Elsevier

Bone

Volume 44, Issue 3, March 2009, Pages 476-484
Bone

The endovanilloid/endocannabinoid system in human osteoclasts: Possible involvement in bone formation and resorption

https://doi.org/10.1016/j.bone.2008.10.056Get rights and content

Abstract

Recent studies suggest a role for the endocannabinoid/endovanilloid anandamide in the regulation of bone resorption/formation balance in mice. Here, we examined the co-expression of the transient receptor potential vanilloid type 1 (TRPV1) and the cannabinoid CB1/CB2 receptors together with N-acylphosphatidylethanolamine-hydrolizing phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), the two enzymes responsible of the synthesis and catabolism of anandamide respectively, in human osteoclasts. Co-expression of TRPV1, CB1/CB2, NAPE-PLD and FAAH was found in both human osteoclast cultures and in native osteoclasts from human bone biopsies. Moreover, agonist-evoked calcium entry indicated that the TRPV1 receptor is functionally active in vitro. Consistently, biomolecular and functional experiments showed that resiniferatoxin (RTX), a selective TRPV1 receptor agonist, increased the expression and the activity of TRAP and cathepsin K, two specific osteoclast biomarkers. The evidence that cannabinoid and vanilloid receptors are co-expressed in human osteoclasts suggests that they might cross-talk to modulate the intrinsic balance of bone mineralization and resorption by different actions of anandamide through TRPV1 and cannabinoid receptors. The presence of the endocannabinoid/endovanilloid proteins in human osteoclasts will likely have implications for the management of bone demineralization associated syndrome (i. e. osteoporosis).

Introduction

Bone is a highly metabolically active tissue and its formation and resorption is at the base of bone remodelling, which continues throughout life [1]. It is generally recognized that removal of bone is the task of osteoclasts [2], [3], while its neo-formation relies on osteoblasts [4], [5]. Bone remodelling serves to adjust bone architecture to meet changing mechanical needs, and it helps to repair micro-damages in bone matrix and to prevent the old bone accumulation. The critical importance of a balanced bone remodelling is demonstrated by human diseases, i.e. osteoporosis, in which a net increase in bone resorption is responsible of skeleton weakening and fracture risk. Osteoclasts have been shown to be implicated in the pathophysiology of osteoporosis and, recently, it has been shown a role for TRPV1 receptor also in bone cancer pain maintenance [6]. Osteoclastic bone resorption is regulated by a complex interplay between circulating calciotropic hormones, like the parathyroid hormone, calcitriol and sex hormones. Moreover, local regulators of bone cell activity like receptor activator of nuclear factor kappa B ligand (RANK-L), macrophage colony stimulating factor (M-CSF) and osteoprotegerin [7], [8], [9], [10], [11], as well as neurotransmitters have been shown to be involved in bone remodelling [12], [13], [14], [15]. More recently, an important role of the endocannabinoid system in the regulation of skeletal remodelling in mice and humans has also been shown [16], [17], [18], [19], [20]. It seems that whereas the cannabinoid receptor (CB) 2 is highly expressed in osteoblasts and osteoclasts with opposite roles in their functions in bone remodelling process (see [21] for review), the CB1 receptor is almost exclusively expressed in noradrenergic fibres innervating the bone tissue and indirectly modulates acute skeletal anabolic function through 2-arachidonoyl-glycerol (2-AG)-mediated activation of prejunctional cannabinoid CB1 receptor [20].

Importantly, new evidence on the cross-talk between CB1/CB2 receptors and transient receptor potential vanilloid type 1 (TRPV1) channels is accumulating. In fact, these receptors are very frequently co-expressed in neural and non-neural cells and they share some of their endogenous agonists, namely anandamide (AEA) and N-arachidonoyl-dopamine [22], [23], [24], [25], [26], [27], [28], [29], [30], [31]. Recently, preliminary studies have shown unexpected roles for some members of the TRP superfamily in bone mass regulation. In particular, there is evidence that TRPV5 receptor knockout mice (TRPV5−/−) exhibit significant disturbances in bone structure, showing a possible involvement of this TRPV1 cognate receptor in bone resorption [32]. Moreover, it is interesting that the mechanisms underlying attenuation of bone resorption following inhibition of osteoclast activity include reduction of the extracellular acidosis and inflammation. Osteoclast activity is accompanied by an increased extracellular accumulation of protons. Such an acidic microenvironment deeply contributes to the worsening of osteoporosis. Acidosis within the osteoporotic tissue may increase TRPV1 receptor activation on primary afferent sensory neurons [33], [34] and this effect supports the idea that TRPV1 serves as a sensor amplifier of several injury-induced chemical stimuli capable to excite and/or sensitize peripheral neural fibers [35], [36]. Also in light of the recent discovery that the CB1/CB2 receptors participate in the bone mass maintenance, similarly to TRPV5 or TRPV6, TRPV1 might be expressed also by osteoclasts, where it might cross-talk with CB2 receptors to modulate the osteoclast proper functioning. The possible presence of the “vanilloid system” in the osteoclast population may pave the way to future studies to further validate the clinical use of selective TRPV1 receptor antagonists for the regulation of bone resorption and, at the same time, the attenuation of bone cancer-induced persistent pain.

Therefore, in this study we considered interesting to investigate the co-expression of CB1/CB2 and TRPV1 receptors in human osteoclasts by performing: 1) biomolecular and immunohistochemical analysis to reveal possible co-expression of CB1, CB2 and TRPV1 receptors in cultured human osteoclasts, as well as in mature osteoclasts from healthy human bone; 2) biomolecular and immunohistochemical analysis in cultured human osteoclasts to reveal the presence of the enzymes NAPE-PLD and FAAH, involved in endocannabinoids synthesis and catabolism, respectively; 3) assays of TRPV1 functional activity as measured by RTX-induced intracellular calcium entry in vitro; 4) measures of the levels of the endocannabinoid/endovanilloid AEA, the endovanilloid N-oleoylethanolamine (OEA) and the endocannabinoid 2-arachydonoylglycerol (2-AG) in osteoclast cultures with and without application of a FAAH blocker 5) the enzymatic and biomolecular assay in vitro for the activity of the specific osteoclast activation biomarker TRAP before and after application of a TRPV1 receptor agonist and antagonist, a CB2 receptor antagonist and a FAAH inhibitor. Moreover, a possible cross-talk between the TRPV1 and the cannabinoid CB2 receptor was evaluated.

Section snippets

Human cell cultures

Osteoclasts were obtained from unfractionated peripheral blood mononuclear cells (PBMCs) of healthy subjects. A signed study subject informed consent was obtained from all healthy subjects with the approval of Ethics Committee of the Second University of Naples and in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki). PBMCs were isolated by centrifugation over

Cell culture and human osteoclast differentiation

Most of the cells were fully differentiated after 20 days of culture. They were identified as osteoclasts by a TRAP-positive cell counts on cultures. RT-PCR analysis on osteoclast RNA extract followed by the cDNA amplification for TRAP and cathepsin K, considered as osteoclastic markers, demonstrated that cells in culture were indeed osteoclasts.

Expression of TRPV1, CB1/CB2 receptor, FAAH and NAPE-PLD mRNAs in human osteoclasts in culture

RT-PCR analysis on osteoclast RNA extract followed by the cDNA amplification showed the presence of mature mRNA for TRPV1, CB1/CB2 receptors and

Discussion

We have shown in this study that human osteoclasts derived by peripheral blood monocytes (PBMC) express TRPV1 and CB1/CB2 receptors together with NAPE-PLD and FAAH, the enzymes responsible of the biosynthesis and catabolism of anandamide, respectively. Interestingly, the blood monocytes we used to differentiate osteoclasts already express the TRPV1 receptor. Even if such a receptor has been suggested to act as a sensor of damage in the blood or of inflammatory conditions at secondary sites [40]

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