Osteoanabolic Effects Research Articles

HPTH(3-34)(29-34) selectively activated PKC and mimicked osteoanabolic effects of hPTH(1-34).

Teriparatide (hPTH(1-34)) exhibits both osteoanabolic and osteocatabolic effects. We generated a novel PTH analog by duplicating the PTH(29-34) domain to hPTH(3-34) (named MY-1), which was identified to activate PKC but not PLC and cAMP/PKA signaling. It increased osteo-differentiation but did not affect osteoclastogenesis and RANKL expression in primary osteoblasts or bone marrow cells. MY-1 and hPTH(1-34) increased the synthesis and decreased the degradation οf β-catenin protein in osteoblasts, while PKC inhibitor blunted such effects. In vivo results indicated that intermittent MY-1 and hPTH(1-34) prevented bone loss in ovariectomized mice, and that MY-1 infusion increased bone volume in normal mice. Histological analysis observed more osteoclasts surrounding the cancellous bone surface in hPTH(1-34), but not MY-1 treated mice. We conclude that MY-1 mimicked the osteoanabolic but not the osteocatabolic effects of hPTH(1-34), which is related to PKC and β-catenin signaling. Such anabolic-only analog provides a new strategy to study PTH's versatile functions and design new medicines to treat osteoporosis and bone defects.

Skeletal restoration by phosphodiesterase 5 inhibitors in osteopenic mice: Evidence of osteoanabolic and osteoangiogenic effects of the drugs.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides and thereby regulate diverse cellular functions. The reports on the skeletal effects of PDE inhibitors are conflicting. Here, we screened 17 clinically used non-xanthine PDE inhibitors (selective and non-selective) using mouse calvarial osteoblasts (MCO) where the readout was osteoblast differentiation. From this screen, we identified sildenafil and vardenafil (both PDE5 inhibitors) having the least osteogenic EC50. Both drugs significantly increased vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expressions in MCO and the nitric oxide synthase inhibitor L-NAME completely blocked VEGF expression induced by these drugs. Sunitinib, a tyrosine receptor kinase inhibitor that also blocks VEGFR2 blocked sildenafil-/vardenafil-induced osteoblast differentiation. At half of their human equivalent doses, i.e. 6.0mg/kg sildenafil and 2.5mg/kg vardenafil, the maximum bone marrow level of sildenafil was 32% and vardenafil was 21% of their blood levels. At these doses, both drugs enhanced bone regeneration at the femur osteotomy site and completely restored bone mass, microarchitecture, and strength in OVX mice. Furthermore, both drugs increased surface referent bone formation and serum bone formation marker (P1NP) without affecting the resorption marker (CTX-1). Both drugs increased the expression of VEGF and VEGFR2 in bones and osteoblasts and increased skeletal vascularity. Sunitinib completely blocked the bone restorative and vascular effects of sildenafil and vardenafil in OVX mice. Taken together, our study suggested that sildenafil and vardenafil at half of their adult human doses completely reversed osteopenia in OVX mice by an osteogenic mechanism that was associated with enhanced skeletal vascularity.

Long acting GLP-1 analog liraglutide ameliorates skeletal muscle atrophy in rodents

BackgroundSkeletal muscle atrophy is characterized by muscle wasting with partial or complete functional loss. Skeletal muscle atrophy severely affects the quality of life and currently, there is no available therapy except for spinal muscular atrophy. ObjectiveDrug repositioning is a promising strategy that reduces cost and time due to prior availability of safety and toxicity details. Here we investigated myogenic and anti-atrophy effects of glucagon-like peptide-1 (GLP-1) analog liraglutide. MethodsWe used several in vitro atrophy models in C2C12 cells and in vivo models in Sprague Dawley rats to study Liraglutide's efficacy. Western blotting was used to assess cAMP-dependent signaling pathways specifically activated by liraglutide. Therapeutic efficacy of liraglutide was investigated by histological analysis of transverse muscle sections followed by morphometry. Myogenic capacity was investigated by immunoblotting for myogenic factors. ResultsLiraglutide induced myogenesis in C2C12 myoblasts through GLP-1 receptor via a cAMP-dependent complex network of signaling events involving protein kinase A, phosphoinositide 3-kinase/protein kinase B, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Liraglutide imparted protection against freeze injury, denervation, and dexamethasone -induced skeletal muscle atrophy and improved muscular function in all these models. In a therapeutic model, liraglutide restored myofibrillar architecture in ovariectomy-induced atrophy. Anti-atrophy actions of liraglutide involved suppression of atrogene expression and enhancement in expression of myogenic factors. ConclusionLiraglutide imparted protection and restored myofibrillar architecture in diverse models of muscle atrophy. Given its potent anti-atrophy, and recently reported osteoanabolic effects, we propose liraglutide's clinical evaluation in skeletal muscle atrophy and musculoskeletal disorders associated with diverse pathologies.

Cyclic adenosine monophosphate-dependent activation of transient receptor potential vanilloid 4 (TRPV4) channels in osteoblast-like MG-63 cells

Parathyroid hormone (PTH) directly interacts with bone remodeling osteoblasts and osteocytes expressing the G-protein coupled receptor PTH receptor 1 (PTH1R), and its osteoanabolic effects mostly involve the cAMP/PKA signaling cascade. Considering that PTH-dependent calcium entry in rat enterocytes is reproduced by the adenylate cyclase agonist forskolin or by cAMP analogues, possible involvement of calcium as a second messenger in PTH-dependent cAMP signaling was investigated in MG-63 cells. First, Ca2+ influx was confirmed in Fluo3-loaded MG-63 cells treated with a cell-permeable cAMP analog. Second, PTH (1–34) and forskolin promoted calcium influxes that were completely abrogated by the PKA inhibitor H-89. Ca2+ entry was not reproduced when PTH (1–34) was combined with the PKC-activating competitor PTH (3–34). Vanilloid transient potential (TRPV) channel inhibitor Ruthenium Red, but not a voltage-dependent calcium channel (VDCC) inhibitor nifedipine, efficiently stunted Ca2+ entry, and comparable abrogation was reproduced in cells treated with TRPV4-selective inhibitor RN-1734 or transfected with TRPV4-specific siRNA. Interestingly, PTH-driven Ca2+ through TRPV4 significantly inhibited MG63 cell migration through a mechanism requiring extracellular Ca2+. In contrast, the inhibitory effects of forskolin on migration were refractory to TRPV4 silencing or to RN-1734. Altogether, our results indicate that single treatment with PTH (1–34) promotes extracellular calcium entry through TRPV4 channels in MG-63 cells through a cAMP/PKA-dependent mechanism, and that this influx affects cell migration.

Lrp4 Mediates Bone Homeostasis and Mechanotransduction through Interaction with Sclerostin In Vivo

SummaryWnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse.

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice.

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation-an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca2+ ] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.

Testosterone replacement in congenital hypogonadotropic hypogonadism maintains bone density but has only limited osteoanabolic effects.

Congenital hypogonadotropic hypogonadism (CHH) is a rare condition characterized by complete sex steroid deficiency. Therefore, CHH is a unique human model to study the impact of long-term testosterone replacement therapy (TRT) on bone. In this single-center retrospective observational study, we assessed the long-term impact of TRT on femoral and lumbar bone mineral density (BMD) in adult CHH men. A total of 25 patients with CHH were included. Femoral and lumbar BMD was assessed by dual-energy X-ray absorptiometry (DEXA) and reported as T-scores. In six patients (treatment-naive group), BMD was measured before start of TRT. The other 19 (pre-treated group) had received TRT for a median duration of 7years (range 1-41years) before first BMD measurement. Age at which TRT was started ranged from 12 to 57years old. Median time between first and last DEXA scan was 11years (range 2-28). At the first DEXA scan, 83% and 61% of CHH patients had lumbar and femoral osteopenia/osteoporosis, respectively. In the treatment-naive group, the increase in lumbar T-score was 2.19±0.13 (mean ± SEM, p<0.01 between first and last DEXA scan) and 1.47±0.29 at femoral level (p<0.001). For the pre-treated group, the increase in lumbar and femoral T-score was 0.77±0.17 (p<0.001) and 0.19±0.12 (p=0.13), respectively. However, lumbar and femoral osteopenia/osteoporosis persisted in 61% and 48% of CHH patients even after several years of continuous TRT. Additionally, BMD clearly decreased in patients who interrupted TRT. Despite modest improvement after starting TRT, BMD remains in the osteopenic/osteoporotic range in most patients with CHH. However, prolonged TRT prevents further bone loss, both at lumbar and femoral level.

An Adiponectin Receptor Agonist Reduces Type 2 Diabetic Periodontitis

Periodontitis is twice as prevalent in diabetics as in nondiabetics, and type 2 diabetes (T2D)–associated periodontitis is severe in many cases due to the altered and aberrant functions of bone cells in hyperglycemic conditions. Therefore, developing an effective method to halt the disease process, as well as restore and regenerate lost alveolar bone to reserve the natural teeth in diabetics, is critically important. In the current study, we applied a newly discovered adiponectin receptor agonist AdipoRon (APR) in experimental periodontitis in diabetic animal models and demonstrated the underlying molecular mechanisms. We found that when APR systemically quenched the blood sugar level in diet-induced obesity (DIO) diabetic mice, it reduced osteoclast numbers and alveolar bone loss significantly due to APR’s inhibition on osteoclast differentiation shown in our in vitro studies. APR also decreased the production of proinflammatory molecules CC chemokine ligand 2 and interleukin 6 in diseased gingival tissues. On the other hand, APR promoted alveolar bone regeneration through enhancing osteogenic differentiation and decreasing stromal cell–derived factor 1 in the bone marrow that facilitates stem cell migration. Same results were achieved by APR treatment of periodontitis induced in adiponectin (APN) knockout mice, indicating the ability of APR to activate the endogenous APN receptors to exert osteoanabolic effects. In summary, our study supports the notion that APR could be used as an effective multipronged approach to target T2D-associated periodontitis.

Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition.

The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.

Intermittent Administration of Parathyroid Hormone 1-34 Enhances Osteogenesis of Human Mesenchymal Stem Cells by Regulating Protein Kinase Cδ.

Human mesenchymal stem cells (hMSCs) can differentiate into osteoblasts and are regulated by chemical cues. The recombinant N-terminal (1–34 amino acids) fragment of the parathyroid hormone (PTH (1–34)) is identified to promote osteogenesis. The osteoanabolic effects of intermittent PTH (1–34) treatment are linked to a complex consisting of signaling pathways; additionally, protein kinase C (PKC) act as mediators of multifunctional signaling transduction pathways, but the role of PKC δ (PKCδ), a downstream target in regulating osteoblast differentiation during intermittent administration of PTH (1–34) is less studied and still remains elusive. The purpose of this study is to examine the role of PKCδ during intermittent and continuous PTH (1–34) administration using osteoblast-lineage-committed hMSCs. Relative gene expression of osteoblast-specific genes demonstrated significant upregulation of RUNX2, type I Collagen, ALP, and Osterix and increased alkaline phosphatase activity in the presence of PTH (1–34). Intermittent PTH (1–34) administration increased PKC activity at day 7 of osteogenic differentiation, whereas inhibition of PKC activity attenuated these effects. In addition, the specific isoform PKCδ was activated upon treatment. These findings demonstrate that intermittent PTH (1–34) treatment enhances the osteogenesis of hMSCs by upregulating osteoblast-specific genes via PKCδ activation.

EZH2 Inhibitor GSK126 Exhibits Osteo-Anabolic Properties in MM Bone Disease and Synergizes with Bortezomib to Inhibit MM Cell Viability

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts (OCL) resulting in severe bone pain, pathological fractures and enhanced mortality. These bone lesions rarely heal even after therapeutic remission due to MM-induced suppression of bone marrow stromal cells (BMSC) differentiation into functional osteoblasts (OB), enhancing their support of MM growth and survival. Although new therapies for MM have greatly improved progression-free survival and overall survival, MM remains incurable for most patients.EZH2is the methyltransferase subunit of the Polycomb Repressive Complex 2 (PRC2) catalyzing the tri-methylation of histone-3 lysine-27 (H3K27me3), which induces gene repression. We previously reported that MM cells induce increased recruitment of EZH2 to the Runx2 gene in the murine pre-OB cell line MC4 resulting in H3K27me3-mediated repression of Runx2, thereby causing repression of OB differentiation. In the present study, we show that the repressive H3K27me3 levels are elevated with reduced H3K9 acetylation on the Runx2 promoter in MM patient derived BMSC. Using the selective small molecule inhibitor GSK126 to block EZH2 activity, we investigated if the MM-induced epigenetic repression of Runx2 is reversible. We found that GSK126 enhances the active architecture at the Runx2 promoter in both BMSC from MM patients and MM-treated MC4 cells, and rescues the osteogenic potential and mineralization capability. 5TGM1 MM cells exhibited reduced adhesion to MC4 pre-OB pre-treated with GSK126 as compared to vehicle in an in vitro adhesion assay, suggesting that EZH2 inhibition will decrease pre-OB support for MM cells.In addition to the direct osteo-anabolic effects of EZH2 inhibition on OB differentiation, we investigated if GSK126 treatment affected the pro-inflammatory, myeloma-amplified OCL differentiation that results in enhanced osteolysis in MM. We reported that expansion of bone marrow monocytes (BMM), which are OCL precursors, in the presence of MM1.S-conditioned media, significantly enhanced formation of TRAP-positive multinucleated OCL. Here we show that MM-conditioned media significantly increased EZH2 and OCL marker genes NFATc1, RANK, OSCAR, Cathepsin K, and DC-STAMP mRNA and enhanced RANKL-induced formation of OCL, which was blocked by GSK126. We found that EZH2 and the corresponding H3K27me3 levels increase during the initial 24h of RANKL-induced osteoclastogenesis and that GSK126 inhibition during the first 24h of RANKL treatment was necessary and sufficient to decrease formation of mature OCL. Thus, GSK126 is likely to both protect the bone from new lytic lesions and help heal the existing osteolytic lesions in MM.We found that GSK126 treatment (48h) inhibited proliferation of human (MM1.S, RPMI, U266, H929, and JJN3) and murine (5TGM1) MM cell lines, and primary 138+MM patient cells. We analyzed the interaction of GSK126 and bortezomib using IC50 constant ratio drug combinations based on the Chou & Talalay method using the CompuSyn program. We found that GSK126 synergizes with bortezomib to inhibit cell viability of these MM cell lines and activated apoptosis via induction of activated caspase-3. We used 5TGM1-GLuc-GFP cells, which secrete Guassia luciferase (GLuc), to measure MM cell viability in a longitudinal drug treatment study. GLuc levels in media correlated with cell proliferation, which was inhibited by single and combination doses of GSK126 and bortezomib, further demonstrating the synergism of these drugs. GSK126 decreased EZH2 mRNA and protein levels in MM cells, which is a cell-type specific effect, since we did not observe EZH2 protein alteration in GSK126 treated OCL progenitors.Our in vitro study suggests that selective inhibition of the epigenetic modifier EZH2 using GSK126 would positively regulate the bone microenvironment by improving the osteogenic potential of MM-exposed BMSC and suppressing OCL formation, and would directly decrease MM cell survival, particularly in combination with bortezomib. These data suggest that targeting EZH2 activity alone or in combination with bortezomib may prove a valuable therapeutic strategy to improve bone health and limit disease progression in MM patients. DisclosuresRoodman:Amgen: Consultancy.

Fibroblast Growth Factor Receptor 3 Deficiency Does Not Impair the Osteoanabolic Action of Parathyroid Hormone on Mice.

Summary: PTH stimulates bone formation in Fgfr3 knockout mice through promotion of proliferation and differentiation in osteoblasts.Introduction: Previous studies showed that endogenous fibroblast growth factor 2 (FGF-2) is required for parathyroid hormone (PTH)-stimulated bone anabolic effects, however, the exact mechanisms by which PTH stimulate bone formation and the function of FGF receptors in mediating these actions are not fully defined. FGF receptor 3 (FGFR3) has been characterized as an important regulator of bone metabolism and is confirmed to cross-talk with PTH/PTHrP signal in cartilage and bone development.Methods: Fgfr3 knockout and wild-type mice at 2-month-old and 4-month-old were intraperitoneally injected with PTH intermittently for 4 weeks and then the skeletal responses to PTH were assessed by dual energy X-ray absorptiometry (DEXA), micro-computed tomography (μCT) and bone histomorphometry.Results: Intermittent PTH treatment improved bone mineral density (BMD) and femoral mechanical properties in both Fgfr3-/- and wild-type mice. Histomorphometric analysis showed that bone formation and bone resorption were increased in both genotypes following PTH treatment. PTH treatment increased trabecular bone volume (BV/TV) in WT and Fgfr3-deficient mice. The anabolic response in Fgfr3-deficient and wild-type bone is characterized by an increase of both bone formation and resorption-related genes following PTH treatment. In addition, we found that Fgfr3 null osteoblasts (compared to wild-type controls) maintained normal abilities to response to PTH-stimulated increase of proliferation, differentiation, expression of osteoblastic marker genes (Cbfa1, Osteopontin and Osteocalcin), and phosphorylation of Erk1/2.Conclusions: Bone anabolic effects of PTH were not impaired by the absence of FGFR3, suggesting that the FGFR3 signaling may not be required for osteoanabolic effects of PTH activities.

Chondroitin sulfate-E mediates estrogen-induced osteoanabolism.

Osteoporosis is an age-related disorder of bone remodeling in which bone resorption outstrips bone matrix deposition. Although anticatabolic agents are frequently used as first-line therapies for osteoporosis, alternative anabolic strategies that can enhance anabolic, osteogenic potential are actively sought. Sex steroid hormones, particularly estrogens, are bidirectional regulators for bone homeostasis; therefore, estrogen-mediated events are important potential targets for such anabolic therapies. Here, we show that estrogen-induced, osteoanabolic effects were mediated via enhanced production of chondroitin sulfate-E (CS-E), which could act as an osteogenic stimulant in our cell-based system. Conversely, estrogen deficiency caused reduced expression of CS-E-synthesizing enzymes, including GalNAc4S-6ST, and led to decreased CS-E production in cultures of bone marrow cells derived from ovariectomized mice. Moreover, Galnac4s6st-deficient mice had abnormally low bone mass that resulted from impaired osteoblast differentiation. These results indicated that strategies aimed at boosting CS-E biosynthesis are promising alternative therapies for osteoporosis.

New Insights Into the Physiology of Bone Regulation: the Role of Neurohormones

Bone metabolism is regulated by interaction between two skeletal cells - osteoclasts and osteoblasts. Function of these cells is controlled by a number of humoral factors, including neurohormones, which ensure equilibrium between bone resorption and bone formation. Influence of neurohormones on bone metabolism is often bimodal and depends on the tissue, in which the hormone is expressed. While hypothalamic beta-1 and beta-2-adrenergic systems stimulate bone formation, beta-2 receptors in bone tissue activate osteoclatogenesis and increases bone resorption. Chronic stimulation of peripheral beta-2 receptors is known to quicken bone loss and alter the mechanical quality of the skeleton. This is supported by the observation of a low incidence of hip fractures in patients treated with betablockers. A bimodal osteo-tropic effect has also been observed with serotonin. While serotonin synthetized in brain has osteo-anabolic effects, serotonin released from the duodenum inhibits osteoblast activity and decreases bone formation. On the other hand, both cannabinoid systems (CB1 receptors in the brain and CB2 in bone tissue) are unambiguously osteo-protective, especially with regard to the aging skeleton. Positive (protective) effects on bone have also been shown by some hypophyseal hormones, such as thyrotropin (which inhibits bone resorption) and adrenocorticotropic hormone and oxytocin, both of which stimulate bone formation. Low oxytocin levels have been shown to potentiate bone loss induced by hypoestrinism in postmenopausal women, as well as in girls with mental anorexia. In addition to reviewing neurohormones with anabolic effects, this article also reviews neurohormones with unambiguously catabolic effects on the skeleton, such as neuropeptide Y and neuromedin U. An important aim of research in this field is the synthesis of new molecules that can stimulate osteo-anabolic or inhibiting osteo-catabolic processes.

The Anabolic Effect of Teriparatide is Undermined by Low Levels of High-Density Lipoprotein Cholesterol

Intermittent parathyroid hormone (PTH) administration has a potent ability to increase bone mass, regardless of underlying conditions or species. A recent study using LDLR(-/-) mice showed that the anabolic effect of PTH was blunted by hyperlipidemia, whereas PTH anabolism was rescued by enhancement of high-density lipoprotein cholesterol (HDL-C) function. We conducted a retrospective longitudinal study to determine whether lipid profiles also affect the anabolic effect of intermittent PTH treatment in humans. Fifty-two patients (8 males and 44 females, ages 38-85 years) with severe osteoporosis who had been treated with teriparatide (TPTD, recombinant human PTH(1-34) for 12 months were studied at Severance Hospital, Yonsei University. C-telopeptide (CTX) and osteocalcin (OCN) were measured at 0, 3, and 12 months; and total cholesterol, triglycerides, and HDL-C were measured at baseline. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry at 0 and 12 months. Lumbar spine BMD increased significantly after 12 months of treatment with TPTD (10.0 ± 9.3%, p < 0.001). Initial 3-month changes in CTX and OCN levels revealed positive correlations with the increase in lumbar BMD (r = 0.546, p = 0.001 and r = 0.500, p = 0.006, respectively). Moreover, percentage change in lumbar BMD at 12 months showed a negative correlation with baseline total cholesterol (r = -0.438, p = 0.009) and a positive correlation with HDL-C (r = 0.498, p = 0.016). A smaller 3-month increase in OCN and a lower HDL-C level at baseline were associated with a smaller lumbar BMD increase after TPTD treatment, even after adjustment for age, sex, and other confounding factors (β = 0.462, p = 0.031 for ΔOCN and β = 0.670, p = 0.004 for HDL-C). Plasma levels of lipids, especially HDL-C, seem to be associated with the extent of osteoanabolic effects of TPTD in humans.

Systemic treatment with the sphingosine‐1‐phosphate analog FTY720 does not improve fracture healing in mice

Sphingosine-1-phosphate (S1P) has recently been recognized as a crucial coupling molecule of osteoclast and osteoblast activity provoking osteoanabolic effects. Targeting S1P receptors could, therefore, be a potential strategy to support bone formation in osteopenic diseases or in fracture repair. Here we investigated whether systemic treatment with the S1P analog FTY720 (Fingolimod) could improve fracture healing. Twelve-week-old, female C57BL/6 mice received an osteotomy of the femur, which was stabilized using an external fixator. The mice received a daily subcutaneous injection of either FTY720 (6 mg/kg) or vehicle from the third postoperative day. Fracture healing was evaluated after 10 and 21 days using biomechanical testing, µ-computed tomography, and histomorphometry. Because FTY720 is supposed to influence osteoclast recruitment, osteoclasts were identified in the fracture callus by staining for tartrate resistant acid phosphatase (TRAP). There were no significant differences in callus mechanical properties, tissue composition and osteoclast number between the groups, suggesting that systemically applied FTY720 did not influence bone regeneration in this model of regular fracture healing. Even if further studies should test the potency of FTY720 under unfavorable healing conditions, we conclude that the effect of systemically applied FTY720 on fracture healing might be inferior compared to other anabolic treatments. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1845-1850, 2013.

Impact of diabetes and its treatments on skeletal diseases

Diabetes mellitus is an enormous menace to public health globally. This chronic disease of metabolism will adversely affect the skeleton if not controlled. Both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are associated with an increased risk of osteoporosis and fragility fractures. Bone mineral density is reduced in T1DM, whereas patients with T2DM have normal or slightly higher bone density, suggesting impaired bone quality is involved. Detrimental effects of T1DM on the skeleton are more severe than T2DM, probably because of the lack of osteo-anabolic effects of insulin and other pancreatic hormones. In both T1DM and T2DM, low bone quality could be caused by various means, including but not limited to hyperglycemia, accumulation of advanced glycosylation end products (AGEs), decreased serum levels of osteocalcin and parathyroid hormone. Risk for osteoarthritis is also elevated in diabetic population. How diabetes accelerates the deterioration of cartilage remains largely unknown. Hyperglycemia and glucose derived AGEs could contribute to the development of osteoarthritis. Moreover, it is recognized that oral antidiabetic medicines affect bone metabolism and turnover as well. Insulin is shown to have anabolic effects on bone and hyperinsulinemia may help to explain the slightly higher bone density in patients with T2DM. Thiazolidinediones can promote bone loss and osteoporotic fractures by suppressing osteoblastogenesis and enhancing osteoclastogenesis. Metformin favors bone formation by stimulating osteoblast differentiation and protecting them against diabetic conditions such as hyperglycemia. Better knowledge of how diabetic conditions and its treatments influence skeletal tissues is in great need in view of the growing and aging population of patients with diabetes mellitus.

Ronacaleret, a calcium-sensing receptor antagonist, increases trabecular but not cortical bone in postmenopausal women

Intermittent injections of parathyroid hormone have osteoanabolic effects that increase bone mineral density (BMD). Ronacaleret is an orally administered calcium-sensing receptor antagonist that stimulates endogenous parathyroid hormone release from the parathyroid glands. Our objective was to compare the effects of ronacaleret and teriparatide on volumetric BMD (vBMD) measured by quantitative computed tomography (QCT). We conducted a randomized, placebo-controlled, dose-ranging trial at 45 academic centers with 31 sites participating in the substudy. Patients included 569 postmenopausal women with low bone mineral density; vBMD was assessed at the spine and hip in a subset of 314 women. Patients were treated for up to 12 months with open-label teriparatide 20 µg subcutaneously once daily or randomly assigned in a double-blind manner to ronacaleret 100 mg, 200 mg, 300 mg, or 400 mg once daily, alendronate 70 mg once weekly, or matching placebos. Ronacaleret increased spine integral (0.49% to 3.9%) and trabecular (1.8% to 13.3%) vBMD compared with baseline, although the increments were at least twofold lower than that attained with teriparatide (14.8% and 24.4%, respectively) but similar or superior to that attained with alendronate (5.0% and 4.9%, respectively). There were small non-dose-dependent decreases in integral vBMD of the proximal femur with ronacaleret (-0.1 to -0.8%) compared with increases in the teriparatide (3.9%) and alendronate (2.7%) arms. Parathyroid hormone (PTH) elevations with ronacaleret were prolonged relative to that seen historically with teriparatide. Ronacaleret preferentially increased vBMD of trabecular bone that is counterbalanced by small decreases in BMD at cortical sites. The relative preservation of trabecular bone and loss at cortical sites are consistent with the induction of mild hyperparathyroidism with ronacaleret therapy.

Tetrahydroquinolines as a novel series of nonsteroidal selective androgen receptor modulators: Structural requirements for better physicochemical and biological properties

A rationally designed tetrahydroquinoline (1) for nonsteroidal selective androgen receptor modulators was modified for the exploration of promising compounds by Grieco three-component condensation using various dienophiles. Based on the in vitro effects and physicochemical properties of the synthesized compounds, compound 4c was selected for further study. Compound 4c increased the femoral bone mineral density as much as DHT, but it reduced the uterus effect compared with DHT in ovariectomized rats. Thus, compound 4c has desirable osteoanabolic effects with weak undesirable effects on the uterus in a female osteoporosis model.

Effects of age on parathyroid hormone signaling in human marrow stromal cells

Human bone marrow stromal cells (hMSCs) have the potential to differentiate into osteoblasts; there are age-related decreases in their proliferation and differentiation to osteoblasts. Parathyroid hormone (PTH), when applied intermittently in vivo, has osteoanabolic effects in a variety of systems. In this study, we compared PTH signaling and osteoanabolic effects in hMSCs from young and old subjects. There were age-related decreases in expression of PTH/PTHrP receptor type 1 (PTHR1) gene (P = 0.049, n = 19) and in PTH activation of CREB (P = 0.029, n = 7) and PTH stabilization of β-catenin (P = 0.018, n = 7). Three human PTH peptides, PTH1-34, PTH1-31C (Ostabolin-C, Leu(27) , Cyclo[Glu(22) -Lys(26) ]-hPTH1-31), and PTH1-84 (10 nm), stimulated osteoblast differentiation with hMSCs. Treatment with PTH1-34 resulted in a significant 67% increase in alkaline phosphatase activity in hMSCs obtained from younger subjects (<50 years old, n = 5), compared with an 18% increase in hMSCs from elders (>55 years old, n = 7). Both knockdown of CREB and treatment with a protein kinase A inhibitor H-89 blocked PTH stimulation of osteoblast differentiation in hMSCs from young subjects. The PTH peptides significantly stimulated proliferation of hMSCs. Treatment with PTH1-34 resulted in an average of twice as many cells in cultures of hMSCs from young subjects (n = 4), but had no effect with hMSCs from elders (n = 7). Upregulation of PTHR1 by 24-h pretreatment with 100 nm dexamethasone rescued PTH stimulation of proliferation in hMSCS from elders. In conclusion, age-related intrinsic alterations in signaling responses to osteoanabolic agents like PTH may contribute to cellular and tissue aging of the human skeleton.