Osteoclast Function To Influence Bone Mass Biology Essay

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It has recently been put forward that GPR55 could have a role in bone biology. The research carried out here shows that the GPR55 receptor could play an inhibitory role in osteoclastogenesis and promote osteopetrosis.1

The skeleton is an active organ that undergoes bone remodelling, a ceaseless process where mineralized bone is broken down by osteoclasts followed by the formation of bone matrix through the osteoblasts that consequently become mineralized. It embodies three phases, resorptive, where osteoclasts digest old bone; reversal, the bone surface is covered in mononuclear cells and finally formation, where the newly resorbed bone is completely replaced by the osteoblasts. Its function is to maintain the bone structure in response to mechanical stress that alters local bone architecture and repair any damages that occur. A dysfunction of bone remodelling can lead to some of the most common diseases such as osteoporosis, arthritis and cancer induced osteolysis. The research carried by Lauren S. Whyte et al. aims to provide the early development of a potential novel mechanism that could someday alleviate the causes of diseases such as these and bring new therapeutics to light.2

GPR55 is a poorly-characterised G-protein coupled receptor that is stimulated by a whole range of cannabinoids and endogenously by LPI (lipophospatidylinositol). There have been a number of recent research reviews on this receptor and several state that this novel receptor should be renamed the CB3 receptor. This study is of particular interest seeing that it has recently been proven that CB1 and CB2 receptors, the established cannabinoid receptors of the endocannabinoid system take part in bone biology which is why Whyte et al. examined the role of GPR55 in bone cells as there has been considerable evidence that non CB1 or CB2 cannabinoid receptors mediate effects physiologically. Knock-out mice lacking CB1 of CB2 receptors have showed defects in bone structure. In addition it was also found that CB1 and CB2 antagonists protected against ovariectomy induced bone loss by inhibiting osteoclastic bone resorption.3,4

Whyte et al. used O-1602 which is a synthetic regioisomer of cannabidiol that is a potent agonist at the GPR55 cannabinoid receptor but fails to elicit responses from both CB1 and CB2 at up to 30μl. They used cannabidiol a compound present in cannabis as an antagonist in the research they conducted for the reason that cannabidiol has minimal affinity for CB1 and CB­2 receptors but can act as an indirect antagonist to cannabinoid agonists.

A wide variety of methods were used to establish whether the GPR55 receptor affected any of the functions of osteoclasts. To investigate whether osteoclasts expressed GPR55, the authors took samples of whole blood from 7 healthy human donors. Differentiated osteoclasts were then generated from peripheral blood monocytes in particular M-CSF monocytes. Using quantitative real-time PCR, it was found that the average osteoclast GPR55 expression was 8 times that of normal monocytes which shows that osteoclast precursors do express GPR55 but that is amplified as the cells become mature. To further support this outcome GPR55 was found to be present by immunostaining multinucleated osteoclasts from both humans and mice. However, mice M-CSF monocytes were not tested to see the quantity of GPR55 expression, although it is clear that mice express GPR55 from the immunostaining. The use of this method is simply to prove that GPR55 is present in osteoclasts as this is the first study involved with GPR55 and bone biology.

Whyte et al. then asked whether GPR55 played a role in osteoclastogenesis by examining osteoclasts in vitro. Primarily they treated human peripheral monocytes with O-1602, the GPR55 agonist, which had no effect on the quantity of osteoclasts. However, when cell cultures were treated with the GPR55 receptor antagonist cannabidiol, it gave a result close to the positive control, thus showing an increase in osteoclast formation. The team proposes that the reason as to why the human osteoclasts did not respond to O-1602 was because humans produce higher concentrations of an unknown endogenous agonist but this is merely a speculation and there is no substantiation behind it. This then followed with a similar method where the authors used osteoclasts from GPR55-/-, CB1-/- and CB2-/- bone marrow macrophages derived from knockout mice. O-1602 and LPI inhibited mice osteoclastogenesis which was not apparent in the CB1-/- and CB2-/- cells while it appeared that the morphology of the bone marrow macrophages from the GPR55-/- mice was abnormal showing that the presence of GPR55 could be needed for the normal function of osteoclasts in mice and the same effects of O-1602 were not witnessed in these cells. These findings give the GPR55 receptor a role in osteoclast generation in response to different substrates. But what exact function does the GPR receptor have?

To answer this query the researchers determined that O-1602 and LPI elicited a stimulatory effect by increasing the proportion of resorptive activity and cell polarization in vitro on human and mice cells and also demonstrated that it is mediated by members of the RhoA family of GTPases and the MAPK/ERK pathway which have been proved to be important in both these actions. The same assay was then carried out on GPR55-/- cells and importantly elicits no response so confirms that GPR55 is undeniably the responsible receptor for these actions. At the same time cells were treated with cannabidiol which showed, compellingly, that alone it inhibits both human osteoclast resorption and polarization. This is valuable for the reason that it indicates that both signalling pathways are mediators of the GPR55 response although there is prior research where the authors could not identify ERK as a GPR55 mediator. In terms of the question they asked, it is constructive to have carried out these assays as it promotes the idea that the GPR55 receptor does affect osteoclast function in vitro.6

To progress their findings Whyte et al. then turned to in vivo testing such as histomorphometry and μCT analysis where they started by proving that GPR55­-/- mice show increased osteopetrotic symptoms revealing a significant increase in the volume and thickness of cancellous bone due to reduction in bone resorption. These findings were shown to be due to defects in osteoclast function rather than excessive osteoblast bone formation as control tests were carried out. However, this was only witnessed in male mice cells and Whyte et al. put an idea forward stating that hormonal balances cannot be kept in vivo and may play a compensatory role in GPR55-/- female mice but this is also hypothetical and the reasons are as yet an unknown.

The authors then tested whether cannabidiol exerted the same effects in vivo as in vitro finding that there was a considerable decrease in the quantity of CTX fragments which is an established clinical biomarker of bone resorption. This testing involved immunoassays in male mice on the basis of the negligible effects upon female mice in previous tests. The purpose behind this is apparent in that it allowed the researchers to address the final part of their question showing that GPR55 signal inhibition causes in vivo bone mass to increase due to an inhibition of resorption.

Whyte et al. have done an ample amount of back up testing to minimise any bias an example of this is that they constantly confirm that GPR55 is always the receptor mediating the effects of any findings. This is imperative since this is totally novel research and any preconceptions would discredit it and there is a lot of research about how CB1 and CB2 receptors have the ability to alter osteoclast function.7

These new findings have opened up new possibilities of research that the effects substrates evoking responses from GPR55 could have on metabolic bone diseases. Cannabidiol has been proven to be a successful anti-arthritic therapeutic in mice. The authors of that research did not know of GPR55 and because the affinity for the cannabinoid receptors is very low they did not rule out the action of a novel receptor. This is supporting the research that was carried out by Whyte et al. Seeing as the majority of therapeutics used in metabolic bone disease is to try and stop bone resorption GPR55 antagonists could represent a promising new class of anti-resoptive for the treatment of osteoporosis and other bone diseases associated with increased osteoclast activity.4

On the other hand, findings have been mixed, and it remains to be seen whether female mice cells will respond to GPR55 agonists. In addition, this is extremely new research and the exact signalling cascade of GPR55 in osteoclasts is still unknown meaning that a vast amount of further in vitro and in vivo testing in humans and mice respectively needs to be done before in vivo testing can even begin in humans. It is too early to promote the ideas put forward by Whyte et al. fully but the research is promising and if the evidence is similar to what has been shown here in future research then this could one day open avenues to new therapeutic approaches. 7,5