Osteogenic growth peptide is a potent anti-inflammatory and bone preserving hormone via cannabinoid receptor type 2.

The endocannabinoid system consists mainly of 2-arachidonoylglycerol and anandamide, as well as cannabinoid receptor type 1 and type 2 (CB2). Based on previous studies, we hypothesized that a circulating peptide previously identified as osteogenic growth peptide (OGP) maintains a bone-protective CB2 tone. We tested OGP activity in mouse models and cells, and in human osteoblasts. We show that the OGP effects on osteoblast proliferation, osteoclastogenesis, and macrophage inflammation in vitro, as well as rescue of ovariectomy-induced bone loss and prevention of ear edema in vivo are all abrogated by genetic or pharmacological ablation of CB2. We also demonstrate that OGP binds at CB2 and may act as both an agonist and positive allosteric modulator in the presence of other lipophilic agonists. In premenopausal women, OGP circulating levels significantly decline with age. In adult mice, exogenous administration of OGP completely prevented age-related bone loss. Our findings suggest that OGP attenuates age-related bone loss by maintaining a skeletal CB2 tone. Importantly, they also indicate the occurrence of an endogenous peptide that signals via CB2 receptor in health and disease.

HU-671, a Novel Oleoyl Serine Derivative, Exhibits Enhanced Efficacy in Reversing Ovariectomy-Induced Osteoporosis and Bone Marrow Adiposity.

Oleoyl serine (OS), an endogenous fatty acyl amide (FAA) found in bone, has been shown to have an anti-osteoporotic effect. OS, being an amide, can be hydrolyzed in the body by amidases. Hindering its amide bond by introducing adjacent substituents has been demonstrated as a successful method for prolonging its skeletal activity. Here, we tested the therapeutic efficacy of two methylated OS derivatives, oleoyl α-methyl serine (HU-671) and 2-methyl-oleoyl serine (HU-681), in an ovariectomized mouse model for osteoporosis by utilizing combined micro-computed tomography, histomorphometry, and cell culture analyses. Our findings indicate that daily treatment for 6 weeks with OS or HU-671 completely rescues bone loss, whereas HU-681 has only a partial effect. The increased bone density was primarily due to enhanced trabecular thickness and number. Moreover, the most effective dose of HU-671 was 0.5 mg/kg/day, an order of magnitude lower than with OS. The reversal of bone loss resulted from increased bone formation and decreased bone resorption, as well as reversal of bone marrow adiposity. These results were further confirmed by determining the serum levels of osteocalcin and type 1 collagen C-terminal crosslinks, as well as demonstrating the enhanced antiadipogenic effect of HU-671. Taken together, these data suggest that methylation interferes with OS's metabolism, thus enhancing its effects by extending its availability to its target cells.

Magel2 Modulates Bone Remodeling and Mass in Prader-Willi Syndrome by Affecting Oleoyl Serine Levels and Activity.

Among a multitude of hormonal and metabolic complications, individuals with Prader-Willi syndrome (PWS) exhibit significant bone abnormalities, including decreased BMD, osteoporosis, and subsequent increased fracture risk. Here we show in mice that loss of Magel2, a maternally imprinted gene in the PWS critical region, results in reduced bone mass, density, and strength, corresponding to that observed in humans with PWS, as well as in individuals suffering from Schaaf-Yang syndrome (SYS), a genetic disorder caused by a disruption of the MAGEL2 gene. The low bone mass phenotype in Magel2-/- mice was attributed to reduced bone formation rate, increased osteoclastogenesis and osteoclast activity, and enhanced trans-differentiation of osteoblasts to adipocytes. The absence of Magel2 in humans and mice resulted in reduction in the fatty acid amide bone homeostasis regulator, N-oleoyl serine (OS), whose levels were positively linked with BMD in humans and mice as well as osteoblast activity. Attenuating the skeletal abnormalities in Magel2-/- mice was achieved with chronic administration of a novel synthetic derivative of OS. Taken together, Magel2 plays a key role in modulating bone remodeling and mass in PWS by affecting OS levels and activity. The use of potent synthetic analogs of OS should be further tested clinically as bone therapeutics for treating bone loss. © 2018 American Society for Bone and Mineral Research.

The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice.

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins.

Smoke carcinogens cause bone loss through the aryl hydrocarbon receptor and induction of Cyp1 enzymes.

Smoking is a major risk factor for osteoporosis and fracture, but the mechanism through which smoke causes bone loss remains unclear. Here, we show that the smoke toxins benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interact with the aryl hydrocarbon receptor (Ahr) to induce osteoclastic bone resorption through the activation of cytochrome P450 1a/1b (Cyp1) enzymes. BaP and TCDD enhanced osteoclast formation in bone marrow cell cultures and gavage with BaP stimulated bone resorption and osteoclastogenesis in vivo. The osteoclastogenesis triggered by BaP or RANK-L was reduced in Ahr(-/-) cells, consistent with the high bone mass noted in Ahr(-/-) male mice. The receptor activator of NF-κB ligand (RANK-L) also failed to induce the expression of Cyp1 enzymes in Ahr(-/-) cells. Furthermore, the osteoclastogenesis induced by TCDD was lower in Cyp1a1/1a2(-/-) and Cyp1a1/1a2/1b1(-/-) cultures, indicating that Ahr was upstream of the Cyp enzymes. Likewise, the pharmacological inhibition of the Cyp1 enzymes with tetramethylsilane or proadifen reduced osteoclastogenesis. Finally, deletion of the Cyp1a1, Cyp1a2, and Cyp1b1 in triple knockout mice resulted in reduced bone resorption and recapitulated the high bone mass phenotype of Ahr(-/-) mice. Overall, the data identify the Ahr and Cyp1 enzymes not only in the pathophysiology of smoke-induced osteoporosis, but also as potential targets for selective modulation by new therapeutics.

Bone marrow oxytocin mediates the anabolic action of estrogen on the skeleton.

Estrogen uses two mechanisms to exert its effect on the skeleton: it inhibits bone resorption by osteoclasts and, at higher doses, can stimulate bone formation. Although the antiresorptive action of estrogen arises from the inhibition of the MAPK JNK, the mechanism of its effect on the osteoblast remains unclear. Here, we report that the anabolic action of estrogen in mice occurs, at least in part, through oxytocin (OT) produced by osteoblasts in bone marrow. We show that the absence of OT receptors (OTRs) in OTR(-/-) osteoblasts or attenuation of OTR expression in silenced cells inhibits estrogen-induced osteoblast differentiation, transcription factor up-regulation, and/or OT production in vitro. In vivo, OTR(-/-) mice, known to have a bone formation defect, fail to display increases in trabecular bone volume, cortical thickness, and bone formation in response to estrogen. Furthermore, osteoblast-specific Col2.3-Cre(+)/OTR(fl/fl) mice, but not TRAP-Cre(+)/OTR(fl/fl) mice, mimic the OTR(-/-) phenotype and also fail to respond to estrogen. These data attribute the phenotype of OTR deficiency to an osteoblastic rather than an osteoclastic defect. Physiologically, feed-forward OT release in bone marrow by a rising estrogen concentration may facilitate rapid skeletal recovery during the latter phases of lactation.

CB2 cannabinoid receptor targets mitogenic Gi protein-cyclin D1 axis in osteoblasts.

CB2 is a Gi protein-coupled receptor activated by endo- and phytocannabinoids, thus inhibiting stimulated adenylyl cyclase activity. CB2 is expressed in bone cells and Cb2 null mice show a marked age-related bone loss. CB2-specific agonists both attenuate and rescue ovariectomy-induced bone loss. Activation of CB2 stimulates osteoblast proliferation and bone marrow derived colony-forming units osteoblastic. Here we show that selective and nonselective CB2 agonists are mitogenic in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures. The CB2 mitogenic signaling depends critically on the stimulation of Erk1/2 phosphorylation and de novo synthesis of MAP kinase-activated protein kinase 2 (Mapkapk2) mRNA and protein. Further downstream, CB2 activation enhances CREB transcriptional activity and cyclin D1 mRNA expression. The CB2-induced stimulation of CREB and cyclin D1 is inhibitable by pertussis toxin, the MEK-Erk1/2 inhibitors PD098059 and U0126, and Mapkapk2 siRNA. These data demonstrate that in osteoblasts CB2 targets a Gi protein-cyclin D1 mitogenic axis. Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical intermediates in this axis.

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.

Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects.

The angiogenic events that accompany bone regeneration function as a "limiting factor" and are the primary regulatory mechanisms that direct the healing process. The general aim of this study was to test whether blood-derived progenitor cells that have endothelial characteristics (EPC), when applied to a large segmental defect, would promote bone regeneration. We established a critical-sized gap platform in sheep tibiae. Our model system takes advantage of the physiological wound healing process that occurs during the first two weeks following injury, and results in the gap being filled with scar tissue. EPC were expanded ex-vivo and 2 x 10(7) cells/0.2 ml were implanted into a wedged-shaped canal excavated in the fibrotic scar tissue. Sham treated sheep served as controls. Bone regeneration was followed every two weeks for three months by X-ray radiography. At the end of the experimental period, the regenerating segments were subjected to micro-computed tomographic (microCT) analysis. While minimal bone formation was detected in sham-treated sheep, six out of seven autologous EPC-transplanted sheep showed initial mineralization already by 2 weeks and complete bridging by 8-12 weeks post EPC transplantation. Histology of gaps 12 weeks post sham treatment showed mostly fibrotic scar tissue. On the contrary, EPC transplantation led to formation of dense and massive woven bone all throughout the defect. The results of this preclinical study open new therapeutic opportunities for the treatment of large scale bone injuries.

Restrain of bone growth by estrogen-mimetic peptide-1 (EMP-1): a micro-computed tomographic study.

Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (microCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new microCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.

Parathyroid hormone 1-34 enhances titanium implant anchorage in low-density trabecular bone: a correlative micro-computed tomographic and biomechanical analysis.

The use of endosseous titanium implants is the standard of care in dentistry and orthopaedic surgery. Nevertheless, implantation in low-density bone has a poor prognosis and experimental studies show delayed implant anchorage following gonadectomy-induced bone loss. Intermittently administered human parathyroid hormone 1-34 [iahPTH(1-34)] is the leading bone anabolic therapy. Hence, this study assessed whether iahPTH(1-34) enhances titanium implant integration in low-density bone. Threaded titanium implants, 0.9 mm in diameter, were inserted horizontally into the proximal tibial metaphysis of 5-month-old rats, 7 weeks postorchiectomy (ORX). Subcutaneous administration of iahPTH(1-34), at 5, 25 and 75 microg/kg/day commenced immediately thereafter and lasted for 8 weeks. Quantitative micro-computed tomography (muCT) at the implantation site was carried out at 15 microm resolution using high energy and long integration time to minimize artifacts resulting from the high implant radiopacity. Osseointegration (OI) was calculated as percent implant surface in contact with bone (%OI) quantified as the ratio of "bone"-to-total voxels in contact with the implant. Additionally, the trabecular bone volume density (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and connectivity density (Conn.D) were measured in the peri-implant bone. All microCT parameters were stimulated by iahPTH(1-34) dose-dependently; the percent maximal enhancement was %OI = 143, BV/TV = 257, Tb.Th = 150, Tb.N = 140 and Conn.D = 193. The maximal values of %OI, BV/TV and Tb.Th in iahPTH(1-34)-treated ORX rats exceeded significantly those measured in the implantation site of untreated sham-ORX controls. The same specimens were then subjected to pullout biomechanical testing. The biomechanical parameters were also enhanced by iahPTH(1-34) dose-dependently, exceeding the values recorded in the sham-ORX controls. The percent iahPTH(1-34)-induced maximal enhancement was: ultimate force = 315, stiffness = 270 and toughness = 395. Except for the BV/TV and Tb.Th, there was no significant difference between the effect of the 25 and 75 microg/kg/day doses. There was a highly significant correlation between the morphometric and biomechanical parameters suggesting the use of quantitative CT as predictive of the implant mechanical properties. These findings demonstrate that iahPTH(1-34) effectively stimulates implant anchorage in low-density trabecular bone and thus the feasibility of administering iahPTH(1-34) to improve the clinical prognosis in low-density trabecular bone sites.

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass.

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.

Osteogenic growth peptide: from concept to drug design.

Recently, the osteogenic growth peptide (OGP) and its C-terminal pentapeptide H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)] have attracted considerable clinical interest as bone anabolic agents and hematopoietic stimulators. They are present in mammalian serum in micromolar concentrations, increase bone formation and trabecular bone density, and stimulate fracture healing when administered to mice and rats. In cultures of osteoblastic and other bone marrow stromal cells, derived from human and other mammalian species, OGP regulates proliferation, alkaline phosphatase activity and matrix mineralization via an autocrine/paracrine mechanism. In vivo it also regulates the expression of type I collagen and the receptor for basic fibroblast growth factor. In addition, OGP and OGP(10-14) enhance hematopoiesis, including the stimulation of bone marrow transplant engraftment and hematopoietic regeneration after ablative chemotherapy. Apparently, the hematopoietic effects of these peptides are secondary to their effect on the bone marrow stroma. Detailed structure-activity relationship study identified the side chains of Tyr(10) and Phe(12) as the principal pharmacophores for OGP-like activity. Recently, it has been demonstrated that several cyclostereoisomers of OGP(10-14), including the analogue retro-inverso (Gly-Gly-D-Phe-Gly-D-Tyr), share the full spectrum of OGP-like bioactivities. Taken together, OGP represents an interesting case of a "housekeeping" peptide that plays an important role in osteogenesis and hematopoiesis, and interacts with its putative macromolecular target via distinct pharmacophores presented in a specific spatial organization.

Bioactive pseudopeptidic analogues and cyclostereoisomers of osteogenic growth peptide C-terminal pentapeptide, OGP(10-14).

The osteogenic growth peptide (OGP) is a key factor in the mechanism of the systemic osteogenic response to local bone marrow injury. When administered in vivo, OGP stimulates osteogenesis and hematopoiesis. The C-terminal pentapeptide OGP(10-14) is the minimal amino acid sequence that retains the full OGP-like activity. Apparently, it is also the physiologic active form of OGP. Residues Tyr(10), Phe(12), Gly(13), and Gly(14) of OGP are essential for the OGP(10-14) activity. The present study explored the functional role of the peptide bonds, carboxyl and amino terminal groups, and conformational freedom in OGP(10-14). Transformations replacing the peptide bonds with surrogates such as Psi(CH(2)NH), Psi(CONMe), and Psi(CH(2)CH(2)) demonstrated that amide bonds do not contribute significantly to OGP(10-14) bioactivity. End-to-end cyclization yielded the fully bioactive cyclic pentapeptide c(Tyr-Gly-Phe-Gly-Gly). The retroinverso analogue c(Gly-Gly-phe-Gly-tyr), a cyclostereoisomer of c(Tyr-Gly-Phe-Gly-Gly), is at least as potent as the parent cyclic pentapeptide. The unique structure-activity relations revealed in this study suggest that the spatial presentation of the Tyr and Phe side chains has a major role in the productive interaction of OGP(10-14) and its truncated and conformationally constrained analogues with their cognate cellular target.