the biosynthesis of N-acylethanolamines. Biochim Biophys Acta 1801:1274–1285PubMedGoogle Scholar
  50. Watanabe K, Kayano Y, Matsunaga T, Yamamoto I, Yoshimura H (1996) Inhibition of anandamide amidase activity in mouse brain microsomes by cannabinoids. Biol Pharm Bull 19:1109–1111PubMedGoogle Scholar
  51. Weiss L, Zeira M, Reich S, Har-Noy M, Mechoulam R, Slavin S, Gallily R (2006) Cannabidiol lowers incidence of diabetes in non-obese diabetic mice. Autoimmunity 39:143–151PubMedCrossRefGoogle Scholar
  52. Wu HY, Jan TR (2010) Cannabidiol hydroxyquinone-induced apoptosis of splenocytes is mediated predominantly by thiol depletion. Toxicol Lett 195:68–74PubMedCrossRefGoogle Scholar
  53. Wu HY, Chu RM, Wang CC, Lee CY, Lin SH, Jan TR (2008) Cannabidiol-induced apoptosis in primary lymphocytes is associated with oxidative stress-dependent activation of caspase-8. Toxicol Appl Pharmacol 226:260–270PubMedCrossRefGoogle Scholar
  54. Wu HY, Chang AC, Wang CC, Kuo FH, Lee CY, Liu DZ, Jan TR (2010) Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes. Toxicol Appl Pharmacol. doi:10.1016/j.physletb.2003.10.071
  55. Yang Q, Liu HY, Zhang YW, Wu WJ, Tang WX (2010) Anandamide induces cell death through lipid rafts in hepatic stellate cells. J Gastroenterol Hepatol 25:991–1001PubMedCrossRefGoogle Scholar
  56. Zimmerman S, Zimmerman AM, Cameron IL, Laurence HL (1977) Delta1-tetrahydrocannabinol, cannabidiol and cannabinol effects on the immune response of mice. Pharmacology 15:10–23PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Neta Rimmerman
    • 1
    • 2
  • Ana Juknat
    • 2
  • Ewa Kozela
    • 2
  • Rivka Levy
    • 1
  • Heather B. Bradshaw
    • 3
  • Zvi Vogel
    • 1
    • 2
  1. 1.The Department of NeurobiologyWeizmann Institute of ScienceRehovotIsrael
  2. 2.The Dr. Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Department of Physiology and Pharmacology, Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael
  3. 3.The Department of Psychological and Brain SciencesIndiana UniversityBloomingtonUSA

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