Thursday, January 31, 2013

Cannabinoid Receptors as Target for Treatment of Osteoporosis: A Tale of Two Therapies

Cannabinoid Receptors as Target for Treatment of Osteoporosis: A Tale of Two Therapies

clipped from article:

THE SKELETAL ENDOCANNABINOID SYSTEM

Cannabinoids and their Receptor Expression in Bone

A number of recent studies reported that endocannabinoids and their metabolising enzymes are present in the skeleton. AEA and 2-AG are present in the bone marrow and within the metabolically-active trabecular compartment, at levels in the same magnitude as the brain [12, 91]. Both osteoblasts and osteoclasts are capable of producing AEA and 2-AG in culture [12, 91, 92]. Complementary to these findings, a number of cell types within the bone micro-environment including osteoblasts, osteoclasts, osteocytes, stromal cells and adipocytes are found to express the endocannabinoid metabolising enzymes NAPE-phospholipase D, fatty acid amide hydrolyse, diacylglycerol lipases and monoacylgycerol lipase (our unpublished data; [12, 93]). The cannabinoid receptors CB1 and CB2 and a number of closely related receptors and channels such as GPR55 and TRPV1 are found in the skeleton. CB1 receptors are known to be expressed on nerve fibres intervening bone [12, 94] and on cells of the immune system within the BM compartment [2, 30]. We and others reported that CB1 receptors are also detected on osteoblasts, osteoclasts and BM derived adipocytes at both protein and mRNA levels [95, 96]. CB2 receptors on the other hand are highly expressed on peripheral blood mononucleated cells and immune cells including macrophage, monocytes, B and T lymphocytes [26, 30, 97-100]. Osteoblasts, osteoclasts and osteocytes also express CB2 receptors at significantly higher level than that reported for CB1 [31, 32, 93, 96]. Recent studies reported that bone cells also express GPR55 and TRPV1 which are known to be targeted by endocannabinoids and synthetic cannabinoid ligands [36, 96, 101, 102, 111].

Cannabinoid Inverse Agonists/Antagonists as AntiResorptive Agents

The prevention and treatment of excessive bone resorption is based on the use of anti-resorptive agents such as Bisphosphonates and calcitonin. Anti-resorptive drugs are a class of therapeutic agents that selectively/specifically target and inhibit osteoclast differentiation and function with minimal direct activity toward osteoblasts (Reviewed in [103, 104]). We have found that CB1 and CB2 expression on osteoclast and their BM precursor cells is highly up regulated in ageing mice and following oestrogen deficiency in adult mice [95]. To determine the relevance of this finding, we studied the effects of CB1 receptor inactivation on bone loss in ovariectomised mice, a well established model of acute bone loss following oestrogen deficiency [105]. We reported that mice lacking CB1 receptors are protected from ovariectomy-induced bone loss and exhibited reduced osteoclast number and bone resorption in comparison to wild type littermates [32]. We also showed that CB1 deficiency in healthy mice results in accelerated bone growth in neonate and high bone mass in adult mice due to reduced osteoclast number and bone resorption [32]. Surprisingly, the number of osteoblasts and all parameters of bone formation remains unaffected by CB1 deficiency during growth and early adulthood [32, 95]. In contrast, CB2 deficient mice of similar age showed no significant changes in bone mass [31, 106]. Based on these findings, it is clear that CB1 receptors regulate osteoclastic bone resorption in adult mice and that under conditions of increased bone turnover these receptors may regulate bone loss. Interestingly, recent studies have reported that adult mice deficient in the orphan receptor GPR55 display increased peak bone mass due to a significant defect on osteoclastogenesis but the number of osteoblasts remains unaffected [107]. The skeletal abnormalities reported in GPR55 KO mice were remarkably similar to those observed in CB1 deficient mice [32]. Bearing in mind that GPR55 is activated by a number of cannabinoids ligands including endocannabinoids and the CB1 selective agonist AM251 [108, 109], it is likely that GPR55 is involved in the regulation of endocannabinoids action in osteoclastic bone resorption.
Over recent years, we have extensively tested whether pharmacological blockage of cannabinoid receptors may be of value in the prevention of acute bone loss. In our studies, we demonstrated that treatment with the CB1 selective inverse agonist/antagonist AM251 and the CB2 selective inverse agonist/antagonist AM630 reduced osteoclast number and bone resorption in vivo and protected against ovariectomy induced bone loss in adult mice [32, 95]. Other workers reported that the novel CB2 selective antagonist Sch.036 prevented inflammation and bone damage in arthritic mice [110]. Interestingly, genetic inactivation of CB2 receptors in adult mice only partially protected from bone loss due to ovariectomy [106]. This suggests that prevention of bone loss following treatment with CB2 selective inverse agonists/antagonists such as AM630 and Sch.036 may occur at least in part by an effect on CB1 receptors. Nevertheless, these findings together confirm the anti-resorptive capabilities of cannabinoid receptor - in particular CB1 - blockage in animal models of acute bone loss (Fig. 33).
Fig. (3)
Hypothetical model for prevention and treatment of postmenopausal osteoporosis using cannabinoid ligands. Cannabinoid receptors play a role in regulating osteoclast and osteoblast differentiation and activity in the ageing skeleton. ...
A number of in vitro studies have recently shed light on the mechanisms by which cannabinoid receptor blockage regulate osteoclastogenesis. For example, the CB1 selective inverse agonists/antagonists AM251 and Rimonabant© and the CB2 selective inverse agonist/antagonist AM630 are capable of exerting direct inhibitory effects on osteoclast formation, fusion, polarisation and activity [32]. Recent studies in our laboratories demonstrated that cannabinoid receptors also regulate osteoclastogenesis by indirectly influencing “osteoblast-osteoclast coupling” (Fig. 22). For example, we showed that osteoclast formation is significantly reduced in osteoblast – bone marrow co-cultures in which the osteoblasts were prepared from CB1KO mice [95, 106]. Further studies showed that osteoblast cultures generated from CB1KO mice express less RANKL therefore confirming the reduced capabilities of these osteoblast to support osteoclast formation normally [95]. Cannabinoid receptor activation using the endocannabinoids AEA and 2-AG, CB1/2 synthetic agonist CP55,940 and CB2 selective agonist JWH133 and HU308 enhance osteoclast number, increase osteoclast size and multinuclearity and stimulate bone resorption [32, 92, 106]. As with CB1 and CB2 selective agonists, TRPV1 and GPR55 receptor agonists are also capable of increasing osteoclast number in human and mouse cultures [96, 107]. A recent study in our laboratories showed that the TRPV1 agonist capsaicin enhances osteoclast formation, whereas the antagonist capsazepine suppressed osteoclast and osteoblast differentiation and function in vitro and inhibited ovariectomy induced bone loss in mice by reducing indices of bone resorption and bone formation [111]. These results together with earlier findings reported by Rossi and colleagues [96] clearly demonstrate that pharmacological blockade of TRPV1 ion channels is capable of inhibiting osteoclastic bone resorption and as a result protects against bone loss in animal model of osteoporosis [96, 111]. Bearing in mind that cannabinoid receptors, TRPV1 and GPR55 are known to co-exist in a number of cells including osteoclasts and osteoblasts [107, 112-115], it is possible that some of cannabinoids actions may actually be mediated via TRPV1, GPR55 and/or other unknown mechanism(s). In keeping with this, we and others found evidence that activation of CB2 – using the CB2 selective agonists HU308 and ajulemic acid - inhibits osteoclast formation under certain conditions by an unknown mechanism(s) [31, 106, 107, 116]. Regardless of this, it is clear that cannabinoid receptor inverse agonists/antagonists show value as anti-resorptive agents for the prevention of osteoporosis and other bone diseases characterised by increased osteoclast activity (Fig. 33).

Endocannabinoids and Synthetic Cannabinoid Agonists as Bone Anabolic Agents

Bone formation plays a critical role in age-related bone loss and the pathogenesis of a number of bone diseases including postmenopausal and drug-induced osteoporosis [51]. In recent years, extensive research into pathways involved in the regulation of osteoblast differentiation and activity has led to the discovery of a number of bone anabolic agents that stimulate bone formation such as exogenous PTH (also known as teriparatide or Forteo©) (Reviewed in [117]). Endocannabinoids and their receptors are involved in the regulation of osteoblast differentiation and bone formation (Fig. 22). The first evidence supporting a potential effect of cannabinoids on bone formation came from two independent studies examining the role of leptin on food intake and energy metabolism. Ducy et al. showed that leptin, acting on the hypothalamus, influences bone remodelling by negatively regulating bone formation [87]. Complementing this finding, Ravinet et al. reported that genetic inactivation of CB1 receptors reduces leptin levels and body weight in experimental animals [118]. Together these studies suggest that CB1 receptors influence - at least in part - the effects of leptin in osteoblast activity and bone formation (Fig. 22). We and others showed that the endocannabinoids AEA and 2-AG, the synthetic CB1/2 agonist CP55,940 and CB2 selective agonists HU308 and JWH133 stimulate early differentiation of BM derived osteoblast precursors and enhance bone nodule formation in osteoblast cultures in vitro (Fig. 22) [31, 93, 119]. Conversely, treatment with the CB receptor inverse agonist/antagonist AM251 suppresses osteoblast number and function acting on CB1 receptors [95, 106, 119]. We and others also showed that BM stromal cells from CB1 and CB2 deficient mice had a significantly reduced capacity to form mineralised bone nodules when cultured in osteogenic medium and had lower expression of the osteoblast specific alkaline phosphatase and core binding factor alpha1 (Cbfa1) [31, 95], indicating that endocannabinoids and their receptors are capable of exerting a cell autonomous effect on osteoblast and their precursors (Fig. 22).
In most of osteoporotic patients, sustained bone loss is mainly due to significant reduction in osteoblast number and bone formation (Fig. 33) [65, 67]. It was reported that CB2 causes accelerated age-related osteoporosis due to enhanced bone turnover [31]. In our studies however we found that bone loss in ageing CB2 deficient mice is associated with elevated bone resorption coupled to a significant reduction in osteoblast number and bone formation [93]. In agreement with this, activation of the peripherally abundant CB2 receptors, using JWH133 or HU308, protected against bone loss in ovariectomised mice by increasing bone formation markers [31, 93]. These findings - together with evidence showing strong association of CB2 polymorphisms with osteoporosis in women [120, 121] – suggest that CB2 agonists show promise for the treatment of osteoporosis as stimulator of bone formation (Fig. 33). However a recent study using the mouse traumatic brain injury model to investigate the role of cannabinoid receptors in bone formation revealed that CB1 – not CB2 – receptor activation is responsible for increased bone formation following brain injury [12]. The authors of this report went on to suggest that activation of CB1 present on presynaptic nerve endings influence new bone formation by suppressing the release of noradrenaline, an inhibitor of osteoblast activity [12, 122]. Taking into account all findings to date, it is clear that cannabinoid receptor - in particular CB1 - activation regulates osteoblast differentiation and function by directly acting on bone cells and/or indirectly influencing the release of systemic mediators of bone formation such as noradrenaline (Fig. 22).
Encouraged by these findings, we recently investigated the effects of pharmacological and genetic modulation of CB1 receptors on osteoblast differentiation and function in ageing osteoporotic mice. We reported that CB1 deficiency profoundly worsen osteoporosis in 12 month old female mice and resulted in marked loss of bone in male mice of similar age [95]. Detailed histological analysis in our studies showed that CB1 deficiency at this age was associated with a significant reduction in osteoblast number and bone formation resulting in a significant bone loss despite of the significant reduction in osteoclast number (Fig. 33) [32, 95]. This has led us to conclude that age-related osteoporosis associated with CB1 deficiency is not due to increased bone resorption, but is instead due to reduced osteoblast differentiation and bone formation. Osteoporosis in CB1 KO mice was also associated with a striking accumulation of adipocytes in the BM compartment [95]. Studies conducted on bone marrow stromal cells (MSC) – a common precursor to adipocyte and osteoblast - revealed that cultures deficient in CB1 receptors showed a significant reduction in osteoblast differentiation mainly due to an increased capacity of MSC to differentiate into adipocytes [95]. This shift in lineage commitment is coupled to a significant down regulation of the osteoblast specific gene Cbfa1 in osteoblasts and upregulation of cAMP response element binding (CREB) phosphorylation in preadipocytes [95]. All these effects were reproduced pharmacologically in wild type cultures by treating with the CB1 selective inverse agonist/antagonist AM251 [95]. However the pharmacological effects of cannabinoid receptor modulation in adipocyte differentiation reported in the literature are difficult to interpret. For example, endocannabinoids are reported to activate the expression of the adipogenic gene peroxisome proliferator-activated receptor gamma (PPARγ), a powerful stimulator of adipocyte differentiation [123, 124]. Conflicting reports showed that the CB1 selective agonist/inverse antagonist Rimonabant© inhibits cell proliferation but increases markers of adipocyte maturation in preadipocyte cultures [125]. In broad agreement with the latter, we showed that treatment with the CB1 selective agonist/inverse antagonist AM251 inhibits stromal cell differentiation but increases adipocyte differentiation and enhances the expression of adipocyte specific genes such as Fatty acid-binding protein 4, Ccaat-enhancer-binding proteins C/EBPβ and C/EBPα ([95] and Idris et al. unpublished data). Whilst these findings raise the possibility that long term use of cannabinoid receptor inverse agonists/antagonists may suppress osteoblast differentiation and enhance adipogenesis in the bone marrow, they also provide an explanation for the stimulatory effect of cannabinoid agonists on osteoblast activity and bone formation.
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