Research Article: Combined deficiency of the Cnr1 and Cnr2 receptors protects against age‐related bone loss by osteoclast inhibition

Date Published: July 28, 2017

Publisher: John Wiley and Sons Inc.

Author(s): Antonia Sophocleous, Silvia Marino, Dilruba Kabir, Stuart H. Ralston, Aymen I. Idris.


The endocannabinoid system plays a role in regulating bone mass and bone cell activity and inactivation of the type 1 (Cnr1) or type 2 (Cnr2) cannabinoid receptors influences peak bone mass and age‐related bone loss. As the Cnr1 and Cnr2 receptors have limited homology and are activated by different ligands, we have evaluated the effects of combined deficiency of Cnr1 and 2 receptors (Cnr1/2−/−) on bone development from birth to old age and studied ovariectomy induced bone loss in female mice. The Cnr1/2−/− mice had accelerated bone accrual at birth when compared with wild type littermates, and by 3 months of age, they had higher trabecular bone mass. They were also significantly protected against ovariectomy‐induced bone loss due to a reduction in osteoclast number. The Cnr1/2−/− mice had reduced age‐related bone loss when compared with wild‐type due to a reduction in osteoclast number. Although bone formation was reduced and bone marrow adiposity increased in Cnr1/2−/− mice, the osteoclast defect outweighed the reduction in bone formation causing preservation of bone mass with aging. This contrasts with the situation previously reported in mice with inactivation of the Cnr1 or Cnr2 receptors individually where aged‐related bone loss was greater than in wild‐type. We conclude that the Cnr1 and Cnr2 receptors have overlapping but nonredundant roles in regulating osteoclast and osteoblast activities. These observations indicate that combined inhibition of Cnr1 and Cnr2 receptors may be beneficial in preventing age‐related bone loss, whereas blockade of individual receptors may be detrimental.

Partial Text

Endogenous cannabinoid (endocannabinoid) ligands and their receptors play important roles in the regulation of bone mineral density in animal models of bone disease (Bab et al., 2009; Idris & Ralston, 2012) and in human (Sophocleous et al., 2017). The endocannabinoid system comprises two known receptors, a family of endogenous ligands and various enzymes that are responsible for ligand synthesis, transport and inactivation. Endocannabinoid ligands and their receptors regulate a wide variety of neurological functions including appetite control, pain perception, motor function and the immune response (Matsuda et al., 1990; Idris & Ralston, 2010). There are two classic cannabinoid receptors: type 1 (Cnr1) and type 2 (Cnr2) receptors, both of which belong to the G protein‐coupled receptor (GPCR) super‐family (Pertwee & Ross, 2002; Demuth & Molleman, 2006). The Cnr1 receptor is strongly expressed in the nervous system (Matsuda et al., 1990; Idris & Ralston, 2010), whereas Cnr2 is predominately expressed in cells of the immune system (Munro et al., 1993). However, both receptors are also expressed in bone cells including osteoclasts, osteoblasts, osteocytes and chondrocytes (Tam et al., 2006, 2008; Idris et al., 2009; Idris & Ralston, 2010; Ofek et al., 2011). The endocannabinoids anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG) and a variety of plant cannabinoids act as an agonist at both Cnr1 and Cnr2 receptors albeit with different degrees of selectivity (Matias et al., 2002; Mechoulam, 2005). The endocannabinoids AEA and 2‐AG are produced within the central nervous system and in the bone microenvironment by osteoblasts and osteoclasts (Tam et al., 2006, 2008; Rossi et al., 2009; Idris & Ralston, 2010; Whyte et al., 2011). Agonists of Cnr1 and 2 receptors inhibit adenylyl cyclase causing reduction in intracellular levels of cyclic adenosine monophosphate and activation of a variety of downstream signalling pathways including ion channels, Nuclear Factor Kappa‐B (NFκB), phosphoinositide kinase, mitogen‐activated protein kinase and modulation of second messengers such as ceramide and intracellular calcium (Demuth & Molleman, 2006; Idris & Ralston, 2010).

Over recent years, there has been increasing interest in the role that endocannabinoids and their receptors play in the regulation of bone metabolism (Bab et al., 2009; Idris & Ralston, 2012). Genetic inactivation of Cnr1on an outbred (CD1) background is associated with increased peak bone mass due to reduced osteoclast number but is associated with lower bone mass with age due to reduced bone formation and accumulation of adipocytes in the bone marrow. Mice deficient in Cnr2 on the same background also have higher peak bone mass than wild‐type but developed lower bone mass on aging, due to reduced bone formation. Unlike Cnr1−/− mice, elderly Cnr2−/− mice do not accumulate fat in the bone marrow. The observations presented here show that combined deficiency in Cnr1 and Cnr2 enhances bone accrual and increases peak bone mass in adult mice due to an effect on osteoclast number and function. In addition, we detected a small reduction in osteoblast number in Cnr1/2−/− mice, but bone formation parameters including bone formation rate and mineral apposition rate remained unchanged. The differences in peak trabecular bone mass and osteoclast and osteoblast numbers were quantitatively similar to those previously observed in single knockout of Cnr1 and 2 of a similar age on the same genetic background (Idris et al., 2009; Sophocleous et al., 2014a,b). Collectively, our results suggest that enhanced bone accrual during skeletal growth and increased levels of trabecular bone mass associated with single or double Cnr1 and 2 deficiencies was most probably driven by a reduction in osteoclast number.

This research was funded by a programme grant from Arthritis Research UK (reference 17713).

Antonia Sophocleous involved in experimental work and data analysis; Silvia Marino involved in experimental work and data analysis; Dilruba Kabir involved in experimental work; Stuart H. Ralston was principal investigator and involved in conception, editing and writing; and Aymen I. Idris involved in conception, experimental, analysis, editing and writing.

A. Sophocleous, S. Marino and D. Kabir report that they have no conflict of interest.




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