1. Anabolic Role of Cannabinoid Receptors in Bone
Targeting Cannabinoid Receptors in Osteoblasts.
Jahan Marcu, Ph.D
February 22nd, 2013
RDdirector@gsdiagnostics.com
2. The Endocannabinoid System (ECS)
• Discovered with the help of phytocannabinoids (Cannabis
Sativa, Voacanga Africana, Rhodenderon Anthpogonoides,
Radula Marginata, and Helichrysum Umbraculigerum)
• Consists of endocannabinoids, cannabinoid receptors (GPCRs
CB1 and CB2), and enzymes for synthesis and catabolism
• “Eat, sleep, relax, forget, and protect”
• Clinical Endocannabinoid Deficiency (CECD; Russo 2004)
3. Cannabinoid Receptor Genetics
• CNR1 encodes CB1 localized on 6q14-q15 (Hoehe et al. 1991)
• CNR2 encodes CB2 localized on 1p36.1 (Valk et al. 1997)
Mutation Description Disease Associations References
CNR1 Schizophrenia, substance abuse Zhang et al., 2004;
Trinucleotide AAT repeat disorders, Parkinson’s disease,
Comings et al., 1997;
repeat in 3’ inverse relation between number Ujike et al., 2002;
UTR of repeats and working memory Barrero et al., 2005,
performance Ruiz-Contreras et al.,
2013
CNR1 SNPs rs6454674; rs806380;
Mutations in cannabinoid receptors may underlie many diseases et al., 2006;
Substance abuse Hopfer
or rs806377; rs1049353; disorders, depression, anxiety Zuo et al., 2009; Zhang
Haplotypes rs806379; rs1535255; not affect amino acid sequence
These mutations do and eating et al., 2004; Juhasz et
rs2023239;rs806368; disorders, obesity, schizophrenia al., 2009; Lazary et al.,
rs806369; rs4707436; the ECSattention deficit disorder to 2009; Ho et al., 2011,
Treating diseases of , may depend on our ability
rs12720071; rs3505747
understand the cannabinoid receptors Okahisha et al., 2011,
Mutombo et al., 2012,
Marcos et al., 2012
CNR2 SNPs rs2502992, rs2501432 Low bone mineral density or Huang et al., 2009;
osteoporosis associated in at Karsak et al., 2005;
least 3 distinct human Karsak et al., 2009;
populations Yamada et al., 2007
4. CB1 and CB2 KO and Bone Loss
A)
Tam 2006 Ofek 2006
5. CB1 and Bone Formation
Tam 2006
Tam 2008
Tam 2008
6. CB Receptor Effects on Cell Proliferation
• Plant cannabinoids increase CFUs
Claims no CB1 expression in MSCs
• Did not look at CB1 specific agonists
• Cannabinoids may be involved in
recruiting MSCs
Scutt 2007
7. CB1 and CB2 KO
• Presence of cannabinoid receptors
• Ablation of cannabinoid receptors disrupts
normal bone development
• Human Studies (Karsak 2005; Karsak 2009;
Yamada 2007)
• CB1CB2 KO Mice
8. Which ‘tools’ can we use to study the effect of
targeting CB1 and/or CB2?
9. Increased Bone Mass with lower levels of proliferation
•CB1CB2 KO mice display increase
bone mass phenotype
•Rat primary cells treated with
SR141716A or SR144528 (A-C)
•WT and CB1CB2 KO primary cells
(D)
How can there be an increase in
the amount of bone, if there is
significantly less cells that provide
bone matrix?
Compensation is a possibility; is
there any evidence of a plausible
mechanism?
10. Hypothetical Signaling Pathway
Perhaps ERK, SMAD, and AKT signaling underlie the observed effects in the bones of
the genetically modified mice (CB1CB2KO).
Adapted from Ross et al. 2005, Kokolus et al. 2009, Liedert et al. 2006, Bikle 2008
11. Hypothesis
Targeting CB1 and CB2 receptors may cause an
increase in the activity of important signaling
molecules, including ERK, SMAD, and AKT
proteins.
12. MC3T3-E1 cells treated with SR141716A, SR144528, and in
combination, assayed for pERK
Bars represent means ± SEM of 3 separate experiments; *P < 0.05 and **P < 0.01.
13. Targeting cannabinoid receptors may cause differential
increases in ERK1 (p42) and ERK2 (p44) activity
No significant difference
between p42 and p44.
Differential MAPK
signaling may not be
directly responsible
14. MC3T3-E1 cells treated with SR141716A, SR144528,
and in combination, assayed for pSMAD158 activity.
15. MC3T3-E1 cells treated with SR141716A, SR144528,
and in combination, assayed for pAKT activity
16. Summary
•The trabecular bone volume of female CB1/2 KO mice is
increased compared to WT, yet the osteoblasts of these
animals proliferate at a significantly lower rate.
•Targeting cannabinoid receptors can significantly alter the
activity of MAP kinases and the SMAD158 complex.
•This suggests that cannabinoids can influence bone
morphogenic signaling pathways, and therefore play a
significant role in osteoblast differentiation and function.
•ERK and SMAD signaling may provide a mechanism for
the observed bone phenotype.
17. Future Directions
• Experiments include using MAPK inhibitors to affect
pSMAD158 activity or cell proliferation, and RT-
PCR analysis could provide reliable data on the levels
of Runx2.
19. Acknowledgements
•Dr. Mary Abood & Lab Members
•Department of Anatomy and Cell Biology
Voacanga Africana •Center for Substance Abuse
•Plant Kingdom Cannabis Sativa
•DA009158 and PA state tobacco funding
Radula Marginata
Rhodenderon Anthpogonoides
Helichrysum Umbraculigerum
As early as 5,000 years ago Cannabis was noted for its effects on the central nervous system. This often manifested in the form of pain relief, appetite stimulation, and sedation (Iverson, 2000). Evidence for the medicinal use of Cannabis goes back to the emperor Chen Nung (the father of Chinese agriculture), a discoverer of medicinal plants, and also taught his people how to cultivate grains. Chen Nung is believed to be the author of the oldest known Chinese pharmacopoeia, in which, he writes about the medical use of Cannabis for rheumatism, menstrual fatigue, malaria, constipation, and absentmindedness. Medicinal Cannabis preparations were widely used in western medicine during the 19th century (at least 200 preparations). At the time it was recognized that preparations of Cannabis distributed through pharmacieswere variable. As the active ingredient was not known, quality control was virtually impossible, and this is in part why the plant fell out of use. One practical issue was that the cannabis, cultivated in Morroco, China, India ,could take as long as a year to reach the west. And storage conditions were not optimal, in say Dutch sailing vessel from the 15th century.During the Victorian era, many alkaloids were extracted from plants, for their unique properties. These plant chemists were successful because the alkaloids they were targeting are water soluble organic bases that form crystalline solids when combined with acids. Among the medicinal compounds isolated in the 19th century were quinine, morphine, and cocaine. These were major advances in plant chemistry. The molecules on the cannabis plant, however, are almost completely insoluble in water. The chemical nature of cannabinoids prevented early Victorian scientists from making efficient extracts of these polar compounds. The active ingredient, delta9-Tetrahydrocannabinol (Δ9-THC), was not isolated and identified until 1964(Mechoulam and Gaoni, 1965). In the 1990s, researchers made discoveries essential for the establishment of the cannabinoid research field. By the end of the decade scientists discovered two distinct cannabinoid receptors (i.e., CB1 and CB2), isolated endogenous cannabinoids (e.g.,Anandamide and 2-Arachidonylglycerol), synthesized a cadre of ligands, and generated cannabinoid receptor knockout mice (i.e., CB1 KO) (Gerard et al., 1990; Matsuda et al., 1990; Zimmer et al., 1999). Efforts to identify and clone the CB1 receptor demonstrate that it is one of the most abundant proteins in the brain. Thus, cannabinoid receptors became an attractive target for drug development. The availability of synthetic THC and novel analogs has allowed researchers to begin characterizing the role of this neuronal G-protein coupled receptor (GPCR). The sum of all knowledge on how cannabinoid receptors and their ligands work together in mammals is referred to as the endocannabinoid system (ECS).Today we know that cannabinoids are found on many plants, THC has not been found on plants, but the stuff of THC-the precurors, such as CBG were found on some of these plants. Additionally, cloves, black pepper also contain what is refferred to as a dietary cannabinoid. Beta-caryophyllene.The reason we have a receptor of cannabinoids, is not because there is some plant out there which makes cannabiniods. That is just a qwuark of nature. We instructions for the building these receptors in genes because it is part of a larger system with a natural THC-like compound synthesized by our bodies.What does the ECS do?Eat: HIV/AIDS and is the basis for pure THC being available by prescription for the cancer and aids patients.Less recognized but still promising are effects we can recognize; sleep, relax, references to sedation and pain relief. Forget: Have you ever forgotten anything? Happened to me once and I am thankful for it because it may indicate I have a healthy brain with a reasonable amount of CBR activation…However, when you really think about, having a system to extinguish Memories or information that is NOT useful or harmful, is just as important as remembering things that are useful. If I remember every face I saw on SEPTA this morning, my brain would burst, trying to remember all that information. This is basis for anecdotal reports and a call for clinical trials with cannabinodis for the treatment of PTSD—it used to be called battle fatigue, before that shell shock. (All quiet on the western front). Protect: Cannabinoids protect neurons from excitotoxicity and other insults. In addition to being wonderful antioxidants, phytocannabinoids and some synthetics can reduce nerve damage in experimental assays involving animal brains being challenged with say nerve gas, blunt trauma, ischemia (heart attack). A clinical endocannabinoid deficiency has been coined…which describes diseases such as fibromyalgia, migraines, and eating disorders—may have an underlying defect in cannabinoid receptors or endocannabinoid production.Many uses:convulsion, menstrual cramps, asthma, childbirth, throat infection, cough, insomnia, migraine, opium withdrawal, appetite, diarrhea,Etc….In the last few years we have made the most astonishing discoveries…And I believe the future of therapeutics, depends heavily on understanding this system.
TOP:C57/Bl6 CB1 KO bone formation and mineralaposition rateBOTTOM: Bone formation and mineral apposition rateModel of CB1 regulation of bone formation by modulating adrenergic signaling. Take home message is that activation of CB1 receptor in bone sympathetic nerve terminals, restrains NE release. Thus, alleviating the tonic supression of bone formation by the sympathetic nervous system.So there are a few things that need to happen when there is a bone injury. Cells need to be turned on. Osteoblasts and Osteoclasts begin differentiating and proliferatingAlso, cells need to migrate to the site to remodel the bone.For instance, in Science I read a study about how MSC travel to sites and differentiate into osteoblasts upon arrival.SO the data we are seeing may be the result of poor osteoblasts differentiation or migration.Research has shown that cannabinoids can affect proliferation and migration.
Scutt 2007: Nonspecific agonist can increase colony forming units of BMCs isolated from Wistar rats. Implying cannabinoids can, perhaps, stimulate the proliferation of bone cells.Treatment of CFU-f cultures with a variety of naturally occurring cannabinoids, including cannabidivarine(CBDV), cannabigerol (CBG), cannabinol (CBN), cannabidiol (CBD), THC, and tetrahydrocannabivarin (THCV), all at 10 lM, produced a stimulation of colonyformation in all cases. This varied from a relatively small stimulation of around 20%in the case of CBG to as muchas 100% after treatment with CBDV or THCV. This increase in colony number was also reflected by a parallelincrease in the number of colonies that were positive for both ALP expression and collagen synthesis
What about THC?An interesting molecule, but it activates both receptors without much discrimination. We need compounds that can seletively target either CB1 or CB2?WIN: We know that indoles are unique compounds but they share a similar characteristic with THC in that they activate both receptors?
Description of assay, part of larger projectMethods?Primary immature osteoblasts were isolated from the calvaria (parietal bones) of 3-5 day old animals by sequential collagenase digestions as previously described (Arnott et al., 2007). Prior to experiments, the cells were cultured in a 75cm2 flasks in standard α-MEM media. The cells were left to adhere and proliferate until the flask was nearly confluent before using in experiments. Proliferation Assays with Primary OsteoblastsPrimary cells from rats isolated from the parietal bones of neonates were plated at 2,000 cells/well in MEM with 10% FBS. After 24 hours the cells were washed with plain media, and the media was replaced with MEM with 1%FBS and cells were treated with 1,3,5,7 and 10 µM of SR141716A, SR144528, or a combination of the two compounds. Cells culture was stopped at 48 hours post treatment. Cell proliferation was measured using a WST-1 proliferation kit from Caymen (Cat# 10008883).Primary cells isolated from parietal bones of WT and CB1/CB2 KO mice were plated at 2,000 cells/well in 96 well plates. After 2 weeks cells numbers were measured using a CyQuant kit from Invitrogen (Cat#C35006). A calibration curve was also generated with WT and CB1/2 KO primary cells to determine cells numbers.If you usually have 12 people to clean a building, and then you have 3. Those people simply work harder. But we need to know how these cells are communicating. How could this increase be occurring?
Figure 1.3. Hypothetical pathway of cannabinoid signaling cascade in osteoblasts. Cannabinoid receptors may influence the activity of MAP kinases, p-SMAD158 complex, and AKT in mesenchymal stem cells (i.e., immature osteoblasts, MC3T3-E1 cell line).Competiting or complimentary hypothesisWhy AKT? survial, proliferationWhy ERK? Proliferation, diffrentiationWhy SMAD158? Differentiation, activity PDK-1 Phosphoinositide-dependent kinase-1F-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls the transcription of DNA.
Figure 3.3. MC3T3-E1 cells treated with SR141716A, SR144528, and in combination, assayed for pERK.A) Representative images from western blot analysis. B) Graph of SR141716A induced ERK activity. C) Graph of SR144528 induced ERK activity. D) Graph of SR141716A and SR144528 combination induced ERK activity. Bars represent means ± SEM of 3 separate experiments; *P < 0.05 and **P < 0.01.
Targeting cannabinoid receptors may cause differential increases in ERK1 (p42) and ERK2 (p44) activity. A) Quantification of SR141716A p42 and p44 activity. B) Quantification of SR144528 p42 and p44 activity. C) Quantification of SR141716 and SR144528 p42 and p44 activity.The MAP kinase pathway plays a central role in cell proliferation control. ERk1/2 necessary for G1 to S phase transition.ERK-1 -/- cells have an inhibition of DNA synthesisERK2 is important for embyronic development, embryonic lethal -/-Functional differences between them remains controversial.An enahcement of ERK2 signaling may impart a growth advantage, while too much ERK1 will lead to cell cycle arrest.Same scale
Figure 3.5. MC3T3-E1 cells treated with SR141716A, SR144528, and in combination, assayed for pSMAD158 activity. A) Representative images from western blot analysis. B) Graph of SR141716A for induced SMAD158 phosphorylation. B) Graph of SR144528 for induced SMAD158 phosphorylation. C) Graph of SR141716A and SR144528 combination for SMAD158 phosphorylation. Bars represent means ± SEM of at least 3 separate experiment*P < 0.05. Same scale!!
MC3T3-E1 cells treated with SR141716A, SR144528, and in combination, assayed for pAKT activity. A) Representative images from western blot analysis. B) Graph of SR141716A 4528 induced pAKT activity. C) Graph of SR144528 induced pAKTphosphorylation. D) Graph of SR141716A and SR144528 induced pAKTphosphorylation. Bars represent means ± SEM of 3 separate experiments; *P < 0.05 and **P < 0.01. Same scale
AKT= proliferationSMAD are important for tissue architectureSignal transduction through BMPRs results in mobilization of members of the SMAD family of proteins. The signaling pathways involving BMPs, BMPRs and Smads are important in the development of the heart, central nervous system, and cartilage, as well as post-natal bone development. And the formation of the endochonral skeleton
Hypothetical pathway of cannabinoid signaling cascade in osteoblasts. Cannabinoid receptors may influence the activity of MAP kinases, p-SMAD158 complex, and AKT in mesenchymal stem cells (i.e., immature osteoblasts, MC3T3-E1 cell line).Fix PKA
New mechanisms on how to manipulate the receptors. New things are still ready/waiting to be discovered.
