The endocannabinoid system consists mainly of 2-arachidonoylglycerol and anandamide, as well as cannabinoid receptor type 1 and type 2 (CB2). Based on previous studies, we hypothesized that a circulating peptide previously identified as osteogenic growth peptide (OGP) maintains a bone-protective CB2 tone. We tested OGP activity in mouse models and cells, and in human osteoblasts. We show that the OGP effects on osteoblast proliferation, osteoclastogenesis, and macrophage inflammation in vitro, as well as rescue of ovariectomy-induced bone loss and prevention of ear edema in vivo are all abrogated by genetic or pharmacological ablation of CB2. We also demonstrate that OGP binds at CB2 and may act as both an agonist and positive allosteric modulator in the presence of other lipophilic agonists. In premenopausal women, OGP circulating levels significantly decline with age. In adult mice, exogenous administration of OGP completely prevented age-related bone loss. Our findings suggest that OGP attenuates age-related bone loss by maintaining a skeletal CB2 tone. Importantly, they also indicate the occurrence of an endogenous peptide that signals via CB2 receptor in health and disease.
By combining pharmacological and mouse genetic genetics strategies the authors show a clear interaction between the cannabinoid receptor CB2 and osteogenic growth peptide (OGP) in the control of bone remodeling and bone mass. They document that OGP attenuates bone loss by maintaining a skeletal CB2 tone, and it does so by allosterically binding to the CB2 receptor. These novel observations should allow further investigations on cannabinoid based strategies for skeletal diseases.
In the past two decades, the endocannabinoid (EC) system was discovered and characterized, after identifying receptors for ∆9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana and hashish. The actions of THC are mediated mainly by the cannabinoid receptor type 1 (CB1) and type 2 (CB2). Both are seven-transmembrane domain, class A G-protein-coupled receptors (GPCRs) sharing 44% overall identity (68% similarity considering the transmembrane regions alone). CB1 is expressed predominantly in neurons and to a lesser extent in several other tissues. In health, CB2 is expressed mainly in immune and bone cells. Owing to the relatively high expression of CB2 in immune cells, it has been hypothesized that this receptor mediates the immunosuppressive effects of phyto- and synthetic cannabinoids. It is widely believed that CB1 and CB2 are principally targeted by two lipid-derived endogenous ligands, N-arachidonoylethanolamine (AEA or anandamide) and 2-arachidonoylglycerol (2-AG). These ECs derive from arachidonic acid and are present mainly in the brain as well as in a variety of peripheral tissues. Quantitative wise, the main EC is 2-AG.
It has been suggested that 2-AG and AEA are synthesized and function ‘on demand’ because (i) non-stimulated CB2-deficient mice have a normal phenotype and (ii) the ECs are rapidly degraded. By contrast, studies in mice and humans have established that the functional activity of CB2 protects the skeleton against age-related bone loss, suggesting the existence of a ‘CB2 tone’ maintained by more stable ligands. Other findings provide room for such an additional agonist. Pharmacological studies dealing with anandamide and 2-AG binding at CB2 reported rather low binding affinities at the µM range, more specifically, one order of magnitude lower than that of THC, suggesting that there could be a more specific and potent endogenous ligand. Furthermore, physiological results from several research groups suggest that anandamide and 2-AG have opposing effects on CB2 signaling. In this study, we aimed at finding a putative additional endogenous specific and potent CB2 peptidic agonist that maintains a CB2 tone throughout life and protects against bone loss.
Many proteins and peptides were shown to be GPCR ligands that play a major role in modulating GPCR activation and expression via direct crosstalk. Targeting of GPCRs and other receptors by non-peptidic lipophilic ligands (e.g. acetylcholine) results in short-term activation, whereas the tonal activity of some of these receptors (e.g. muscarinic and nicotinic) is maintained by protein or peptide ligands with higher binding affinities. In GPCRs, small-molecule lipophilic ligands have their binding pocket in transmembrane loops, whereas proteins and peptides bind to the extracellular domains. Indeed, previous studies have shown the existence of a series of endogenous peptide cannabinoid ligands, termed ‘pepcans’. One of them, ‘pepcan-12,’ is a 12-amino-acid peptide exhibiting a binding affinity to CB1 in the nanomolar range. Consistent with the binding of peptides and lipophilic ligands to different domains, pepcan-12 potently targets an allosteric site at CB1 and interacts rather weakly with the ‘classical’ CB1 binding pocket, characterized by the high-affinity binding of the synthetic cannabinoids CP55940 and WIN55212–2. This same peptide was later identified as a CB2 positive allosteric modulator (PAM). Pepcan-12 has been identified at significant levels in the brain, peripheral tissues, and plasma following ischemia/reperfusion injury, suggesting its pathophysiological role in modulating CB2 activation. However, pepcan-12 has no reported actions in bone cells and does not seem to decline with age, thus bringing into question its involvement in preserving bone mass during aging.
To assess the possibility that the skeletal protective actions of CB2 are maintained by an endogenous peptide agonist, we performed an in-depth literature search, looking for a peptide/protein that can bind to CB2 or to any other GPCR in bone cells. In January 2017, a search in PubMed looking for ‘(peptide or protein) and (‘Gi protein’ or ‘G(i)’ or ‘GPCR’ or ‘G*coupled receptor’) and (osteoblast* or osteoclast* or osteocyt*)’ resulted in 96 articles. Out of these 96 articles, there were only 26 Gi agonists and 10 were endogenous proteins/peptides. Furthermore, 4 out of the 10 endogenous peptides/proteins were reported to bind to an unknown Gi or to CB2 in bone cells. Out of these four remaining peptides/proteins, two were affected by aging and only one, named osteogenic growth peptide (OGP), played a positive role in maintaining bone mass. We could not find any report on the putative receptor for OGP. We therefore proceeded with this peptide to assess whether it binds to and activates CB2.
In 1992, we reported that a C-terminal 14-amino acid peptide derived from the gene for histone H4, H4(90–103) in mice and H4(89–102) in humans, is mitogenic to osteoblasts, stimulates bone formation in rodents and rescues ovariectomy-induced bone loss. This peptide was therefore called OGP(1-14). OGP(1-14) is physiologically present in the serum at nano- to micromolar concentrations, with most of it complexed to α2-macroglobulin. Upon dissociation from this complex, it is proteolytically cleaved, thus generating its 5-amino-acid cellular activator OGP referred to here as OGP. This pentapeptide targets a pertussis toxin-sensitive GPCR and consequently activates a Gi-protein – Erk1/2 – Mapkapk2 – CREB cascade. Although OGP has never been attributed anti-inflammatory properties (a hallmark of CB2 agonists), there is a striking resemblance between the signal transduction triggered by this pentapeptide and that of CB2 agonists in bone cells. Moreover, CB2 agonists and OGP have an identical mitogenic dose-response activity in osteoblasts and can stimulate bone formation and bone mass in intact rats and osteoporotic mice. Interestingly, the serum levels of biologically active OGP decline with age during adulthood. We therefore hypothesized that OGP is a CB2 agonist, and that its serum levels drive an age-related reduction in CB2 tone, thus alleviating the protective role of CB2 during aging. Indeed, in a series of ex vivo and in vivo skeletal and inflammatory experiments, we show here that OGP shares the activities of highly potent CB2 agonists and that these actions depend on the presence of an active CB2 receptor. Our present results suggest for the first time that OGP is an endogenous peptide agonist at CB2.
Bone loss is an inherent consequence of aging in both mice and humans. Because CB2 expression was shown to protect against age-related bone loss, we first investigated whether age induces a decline in CB2 expression or the levels of its classical endogenous agonists in wild type (WT) animals. We therefore examined the skeletal changes in male and female mice between the ages of 3 and 6 months, when a dramatic age-related bone loss occurs, and measured the bone levels of AEA, 2AG, and arachidonic acid as well as CB2 expression in 3 vs. 6-month-old male and female mice. As expected, our results show a significant age-related decline in bone mass in both sexes. In 3 months, the trabecular BV/TV decreased by 61 and 75% in males and females, respectively, due to significant 64 and 70% decrease in the bone volume. Importantly, we did not find any difference in CB2 expression or in AEA, 2AG, and arachidonic acid tissue levels in either gender. These results support our working hypothesis that another endogenous agonist is responsible for maintaining long-term CB2 activation, and that age-related bone loss is caused by a decline in the levels of this agonist.
Attenuation of forskolin (FSK)-stimulated cAMP levels is a hallmark of Gi-c
