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

The central nervous system plays an important role in regulating bone metabolism in health and in disease with a number of neurotransmitters been reported to influence bone cell activity through a central relay. In keeping with this, recent studies demonstrated that endocannabinoids and their receptors are involved in the pathogenesis of osteoporosis. The endocannabinoids anandamide and 2-arachidonylglycerol are found in the skeleton and numerous studies also showed that bone cells express the cannabinoid receptors CB1 and CB2 and the orphan receptor GPR55. Pharmacological and genetic inactivation of CB1, CB2 and GPR55 in adult mice suppress bone resorption, increase bone mass and protect against bone loss, suggesting that inverse agonists/antagonists of these receptors may serve as anti-resorptive agents. In the ageing skeleton however CB1 and CB2 receptors have a protective effect against age-dependent bone loss in both male and female mice. CB1 receptor deficiency in aged mice results in accelerated age-dependent osteoporosis due to marked increase in bone resorption and significant reduction in bone formation coupled to enhanced adipocyte accumulation in the bone marrow compartment. Similar acceleration of bone loss was also reported in CB2 deficient mice of similar age but found to be associated with enhanced bone turnover. This review summarises in vitro and in vivo findings relating to the influence of cannabinoid ligands on bone metabolism and argues in favour of the exploitation of cannabinoid receptors as targets for both anabolic and anti-resorptive therapy for treatment of complex multifaceted bone diseases such as osteoporosis

Role of cannabinoids in the regulation of bone remodeling

The endocannabinoid system plays a key role in regulating a variety of physiological processes such as appetite control and energy balance, pain perception, and immune responses. Recent studies have implicated the endocannabinoid system in the regulation of bone cell activity and bone remodelling. These studies showed that endogenous cannabinoid ligands, cannabinoid receptors and the enzymes responsible for ligand synthesis and breakdown all play important roles in bone mass and in the regulation of bone disease. These findings suggest that the endocannabinoid pathway could be of value as a therapeutic target for the prevention and treatment of bone diseases. Here, we review the role of the skeletal endocannabinoid system in the regulation of bone remodelling in health and disease

Pharmacologic inhibitors of IκB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo

The NF-κB signaling pathway is known to play an important role in the regulation of osteoclastic bone resorption and cancer cell growth. Previous studies have shown that genetic inactivation of IκB kinase (IKK), a key component of NF-κB signaling, inhibits osteoclastogenesis, but the effects of pharmacologic IKK inhibitors on osteolytic bone metastasis are unknown. Here, we studied the effects of the IKK inhibitors celastrol, BMS-345541, parthenolide, and wedelolactone on the proliferation and migration of W256 cells in vitro and osteolytic bone destruction in vivo. All compounds tested inhibited the growth and induced apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. Celastrol, BMS-345541, and parthenolide abolished IL1β and tumor necrosis factor α–induced IκB phosphorylation and prevented nuclear translocation of NF-κB and DNA binding. Celastrol and parthenolide but not BMS-345541 prevented the activation of both IKKα and IKKβ, and celastrol inhibited IKKα/β activation by preventing the phosphorylation of TAK1, a key receptor–associated factor upstream of IKK. Celastrol and parthenolide markedly reduced the mRNA expression of matrix metalloproteinase 9 and urinary plasminogen activator, and inhibited W256 migration. Administration of celastrol or parthenolide at a dose of 1 mg/kg/day suppressed trabecular bone loss and reduced the number and size of osteolytic bone lesions following W256 injection in rats. Histomorphometric analysis showed that both compounds decreased osteoclast number and inhibited bone resorption. In conclusion, pharmacologic inhibitors of IKK are effective in preventing osteolytic bone metastasis in this model and might represent a promising class of agents to the prevention and treatment of metastatic bone disease associated with breast cancer

Raman Spectroscopy as a Predictive Tool for Monitoring Osteoporosis Therapy in a Rat Model of Postmenopausal Osteoporosis

Pharmacological therapy of osteoporosis reduces bone loss and risk of fracture in patients. Modulation of bone mineral density cannot explain all effects. Other aspects of bone quality affecting fragility and ways to monitor them need to be better understood. Keratinous tissue acts as surrogate marker for bone protein deterioration caused by oestrogen deficiency in rats. Ovariectomised rats were treated with alendronate (ALN), parathyroid hormone (PTH) or estrogen (E2). MicroCT assessed macro structural changes. Raman spectroscopy assessed biochemical changes. Micro CT confirmed that all treatments prevented ovariectomy-induced macro structural bone loss in rats. PTH induced macro structural changes unrelated to ovariectomy. Raman analysis revealed ALN and PTH partially protect against molecular level changes to bone collagen (80% protection) and mineral (50% protection) phases. E2 failed to prevent biochemical change. The treatments induced alterations unassociated with the ovariectomy; increased beta sheet with E2, globular alpha helices with PTH and fibrous alpha helices with both ALN and PTH. ALN is closest to maintaining physiological status of the animals, while PTH (comparable protective effect) induces side effects. E2 is unable to prevent molecular level changes associated with ovariectomy. Raman spectroscopy can act as predictive tool for monitoring pharmacological therapy of osteoporosis in rodents. Keratinous tissue is a useful surrogate marker for the protein related impact of these therapies. The results demonstrate utility of surrogates where a clear systemic causation connects the surrogate to the target tissue. It demonstrates the need to assess broader biomolecular impact of interventions to examine side effects. [Figure not available: see full text.]

Raman Spectroscopy Predicts the Link between Claw Keratin and Bone Collagen Structure in a Rodent Model of Oestrogen Deficiency

Osteoporosis is a common disease characterized by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures; however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes

Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors

Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and to the development of new treatments. Here we show that mice with inactivation of cannabinoid type 1 (CB(1)) receptors have increased bone mass and are protected from ovariectomy induced bone loss. Pharmacological antagonists of CB(1) and CB(2) receptors prevented ovariectomy induced bone loss in vivo and caused osteoclast inhibition in vitro by promoting osteoclast apoptosis and inhibiting production of several osteoclast survival factors. These studies show that the CB(1) receptor plays a role in the regulation of bone mass and ovariectomy induced bone loss and that CB(1) and CB(2) selective cannabinoid receptor antagonists are a novel class of osteoclast inhibitors that may be of value in the treatment of osteoporosis and other bone diseases

A comparison between the effects of hydrophobic and hydrophilic statins on osteoclast function in vitro and ovariectomy-induced bone loss in vivo

Statins potently inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, blocking downstream biosynthesis of isoprenoid lipids and causing inhibition of protein prenylation. Prenylated signaling molecules are essential for osteoclast function, consistent with our previous observation that mevastatin can inhibit osteoclast activity in vitro. Several reports suggest that statins may also have an anabolic effect on bone and stimulate osteoblast differentiation. This study sought to determine the effects of both hydrophobic and hydrophilic statins, particularly rosuvastatin (RSV), on osteoclast function in vitro and in vivo. All statins tested (RSV, pravastatin [PRA], cerivastatin [CER], and simvastatin [SIM]) caused accumulation of unprenylated Rap-1A in rabbit osteoclast-like cells and J774 macrophages in vitro and inhibited osteoclast-mediated resorption. The order of potency for inhibiting prenylation in vitro (at concentrations of 0.01-50 muM) was CER&gt;SIM&gt;RSV&gt;PRA. The most potent hydrophilic statin (CER, 0.05 and 0.3 mg/kg) inhibited prenylation in rabbit osteoclasts 24 hours after a single subcutaneous (s.c.) injection more effectively than the most potent hydrophobic statin (RSV, 20 mg/kg). However, in a mouse model of osteoporosis, s.c. 0.05 mg/kg/day CER and 2 or 20 mg/kg/day RSV for 3 weeks only mildly prevented loss of cortical and trabecular bone induced by ovariectomy. No increase in bone formation rate was observed with statin treatment, suggesting that this effect was due to inhibition of osteoclast-mediated resorption rather than increased bone formation.</p

Role of type 2 cannabinoid receptor in bone metabolism

Cannabinoid receptors play an important role in regulating bone mass and bone turnover. Studies in our laboratories have shown that young mice lacking type 1 cannabinoid receptor (CNR1-/-) had increased bone mass and were resistant to ovariectomy-induced bone loss. Other workers have reported that type 2 cannabinoid receptor knockout mice (CNR2-/-) develop age-related osteoporosis. The aim of this PhD thesis was to further investigate the role of CNR2 in bone metabolism in vitro and in vivo, using genetic and pharmacological approaches. This study showed that CNR2-/- mice had normal bone mass and bone turnover at 3 months of age, but following ovariectomy, CNR2-/- mice were partially protected from bone loss, because of a mild defect in osteoclast formation and bone resorption. In keeping with this, studies in vitro showed that RANKL-stimulated bone marrow cultures from CNR2-/- mice had fewer osteoclasts than cultures from wild type littermates. The CNR2-selective antagonist/inverse agonist AM630, inhibited osteoclast formation in wild type bone marrow cultures in vitro and prevented ovariectomy-induced bone loss in wild type mice in vivo. In contrast, osteoclast cultures from CNR2-/- mice were resistant to the inhibitory effects of AM630 at low concentrations and CNR2-/- ovariectomised mice did not respond to its protective effects at low doses, consistent with a CNR2- mediated effect. These results indicate that CNR2 regulates bone loss under conditions of increased bone turnover, such as ovariectomy, by affecting osteoclast differentiation and function. CNR2-deficient mice developed accelerated age-related osteoporosis and by 12 months of age they had a significant reduction in osteoblast numbers and bone formation, whereas osteoclast numbers remained comparable to wild type littermates. In agreement with this, osteoblasts derived from bone marrow of CNR2-/- mice had reduced PTHstimulated alkaline phosphatase activity and ability to form bone nodules, when compared with wild type cultures. The CNR2-selective agonist, HU308, stimulated bone nodule formation in wild type calvarial osteoblast cultures in vitro and reversed ovariectomy-induced bone loss in wild type mice in vivo. HU308 had blunted effects on bone nodule formation in cultures from CNR2-/- mice and no significant effects on ovariectomy-induced bone loss in CNR2-/- mice, indicating a CNR2-mediated effect. These studies demonstrate that CNR2 protects against age-related bone loss by mainly enhancing osteoblast differentiation and bone formation. In conclusion, type 2 cannabinoid receptors protect from bone loss by maintaining bone remodelling at balance. In addition, type 2 cannabinoid receptor agonists show evidence of anabolic activity, whereas antagonists/inverse agonists show evidence of antiosteoclastic activity in vitro and in vivo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo