In the past two decades, the endocannabinoid (EC) system was discovered and characterized, after identifying receptors for ∆⁹-tetrahydrocannabinol (THC), the major psychoactive component of marijuana and hashish. The actions of THC are mediated mainly by the cannabinoid receptor type 1 (CB1) and type 2 (CB2) (Mackie, 2006). Both are seven-transmembrane domain, class A G-protein-coupled receptors (GPCRs) sharing 44% overall identity (68% similarity considering the transmembrane regions alone) (Munro et al., 1993). CB1 is expressed predominantly in neurons and to a lesser extent in several other tissues. In health, CB2 is expressed mainly in immune and bone cells (Mackie, 2005). Owing to the relatively high expression of CB2 in immune cells, it has been hypothesized that this receptor mediates the immunosuppressive effects of phyto- and synthetic cannabinoids (Munro et al., 1993). It is widely believed that CB1 and CB2 are principally targeted by two lipid-derived endogenous ligands, N-arachidonoylethanolamine (AEA or anandamide) and 2-arachidonoylglycerol (2-AG) (Bab et al., 2008). These ECs derive from arachidonic acid and are present mainly in the brain as well as in a variety of peripheral tissues (Bab et al., 2009). Quantitative wise, the main EC is 2-AG.

It has been suggested that 2-AG and AEA are synthesized and function ‘on demand’ (Hashimotodani et al., 2013) because (i) non-stimulated CB2-deficient mice have a normal phenotype (Buckley et al., 2000) and (ii) the ECs are rapidly degraded (Blankman and Cravatt, 2013). By contrast, studies in mice and humans have established that the functional activity of CB2 protects the skeleton against age-related bone loss (Ofek et al., 2006; Idris et al., 2008; Karsak et al., 2005), suggesting the existence of a ‘CB2 tone’ maintained by more stable ligands. Other findings provide room for such an additional agonist. Pharmacological studies dealing with anandamide and 2-AG binding at CB2 reported rather low binding affinities at the µM range, more specifically, one order of magnitude lower than that of THC (Munro et al., 1993; Mechoulam et al., 1995), suggesting that there could be a more specific and potent endogenous ligand. Furthermore, physiological results from several research groups suggest that anandamide and 2-AG have opposing effects on CB2 signaling (Tam et al., 2008; Gonsiorek et al., 2000). In this study, we aimed at finding a putative additional endogenous specific and potent CB2 peptidic agonist that maintains a CB2 tone throughout life and protects against bone loss.

Many proteins and peptides were shown to be GPCR ligands that play a major role in modulating GPCR activation and expression via direct crosstalk. Targeting of GPCRs and other receptors by non-peptidic lipophilic ligands (e.g. acetylcholine) results in short-term activation, whereas the tonal activity of some of these receptors (e.g. muscarinic and nicotinic) is maintained by protein or peptide ligands with higher binding affinities (Alreja et al., 2000; Wu and Yeh, 2005; Miwa et al., 2012). In GPCRs, small-molecule lipophilic ligands have their binding pocket in transmembrane loops, whereas proteins and peptides bind to the extracellular domains (Hurst et al., 2013). Indeed, previous studies have shown the existence of a series of endogenous peptide cannabinoid ligands, termed ‘pepcans’ (Bauer et al., 2012). One of them, ‘pepcan-12,’ is a 12-amino-acid peptide exhibiting a binding affinity to CB1 in the nanomolar range. Consistent with the binding of peptides and lipophilic ligands to different domains, pepcan-12 potently targets an allosteric site at CB1 and interacts rather weakly with the ‘classical’ CB1 binding pocket, characterized by the high-affinity binding of the synthetic cannabinoids CP55940 and WIN55212–2 (Bauer et al., 2012). This same peptide was later identified as a CB2 positive allosteric modulator (PAM) (Petrucci et al., 2017). Pepcan-12 has been identified at significant levels in the brain, peripheral tissues, and plasma following ischemia/reperfusion injury, suggesting its pathophysiological role in modulating CB2 activation (Petrucci et al., 2017). However, pepcan-12 has no reported actions in bone cells and does not seem to decline with age, thus bringing into question its involvement in preserving bone mass during aging.

To assess the possibility that the skeletal protective actions of CB2 are maintained by an endogenous peptide agonist, we performed an in-depth literature search, looking for a peptide/protein that can bind to CB2 or to any other GPCR in bone cells. In January 2017, a search in PubMed looking for ‘(peptide or protein) and (‘Gi protein’ or ‘G(i)’ or ‘GPCR’ or ‘G*coupled receptor’) and (osteoblast* or osteoclast* or osteocyt*)’ resulted in 96 articles (Figure 1A). Out of these 96 articles, there were only 26 Gi agonists and 10 were endogenous proteins/peptides (Supplementary file 1). Furthermore, 4 out of the 10 endogenous peptides/proteins were reported to bind to an unknown Gi or to CB2 in bone cells (Supplementary file 1). Out of these four remaining peptides/proteins, two were affected by aging and only one, named osteogenic growth peptide (OGP), played a positive role in maintaining bone mass (Spreafico et al., 2006; Bab et al., 1992; Greenberg et al., 1993; Greenberg et al., 1995). We could not find any report on the putative receptor for OGP. We therefore proceeded with this peptide to assess whether it binds to and activates CB2.

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BV/TV female and male mice 6 and 3 month old.

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Cnr2 expression male and female 3 and 6 month mice.

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Endocannabinoid levels in 3 and 6 month old mice.

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In 1992, we reported that a C-terminal 14-amino acid peptide derived from the gene for histone H4, H4(90–103) in mice and H4(89–102) in humans, is mitogenic to osteoblasts (Spreafico et al., 2006; Bab et al., 1992; Greenberg et al., 1993), stimulates bone formation in rodents and rescues ovariectomy-induced bone loss (Bab et al., 1992; Chen et al., 2000). This peptide was therefore called OGP(1-14). OGP(1-14) is physiologically present in the serum at nano- to micromolar concentrations, with most of it complexed to α2-macroglobulin (Greenberg et al., 1995; Gavish et al., 1997; Bab et al., 1999). Upon dissociation from this complex, it is proteolytically cleaved, thus generating its 5-amino-acid cellular activator OGP (Idris et al., 2008; Karsak et al., 2005; Mechoulam et al., 1995; Tam et al., 2008; Gonsiorek et al., 2000) referred to here as OGP (Bab et al., 1999). This pentapeptide targets a pertussis toxin-sensitive GPCR and consequently activates a Gi-protein – Erk1/2 – Mapkapk2 – CREB cascade (Miguel et al., 2005). Although OGP has never been attributed anti-inflammatory properties (a hallmark of CB2 agonists), there is a striking resemblance between the signal transduction triggered by this pentapeptide and that of CB2 agonists in bone cells (Ofek et al., 2011). Moreover, CB2 agonists and OGP have an identical mitogenic dose-response activity in osteoblasts and can stimulate bone formation and bone mass in intact rats and osteoporotic mice (Idris et al., 2008; Bab et al., 1992; Chen et al., 2000; Ofek et al., 2011). Interestingly, the serum levels of biologically active OGP decline with age during adulthood (Greenberg et al., 1995). We therefore hypothesized that OGP is a CB2 agonist, and that its serum levels drive an age-related reduction in CB2 tone, thus alleviating the protective role of CB2 during aging. Indeed, in a series of ex vivo and in vivo skeletal and inflammatory experiments, we show here that OGP shares the activities of highly potent CB2 agonists and that these actions depend on the presence of an active CB2 receptor. Our present results suggest for the first time that OGP is an endogenous peptide agonist at CB2.

The CB2 tone has been emerging as a key theme in the homeostatic maintenance of the skeletal and possibly other physiological systems. Insights derived from our studies suggest that the actions of OGP on CB2-regulated processes are at least equally important to those of the arachidonic acid-derived ECs: 2-AG and AEA. Moreover, our data suggest that OGP rather than the classical ECs is essential for maintaining the CB2 tone in bone during aging.

Several studies in the last two decades demonstrated a major role for CB2 in suppressing inflammatory responses and related conditions such as neuropathic pain, ischemia/reperfusion injury, and atherosclerosis, as well as protection against age-related bone loss (Ofek et al., 2006; Racz et al., 2008; Prime et al., 2009; Steffens et al., 2005). This role has been revealed primarily by using phyto- or synthetic cannabinoids rather than ECs, which are unstable, short-lived neurotransmitter-like compounds. The activity of such agonists is inconsistent with the continuous tone required for attenuating age-related bone loss. Rather, we hypothesized that circulating peptide agonists at appropriate concentrations fulfill these functions.

We demonstrated here that the bone loss that started during adulthood in mice is neither accompanied by age-related changes in the levels of the two classical ECs nor in CB2 expression levels in bone tissue (Bab et al., 2007, Figure 1). These results suggest that another physiological CB2 agonist declines with time, thus explaining this age-related bone loss. Our literature survey, performed in 2017, identified OGP(1-14)/OGP as a likely candidate (Figure 1A) that is present in the circulation at the micromolar level under physiological conditions (Bab et al., 1992; Greenberg et al., 1995; Bab and Chorev, 2002). The receptor for this peptide has remained unknown for almost three decades, although we showed in separate studies that OGP and CB2 receptor cannabinoid ligands trigger the same intracellular signaling pathway (Miguel et al., 2005; Ofek et al., 2011). Here, we demonstrate for the first time that OGP binds to and activates CB2 using several in vitro and in vivo assays in the skeletal and immune systems. Interestingly, we previously showed a relative age-related decline of free OGP in human male subjects (Greenberg et al., 1995), and here, we show in premenopausal women a significant age-related reduction in the total serum OGP(1-14) and OGP levels (Figure 8A).

Virtually all living cells may be able to secrete certain amounts of endogenous AEA or 2-AG, and the serum used in in vitro assays may contain small amounts of ECs. Our quick assays performed in CHO cells with no serum (Figure 2B) may suggest that OGP can induce CB2 signaling by itself. Because of its hydrophilic nature and dramatically different molecular structure, OGP is less likely to compete with lipophilic cannabinoid ligands (e.g. AEA and 2-AG) at the classical transmembranous binding site under normal physiological conditions. This lack of competition would suggest that the allosteric binding of OGP at CB2 can modulate the orthosteric binding of lipophilic agonists. Indeed, our docking simulation together with our competitive binding assays between OGP and tritiated CP55940 demonstrate that OGP binds at an allosteric site and modulates the level of CB2 activation by the orthosteric agonist (Figure 3). From these results, it is still unclear whether OGP increases CP55940 binding at CB2 or merely the CB2 functional response to CP55940. Be that as it may, these results further support the notion that OGP acts as a PAM.

Computational docking and MD studies suggest a possible allosteric site for OGP binding at the CB2 receptor. While not ruling out the possibility of binding at the orthosteric site, our simulations imply that OGP can also bind to the ECL region and that this complex can stably last for a prolonged time. Docking to both receptor models generated resembling binding and involved similar interactions with residues from the ECL1, ECL2, and the N-terminal. The exact binding mode remains uncertain as the exact structure of the N-terminal, which is crucial to the exact binding mode of an allosteric site, is hard to predict. Notably, the peptide is very flexible and its conformation is therefore rather elusive. In addition, in the case of the homology models, the overall structure of the receptor should also be considered with care. Hence, our results suggest that OGP can bind to the ECL region, although the exact binding mode may be slightly different than that obtained from our calculations. Our findings are supported by recent studies of Pandey et al. who also suggested a possible allosteric site for dihydro-gambogic acid and trans-β-caryophyllene within the CB2 receptor (Pandey et al., 2020). However, their study suggested a binding site that involved ECL1, ECL2, and the TM region, while our allosteric site for OGP involved only the ECL regions. These differences may arise from differences in the chemical character and activity of the studied ligands. More comprehensive studies are needed to determine whether OGP is a full agonist at CB2 or a PAM in conjunction with bound orthosteric agonists.

Many cannabinoid agonists bind to both CB1 and CB2, likely due to the moderate homology between CB1 and CB2. Following the recently reported crystal structure of CB2 (47), we estimate that these two receptors share a 30% identity (43% similarity) over the full sequence, 44% identity (62% similarity) without the N- and C-terminal regions, and 51% identity (69% similarity) if only the transmembrane regions are considered (Supplementary file 3; Munk et al., 2019). Assuming that the allosteric binding at CB2 involves the extracellular regions where the homology between the two receptors drops to 38% identity (or even 10% if we consider the N-terminal region) reduces the likelihood that OGP also binds to CB1. Indeed, using our cAMP assay in hCB1-transfected HEK293 cells, OGP failed to activate CB1 (Figure 2—figure supplement 2). However, we cannot rule out the possibility that under certain conditions OGP binds to CB1. More experiments are needed to elucidate this issue.

In vitro, all our assays showed a biphasic dose-response, where below the optimal dose, the effect increases with the dose, but after this optimal dose, the effect diminishes gradually to reach the no treatment levels (Figure 4A and B) and sometimes even inverses the effect (Figure 4—figure supplement 1). This biphasic effect has been repeatedly reported for OGP, other CB2 agonists, and agonists of other GPCRs (Ofek et al., 2006; Miguel et al., 2005; Ofek et al., 2011; Bab and Chorev, 2002; Smoum et al., 2015; Schattauer et al., 2019). The reversal of the effect at high doses likely results from desensitization of the receptor (Kelly et al., 2008). Interestingly, such a biphasic effect in vivo was also reported for another CB2 agonist (Smoum et al., 2015), although the most common observation is a plateau after the optimal dose is reached (Bab et al., 1992; Schattauer et al., 2019).

The presence of such a stable and selective CB2 agonist and PAM is of great significance since the known EC ligands (AEA and 2-AG) are prone to fast enzymatic breakdown, bind to both CB1 and CB2 and elicit different effects on each receptor. This study portrays OGP as a third endogenous cannabinoid with long-lasting effects and CB2 selectivity. The latter is particularly relevant in the CNS thus avoiding the psychoactive effect associated with CB1 EC agonists. Because CB2 has been primarily associated with the immune and skeletal systems in mice and men, it suggests that the main roles of OGP (and its potential therapeutic effects) are in these two systems.

Ideally, genetic ablation of OGP could be used to assess the physiological role of OGP. However, these loss-of-function approaches to study the physiological significance of OGP are problematic. First, molecular targeting of OGP, such as by gene ablation or RNA interference, would also target histone H4 expression, which is indispensable for life. Second, gene replacement studies (knock-in) for histones are unrealistic, given their multiple copies in mammalian genomes and that deletion of all these genes is not compatible with life (Smith et al., 2005). Therefore, in our approach for testing the OGP bone protective effect, we employed an experiment involving long-term treatment of OGP, supposedly compensating for the age-related decline of the endogenous peptide in the circulation. Using this approach, we were able to prevent age-related bone loss in mice, thus confirming the skeletal protective role of OGP. These data strengthen the assumption that the reduced levels of OGP are at least partially responsible for the slow decline in bone mass with age, starting after the age of peak bone mass.

An important part of this study was to assess the link between OGP and age-related bone loss. A link between OGP levels and age in men has already been published (Greenberg et al., 1995), as well as in non-OVX female mice overexpressing OGP (Smith et al., 2005). We show here new data on premenopausal women as well as OVX female and naive male mice treated with exogenous OGP (Figures 5 and 8, Figure 5—figure supplement 1 and Figure 8—figure supplement 1) during an age range associated with age-related bone loss in both humans and mice (Kralick and Zemel, 2020 and Figure 1). Collectively, the published literature (Greenberg et al., 1995; Smith et al., 2005) and this study indicate a link between age and endogenous OGP levels in men and women, and demonstrate that increasing OGP levels increases bone mass in both male and female mice.

Our findings prompted us to envision additional experiments to assess their translational potential. Since the biosynthetic pathway of OGP is relatively well defined, this pathway could be modulated (e.g. stimulate H4 leaky ribosomal scanning, enhance OGP[1-14] proteolytic activation) to maintain optimal OGP levels. The development of a controlled-release device that would normalize the serum levels of OGP throughout aging is also a realistic approach in managing and minimizing age-related bone loss. The specificity of this peptide agonist for CB2, if confirmed, would imply the absence of undesirable psychotropic effects shared by many of the non-specific cannabinoid receptor ligands that, in addition to CB2, activate neuronal CB1 receptors. Moreover, the results of our study will potentially pave the way for OGP-based treatments for a vast array of skeletal and inflammatory conditions. On a broader perspective, OGP is secreted by osteoblasts (Bab et al., 1999) and this is one of the very few examples attributing a systemic endocrine role to osteoblasts. From an osteoimmunological standpoint, previous studies showed mechanisms related to the effects of inflammation and immune cells on bone cells (Bar-Shavit, 2008; Blaschke et al., 2018), some show interactions between osteoclasts and hematopoietic/immune cells (Kollet et al., 2006), or cytokine expression in osteoblasts (e.g. Patil et al., 2004). However, whether osteoblasts may suppress inflammatory processes remains an open question. OGP may be the first example of an anti-inflammatory hormone secreted by osteoblasts and this study prompts future research on the pathophysiological and clinical relevance of this putative immunomodulatory role of bone cells.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 and 2 and 4–8.

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Decision letter after peer review:

Thank you for submitting your article "Osteogenic growth peptide is a potent anti-inflammatory and bone preserving hormone via cannabinoid receptor type 2" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Summary:

This is a well-written manuscript exploring the function and receptor for a peptide called Osteogenic Growth Peptide (OGP), which is derived from the histone H4 gene. The authors provide convincing evidence that the CB2 cannabinoid receptor serves as the cellular target for osteoblasts and osteoclasts in mediating the bone mass-promoting effects of this peptide.

Essential Revisions:

1. The manuscript needs to be better organized. The introduction needs to be revised to help readers understand the significance of the study and the role of CB2 on bone protection.

2. For Figure 1A, detailed descriptions of "in-depth literature search" should be included in the manuscript, such as the source of literatures or summary of 96 proteins to justify the importance of OGP.

3. The authors find the BV/TV of mouse trabecular bones at 6 months old is significantly lower than the one at 3 months old. The bone mass peak is achieved around 4-6 months of age in most mouse strains (Jilka 2013, Bernard 2002, Mouse Phenome Database, http://phenome.jax.org/). The decreased BV/TV may be the result of enlarged TV, not BV. Also, BV/TV of trabecular bones is the only parameter to evaluate bone phenotype throughout this study. Therefore, the existing data are not sufficient to support the conclusion of the regulation of CB2 signaling on bone loss and bone protection. More measurements, such as BV, trabecular bone number, trabecular thickness, bone mineral density of both trabecular bones and cortical bones, should be included to make reliable conclusions.

4. It is not sufficient to validate OGP binding to CB2 by molecular dynamics simulation alone. The binding should be at least validated by CO-IP assay etc.

5. It is not well accepted to use the number of cells to validate cell proliferation. Moreover, the authors have not indicated whether the "% control" refers to live cells or total cells. Trypan blue staining or other cell viability assays should be included. Other assays such as MTT, BrdU etc., would be better than cell counting to assess cell proliferation.

6. Low concentrations of OGP show the effects on osteoblast proliferation and anti-inflammatory processes. But high concentrations reverse these phenotypes. More explanations need to be provided.

7. The bone and anti-inflammatory phenotypes in CB2-/- mice should be validated in this study.

8. The authors demonstrate that anti-inflammatory activity is a hallmark of CB2 agonists. But the anti-inflammatory effects of CB2 signaling is not provided in the experiment. The comparison between CB2 -/- and WT cells should be included in the experiment. The conclusion would be more convincing if protein levels are measured.

9. For Figure 7, the authors measure the OGP (1-14) levels in women at 18-49. But they have not evaluated bone volume and density in those women. And also, they use 3-month old and 6-month old male mice to validate the skeletal effect. Therefore, age and sex groups of humans are not comparable to the groups of mice. A human at the age of 50 is approximately equivalent to a mouse at the age of 15 months (Dutta and Sengupta 2016). Therefore, the equivalent age and sex of mice and humans would be better to validate the beneficial effects of OGP on bones.

10. For Figure 1, OGP should have been measured in the aging mice as a control to show significant declines relative to other CB2 agonists.

11. For figure 7, where are the complete set of micro CT data parameters? Moreover, bone histomorphometric parameters with MAR, etc. should have been performed.

12. For figure 7, a great control given that the CB2 KO mice have no bone phenotype would have been to give OGP to them and demonstrate a failure of bone mass increase.Reviewer #1 (Recommendations for the authors):

Raphael-Mizrahi et al., investigate the role of osteogenic growth peptide (OGP) as cannabinoid receptor type 2 (CB2) agonist on bone and anti-inflammatory processes by clinical data, in-vitro, and in-vivo experiments. The authors show that OGP stimulates osteoblast proliferation at low concentrations and inhibits osteoclastogenesis via CB2. It also alleviates the acute inflammatory response by using the ear-swelling mouse model. This study suggests OGP is a potential candidate to treat inflammation and age-related bone loss. These data provide a possible alternative therapy for osteoporosis.

The conclusions of this study, to some extent, are supported by data, but some concerns need to be considered and clarified.

1. The authors demonstrated in the introduction that the selection of OGP as a CB2 agonist is through an in-depth literature search, but there is no further description of "search" in the manuscript.

2. The main findings of this study is that OGP activates CB2 signaling to rescue age-related bone loss. This conclusion is based on the premise that the activation of CB2 signaling protects bones. However, the bone phenotype of CB2-/- mice is not included in the study. Only BV/TV is not sufficient to indicate bone volume and quality. Thus, the major finding is not fully supported by the existing data.

3. The authors claim that OGP is CB2 agonist and binds to CB2, but only molecular dynamics simulation is weak to substantiate this conclusion.

4. The authors conclude OGP affects osteoblast proliferation. However, the method of cell counting is not sufficient to assess cell proliferation. Also, the method is not applied rigorously. It is not clear whether "% Control" refers to live cells only or total cells including dead cells. Therefore, it is difficult to assess this conclusion.

5. CB2 signaling affects anti-inflammatory processes. However, the conclusion is not reliable due to the lack of comparison between CB2 -/- and WT cells in the experiment. Moreover, the high standard deviation in three independent experiments makes it hard to assess the reproducibility of the conclusion.

6. It is not clear the underlying relationship between bone protection and anti-inflammatory functions of OGP.Reviewer #2 (Recommendations for the authors):

Overall this is a well-written manuscript of a research study involving in silico investigations, in vitro investigations, in vivo studies in mice, as well as ex vivo studies from human samples. The main premise sought to explore the bone effects of a peptide called Osteogenic Growth Peptide (OGP), which is derived from the histone H4 gene.

The authors provide convincing evidence that the CB2 cannabinoid receptor serves as the cellular target for osteoblasts and osteoclasts in mediating the bone mass-promoting effects of this peptide. Much of this experimentation takes advantage of CB2 knockout mice, which interesting by themselves have no phenotype. Here their initial experimentation could use strengthening as traditional evaluations such as CFU-OB assays or the use of CB2 KO mice for OGP injection experiments is currently lacking.

The manuscript goes on in its investigation to demonstrate that OGP has more "traditional" anti-inflammatory actions, such as the suppression of inflammatory cytokines or tissue inflammation. The data presented in that regard was sufficient.

This reviewer remains positive about this paper and overall finds the topic fascinating. With some revision to strengthen experimentation, the manuscript would reach an appropriate level warranting publication.

Essential Revisions:

1. The manuscript needs to be better organized. The introduction needs to be revised to help readers understand the significance of the study and the role of CB2 on bone protection.

We added a paragraph describing the significance of this research.

2. For Figure 1A, detailed descriptions of "in-depth literature search" should be included in the manuscript, such as the source of literatures or summary of 96 proteins to justify the importance of OGP.

We modified the text in the introduction to make it clearer that the initial literature search generated 96 articles (not proteins). We made a new table (Supplementary File 1) where all the 26 Gi-GPCR agonists described in these 96 articles are listed (with references) and emphasized the 10 that are also endogenous peptides/proteins.

3. The authors find the BV/TV of mouse trabecular bones at 6 months old is significantly lower than the one at 3 months old. The bone mass peak is achieved around 4-6 months of age in most mouse strains (Jilka 2013, Bernard 2002, Mouse Phenome Database, http://phenome.jax.org/). The decreased BV/TV may be the result of enlarged TV, not BV. Also, BV/TV of trabecular bones is the only parameter to evaluate bone phenotype throughout this study. Therefore, the existing data are not sufficient to support the conclusion of the regulation of CB2 signaling on bone loss and bone protection. More measurements, such as BV, trabecular bone number, trabecular thickness, bone mineral density of both trabecular bones and cortical bones, should be included to make reliable conclusions.

The peak trabecular BV/TV in the distal femur of the C57Bl/6J mouse strain is achieved at around 3 months in males and even earlier in females, based on our own published data as well as in an additional independent study using the same analysis protocol (1, 2). The main decline is shown until 6 months of age. After that, bone mass continues to decline but at a slower pace (1, 2). To address the possible bias of changes in TV rather than BV, we looked at the data and added the following sentence in the Results section:

“In 3 months, the trabecular BV/TV decreased by 61% and 75% in males and females, respectively, due to significant 64% and 70% decrease in the bone volume.” (line 145)

As suggested, we also added a more comprehensive microCT analysis to the supplemental data (Figure 1 —figure supplement 1).

4. It is not sufficient to validate OGP binding to CB2 by molecular dynamics simulation alone. The binding should be at least validated by CO-IP assay etc.

Molecular dynamics simulation alone is indeed not sufficient to validate that OGP binds to CB2. In this study, our conclusion is mainly supported by our several cAMP inhibition assays (Figure 2 and Figure 2 —figure supplementary 2 from the original manuscript, now Figure 2 —figure supplementary 1 and more data included in the revised version). This assay is widely considered a binding assay, i.e. the inhibition of cAMP demonstrates agonist binding to a Gi-GPCR. To further strengthen our conclusion, we now add data from a competitive binding assay between OGP and tritiated CP55940 and an assay that shows the effect of OGP on the binding of ^[35S]GTPγS that is CP55940-induced (Figure 3). In these assays we show that OGP and CP55940 do not compete on the same binding site and that OGP has a positive allosteric effect via its binding to the receptor. Results from these assays further indicate that OGP binds to CB2 and strengthens the evidence that OGP is a positive allosteric modulator as suggested from the molecular dynamics findings. It is worth mentioning that we could not perform a CO-IP assay since there are no available antibodies against the 5-amino acid OGP. The antibodies that were used to produce the human data were custom-made against the 14-amino acid OGP and required a large amount of serum that couldn’t be extracted from a mouse (as mentioned in line 265-269 in the main manuscript). Work is in progress to develop a good antibody that will detect OGP in small amounts of serum and can be used for immunofluorescence.

5. It is not well accepted to use the number of cells to validate cell proliferation. Moreover, the authors have not indicated whether the "% control" refers to live cells or total cells. Trypan blue staining or other cell viability assays should be included. Other assays such as MTT, BrdU etc., would be better than cell counting to assess cell proliferation.

Indeed, the % control are referred to live cells only since the dead cells are washed out and only adherent live cells are counted. The data were normalized so that the control number of live cells are 100% and all data is in ratio to the control. Additionally, we performed BRDU assays and added the results to the supplemental data (Figure 4 —figure supplement 1) with reference to this assay and results in the text.

6. Low concentrations of OGP show the effects on osteoblast proliferation and anti-inflammatory processes. But high concentrations reverse these phenotypes. More explanations need to be provided.

GPCR are known to have a biphasic effect when a vast range of agonist concentration are used (3). This biphasic effect was demonstrated many times before in response to high vs. low concentrations of ligands of the endocannabinoid system (4). More explanation is also added to the main manuscript in the discussion (paragraph starting with “in vitro, all our assays showed a biphasic dose-response…”).

7. The bone and anti-inflammatory phenotypes in CB2-/- mice should be validated in this study.

The increased inflammatory phenotype in Cnr2^-/- has been repeatedly demonstrated (reviewed in (5)). The bone phenotype of Cnr2^-/- mice has been comprehensively studied in our and others’ published articles (6-8). Here we show that CB2 agonists strongly suppress inflammation in the ear edema in vivo model and in macrophages in vitro, via the CB2 receptor. Regarding the bone phenotype, here we show that OGP affects osteoblast proliferation and osteoclast differentiation via CB2. In this revised version, we now add data on an ovariectomy-induced osteoporosis experiment in WT and Cnr2^-/- mice. The results show that OGP rescues OVX-induced bone loss via CB2 (see Results, Figure 5 and Figure 5 —figure supplement 5).

8. The authors demonstrate that anti-inflammatory activity is a hallmark of CB2 agonists. But the anti-inflammatory effects of CB2 signaling is not provided in the experiment. The comparison between CB2 -/- and WT cells should be included in the experiment. The conclusion would be more convincing if protein levels are measured.

This study does not demonstrate that anti-inflammatory activity is a hallmark of CB2 agonists (see Introduction). This has been demonstrated by many others and is now a well-established evidence. In line with this established fact, here we show that CB2 agonists (OGP and HU910) strongly suppress inflammation in the ear edema in vivo model. We also show that OGP has an anti-inflammatory effect (decreased expression of TNFα and IL1β) in LPS-treated macrophages in vitro, via the CB2 receptor.

9. For Figure 7, the authors measure the OGP (1-14) levels in women at 18-49. But they have not evaluated bone volume and density in those women. And also, they use 3-month old and 6-month old male mice to validate the skeletal effect. Therefore, age and sex groups of humans are not comparable to the groups of mice. A human at the age of 50 is approximately equivalent to a mouse at the age of 15 months (Dutta and Sengupta 2016). Therefore, the equivalent age and sex of mice and humans would be better to validate the beneficial effects of OGP on bones.

The main purpose of the mouse and human experiments is to assess the link between OGP and age-related bone loss. A link between OGP levels and age in men has already been published (9); as well as in female mice overexpressing OGP (10). We therefore show here new data on pre-menopausal women and male mice treated with exogenous OGP (new Figure 8). We also added in this revised manuscript, data on OVX females treated with OGP (new figure 5). Collectively, the published literature (9, 10) and this study (Figure 8) indicate a link between age and endogenous OGP levels during the period associated with slow age-related bone loss in both men and women (11), and demonstrate that increasing OGP levels increases bone mass (Figure 8) during a period associated with age-related bone loss in both male and female mice (Figure 1). In our mouse strain, using older animals is problematic as beyond 6 months in C57BL/6J-Rcc mice, there is almost no trabecular bone that would allow for a meaningful evaluation of further age-related bone loss.

We added a paragraph to address this question in the discussion of the revised manuscript (one before last paragraph in the discussion, “An important part…”).

10. For Figure 1, OGP should have been measured in the aging mice as a control to show significant declines relative to other CB2 agonists.

We agree that this would have been preferable. Unfortunately, what was done in humans was made possible with a large volume of serum that could not be obtained from mice. All our attempts to develop a new anti-serum against OGP did not reach a high enough sensitivity for mouse serum. See also answer to Comment 4.

11. For figure 7, where are the complete set of micro CT data parameters? Moreover, bone histomorphometric parameters with MAR, etc. should have been performed.

In the revised manuscript we added a more comprehensive morphometric analysis of the cortical and trabecular parameters (for both the age-related and the OVX experiments, Figures 5, 8, Figure 5 —figure supplement 1 and figure 8 —figure supplement 1). Re histomorphometry, we did not inject calcein as this has been repeatedly examined in past publications (10, 12). At this point, we therefore cannot provide the dynamic histomorphometry without repeating the entire experiment and sacrificing a large number of animals.

12. For figure 7, a great control given that the CB2 KO mice have no bone phenotype would have been to give OGP to them and demonstrate a failure of bone mass increase.

We added an ovariectomy (OVX) induced bone loss experiment to show in vivo that OGP has no bone effect on Cnr2^-/- mice. The manuscript provides ample evidence in vivo and in vitro to demonstrate that all the bone protective and anti-inflammatory effect of OGP are dependent on CB2, including lack of binding and effect in CHO cells, osteoblasts, osteoclasts, macrophages and in vivo using inflammation (ear edema) and OVX-induced bone loss models. We hope the reviewers will agree that given the additional data provided in the revised manuscript, there is little added value to show again Cnr2^-/- mice treated with OGP at this point of the study.

Reviewer #1 (Recommendations for the authors):

Raphael-Mizrahi et al., investigate the role of osteogenic growth peptide (OGP) as cannabinoid receptor type 2 (CB2) agonist on bone and anti-inflammatory processes by clinical data, in-vitro, and in-vivo experiments. The authors show that OGP stimulates osteoblast proliferation at low concentrations and inhibits osteoclastogenesis via CB2. It also alleviates the acute inflammatory response by using the ear-swelling mouse model. This study suggests OGP is a potential candidate to treat inflammation and age-related bone loss. These data provide a possible alternative therapy for osteoporosis.

The conclusions of this study, to some extent, are supported by data, but some concerns need to be considered and clarified.

It is not clear the underlying relationship between bone protection and anti-inflammatory functions of OGP.

That’s an important question for which part of the answer might be philosophical. Still, we added two paragraphs in the discussion (last one and 4^th before last) to address this aspect from a biological and clinical perspective:

“The presence of such a stable and selective CB2 agonist […] Because CB2 has been primarily associated with the immune and skeletal systems in mice and men, it suggests that the main roles of OGP (and its potential therapeutic effects) are in these 2 systems.”

“On a broader perspective, OGP is secreted by osteoblasts (13) and this is one of the very few examples attributing a systemic endocrine role to osteoblasts. From an osteoimmunological standpoint, previous studies showed mechanisms relating to the effects of inflammation and immune cells on bone cells (14, 15), some show interactions between osteoclasts and hematopoietic/immune cells (16), or cytokine expression in osteoblasts (e.g. (17)). However, whether osteoblasts may suppress inflammatory processes remains an open question. OGP may be the first example of an anti-inflammatory hormone secreted by osteoblasts and this study prompts future research on the pathophysiological and clinical relevance of this putative immunomodulatory role of bone cells.”

Reviewer #2 (Recommendations for the authors):

Overall this is a well-written manuscript of a research study involving in silico investigations, in vitro investigations, in vivo studies in mice, as well as ex vivo studies from human samples. The main premise sought to explore the bone effects of a peptide called Osteogenic Growth Peptide (OGP), which is derived from the histone H4 gene.

The authors provide convincing evidence that the CB2 cannabinoid receptor serves as the cellular target for osteoblasts and osteoclasts in mediating the bone mass-promoting effects of this peptide. Much of this experimentation takes advantage of CB2 knockout mice, which interesting by themselves have no phenotype. Here their initial experimentation could use strengthening as traditional evaluations such as CFU-OB assays or the use of CB2 KO mice for OGP injection experiments is currently lacking.

Please see answers to comments 14 and 16 to the editor’s summary

The manuscript goes on in its investigation to demonstrate that OGP has more "traditional" anti-inflammatory actions, such as the suppression of inflammatory cytokines or tissue inflammation. The data presented in that regard was sufficient.

This reviewer remains positive about this paper and overall finds the topic fascinating. With some revision to strengthen experimentation, the manuscript would reach an appropriate level warranting publication.

The authors wish to thank the Reviewers and the Editor for their thorough review of our work and the constructive comments that we feel significantly improved the quality of our manuscript.

References

1. Bab I, Müller R, Hajbi-Yonissi C, and Gabet Y. Micro-Tomographic Atlas of the Mouse Skeleton. New York: Springer; 2007.

2. Glatt V, Canalis E, Stadmeyer L, and Bouxsein ML. Age-related changes in trabecular architecture differ in female and male C57BL/6J mice. J Bone Miner Res. 2007;22(8):1197-207.

3. Schattauer SS, Bedini A, Summers F, Reilly-Treat A, Andrews MM, Land BB, et al. Reactive oxygen species (ROS) generation is stimulated by kappa opioid receptor activation through phosphorylated c-Jun N-terminal kinase and inhibited by p38 mitogen-activated protein kinase (MAPK) activation. J Biol Chem. 2019;294(45):16884-96.

4. Smoum R, Baraghithy S, Chourasia M, Breuer A, Mussai N, Attar-Namdar M, et al. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency. Proc Natl Acad Sci U S A. 2015;112(28):8774-9.

5. Turcotte C, Blanchet MR, Laviolette M, and Flamand N. The CB2 receptor and its role as a regulator of inflammation. Cell Mol Life Sci. 2016;73(23):4449-70.

6. Idris AI, van 't Hof RJ, Greig IR, Ridge SA, Baker D, Ross RA, et al. Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat Med. 2005;11(7):774-9.

7. Idris AI, Sophocleous A, Landao-Bassonga E, van't Hof RJ, and Ralston SH. Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor. Endocrinology. 2008;149(11):5619-26.

8. Ofek O, Karsak M, Leclerc N, Fogel M, Frenkel B, Wright K, et al. Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc Natl Acad Sci U S A. 2006;103(3):696-701.

9. Greenberg Z, Chorev M, Muhlrad A, Shteyer A, Namdar-Attar M, Casap N, et al. Structural and functional characterization of osteogenic growth peptide from human serum: identity with rat and mouse homologs. J Clin Endocrinol Metab. 1995;80(8):2330-5.

10. Smith E, Meyerrose TE, Kohler T, Namdar-Attar M, Bab N, Lahat O, et al. Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo. Nucleic Acids Res. 2005;33(4):1298-308.

11. Kralick AE, and Zemel BS. Evolutionary Perspectives on the Developing Skeleton and Implications for Lifelong Health. Front Endocrinol (Lausanne). 2020;11:99.

12. Bab I, Gazit D, Chorev M, Muhlrad A, Shteyer A, Greenberg Z, et al. Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity. EMBO J. 1992;11(5):1867-73.

13. Bab I, Gavish H, Namdar-Attar M, Muhlrad A, Greenberg Z, Chen Y, et al. Isolation of mitogenically active C-terminal truncated pentapeptide of osteogenic growth peptide from human plasma and culture medium of murine osteoblastic cells. J Pept Res. 1999;54(5):408-14.

14. Bar-Shavit Z. Taking a toll on the bones: regulation of bone metabolism by innate immune regulators. Autoimmunity. 2008;41(3):195-203.

15. Blaschke M, Koepp R, Cortis J, Komrakova M, Schieker M, Hempel U, et al. IL-6, IL-1beta, and TNF-α only in combination influence the osteoporotic phenotype in Crohn's patients via bone formation and bone resorption. Adv Clin Exp Med. 2018;27(1):45-56.

16. Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, et al. Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med. 2006;12(6):657-64.

17. Patil C, Zhu X, Rossa C, Jr., Kim YJ, and Kirkwood KL. p38 MAPK regulates IL-1beta induced IL-6 expression through mRNA stability in osteoblasts. Immunol Invest. 2004;33(2):213-33.

Author details

1. Bitya Raphael-Mizrahi

   Department of Anatomy & Anthropology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review and editing

   For correspondence

   bityar@tau.ac.il

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8629-1088

2. Malka Attar-Namdar

   Bone Laboratory, Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

   Contribution

   Formal analysis, Investigation, Methodology, Project administration

   Competing interests

   No competing interests declared

3. Mukesh Chourasia

   Institute for Drug Research, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

   Contribution

   Formal analysis, Methodology, Software, Visualization

   Competing interests

   No competing interests declared

4. Maria G Cascio

   Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

5. Avital Shurki

   Institute for Drug Research, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision

   Competing interests

   No competing interests declared

6. Joseph Tam

   Institute for Drug Research, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

   Contribution

   Formal analysis, Methodology, Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0948-0093

7. Moshe Neuman

   Bone Laboratory, Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

8. Neta Rimmerman

   Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   Formal analysis, Investigation, Methodology, Validation

   Competing interests

   No competing interests declared

9. Zvi Vogel

   Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   Investigation, Resources, Supervision

   Competing interests

   No competing interests declared

10. Arie Shteyer

    Department of Oral and Maxillofacial Surgery, Hadassah-Hebrew University Hospital, Jerusalem, Israel

    Contribution

    Resources, Supervision

    Competing interests

    No competing interests declared

11. Roger G Pertwee

    Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom

    Contribution

    Formal analysis, Methodology, Resources, Supervision

    Competing interests

    No competing interests declared

12. Andreas Zimmer

    Institute of Molecular Psychiatry, University of Bonn, Bonn, Germany

    Contribution

    Investigation, Methodology, Supervision

    Competing interests

    No competing interests declared

13. Natalya M Kogan

    Bone Laboratory, Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

    Contribution

    Formal analysis, Investigation, Methodology, Supervision

    Contributed equally with

    Itai Bab and Yankel Gabet

    Competing interests

    No competing interests declared

14. Itai Bab

    Bone Laboratory, Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

    Contribution

    Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - original draft

    Contributed equally with

    Natalya M Kogan and Yankel Gabet

    Competing interests

    No competing interests declared

15. Yankel Gabet

    Department of Anatomy & Anthropology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing

    Contributed equally with

    Natalya M Kogan and Itai Bab

    For correspondence

    yankel@tauex.tau.ac.il

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7494-0631

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