Tim Skerry

The response of bone to mechanical loading and disuse: fundamental principles and influences on osteoblast/osteocyte homeostasis.

Bones response to increased or reduced loading/disuse is a feature of many clinical circumstances, and our daily life, as habitual activities change. However, there are several misconceptions regarding what constitutes loading or disuse and why the skeleton gains or loses bone. The main purpose of this article is to discuss the fundamentals of the need for bone to experience the effects of loading and disuse, why bone loss due to disuse occurs, and how it is the target of skeletal physiology which drives pathological bone loss in conditions that may not be seen as being primarily due to disuse. Fundamentally, if we accept that hypertrophy of bone in response to increased loading is a desirable occurrence, then disuse is not a pathological process, but simply the corollary of adaptation to increased loads. If adaptive processes occur to increase bone mass in response to increased load, then the loss of bone in disuse is the only way that adaptation can fully tune the skeleton to prevailing functional demands when loading is reduced. The mechanisms by which loading and disuse cause bone formation or resorption are the same, although the direction of any changes is different. The osteocyte and osteoblast are the key cells involved in sensing and communicating the need for changes in mass or architecture as a result of changes in experienced loading. However, as those cells are affected by numerous other influences, the responses of bone to loading or disuse are not simple, and alter under different circumstances. Understanding the principles of disuse and loading and the mechanisms underlying them therefore represents an important feature of bone physiology and the search for targets for anabolic therapies for skeletal pathology.

Interruption of disuse by short duration walking exercise does not prevent bone loss in the sheep calcaneus

The effect on calcaneal bone loss of interrupting immobilization by daily periods of normal walking was investigated in adult female sheep. The left calcaneus of 28 sheep was protected from normal loading by placing an external fixator across the hock joint, from the tibia to the metatarsus. In vivo strain gauge recordings from 3 animals showed that, at a test location on the calcaneus, this resulted in a significant reduction in principal strain magnitude during walking at 1.5 m s-1 from a peak of -228 x 10(-6) before application of the fixator, to 71 x 10(-6) with the fixator in place. In addition to reducing peak strains during normal activity, the fixator also abolished the high magnitude transient strains (peak -1147 x 10(-6)) normally experienced when the sheep made sudden movements. Thirteen animals (group 1) had fixators applied, and were given no further treatment. In a further 6 animals (group 2), the fixator was removed for 20 min per day, during which they walked on a motorized treadmill at 1.5 m s-1. In the remaining 6 sheep (group 3), incomplete fixator bars were applied to allow uninterrupted joint movement. Over the 12-week period of the experiment, dual photon absorptiometric scans showed that the bone mineral content (BMC) of the calcanei of group 1 fell by 22%. In group 2, where immobilization was interrupted by daily walking, the BMC fell by 21%. In group 3, with incomplete bars, there was no significant reduction in BMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1tr/tr) have ~50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1tr/tr osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1tr/tr cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.

Reduced Bone Turnover in Mice Lacking the P2Y13 Receptor of ADP

Osteoporosis is a condition of excessive and uncoupled bone turnover, in which osteoclastic resorption exceeds osteoblastic bone formation, resulting in an overall net bone loss, bone fragility, and morbidity. Although numerous treatments have been developed to inhibit bone loss by blocking osteoclastic bone resorption, understanding of the mechanisms behind bone loss is incomplete. The purinergic signaling system is emerging to be a pivotal regulator of bone homeostasis, and extracellular ADP has previously been shown to be a powerful osteolytic agent in vitro. We report here that deletion of the P2Y(13) receptor, a G protein-coupled receptor for extracellular ADP, leads to a 40% reduction in trabecular bone mass, 50% reduction in osteoblast and osteoclast numbers in vivo, as well as activity in vitro, and an overall 50% reduction in the rate of bone remodeling in mice in vivo. Down-regulation of RhoA/ROCK I signaling and a reduced ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin observed in osteoblasts from P2Y(13)R(-/-) mice might explain this bone phenotype. Furthermore, because one of the main causes of osteoporosis in older women is lack of estrogen, we examined the effect of ovariectomy of the P2Y(13)R(-/-) mice and found them to be protected from ovariectomy-induced bone loss by up to 65%. These data confirm a role of purinergic ADP signaling in the skeleton, whereby deletion of the P2Y(13) receptor leads to reduced bone turnover rates, which provide a protective advantage in conditions of accelerated bone turnover such as oestrogen deficiency-induced osteoporosis.

Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)

Background: The glucagon and glucagon-like peptide-1 (GLP-1) receptors are important targets for treating type 2 diabetes. Results: We describe novel glucagon receptor pharmacology, through interaction with the receptor activity-modifying protein-2 (RAMP2). Conclusion: RAMP2 regulates both ligand binding and G protein selectivity of the glucagon receptor. Significance: The effect of RAMP2 should be considered when designing anti-diabetic treatments. The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.

Intramembranous trabecular generation in normal bone.

The ability of trabeculae to reform following localized ablation may provide further insight into the sequence of events in cancellous regeneration. Histological features of cancellous repair were examined in the iliac crest of aged female sheep at intervals after removal of a 1-cm diameter biopsy. Comparison was made with normal intramembranous trabecular formation in the foetal lamb. The first immature trabeculae to form in the defects within 3 weeks were exclusively intramembranous, not endochondral, and the systematic process was indistinguishable from that in the intact growing foetal lamb. In both the young and old skeleton, two features were prominent. First, the damaged endosteum of the sheep functioned like the intact periosteum of the lamb to produce orderly migrating arrays of discrete coarse collagenous fibres, 5-25 microns thick, which penetrated the surrounding soft tissues to form a polarised preliminary framework. Without this structure, primary trabecular development did not take place. Throughout subsequent bone apposition the preliminary framework, which bonded hard to soft tissues and new bone to old, remained largely unmineralised. Second, intratrabecular resorption channels divided the established, thickened primary bars into networks of mature secondary trabeculae. It is concluded that the two features are central and universal to trabecular proliferation and may provide a morphological basis for future trabecular restitution of the depleted elderly skeleton.

Research Resource: Haploinsufficiency of Receptor Activity-Modifying Protein-2 (Ramp2) Causes Reduced Fertility, Hyperprolactinemia, Skeletal Abnormalities, and Endocrine Dysfunction in Mice

Receptor activity-modifying protein-2 (RAMP2) is a single-pass transmembrane protein that can regulate the trafficking, ligand binding, and signaling of several G protein-coupled receptors (GPCR). The most well-characterized role of RAMP2 is in the regulation of adrenomedullin (AM) binding to calcitonin receptor-like receptor (CLR), and our previous studies using knockout mouse models support this canonical signaling paradigm. For example, Ramp2(-/-) mice die at midgestation with a precise phenocopy of the AM(-/-) and Calcrl(-/-) mice. In contrast, Ramp2(+/-) mice are viable and exhibit an expanded variety of phenotypes that are distinct from those of Calcrl(+/-) mice. Using Ramp2(+/-) female mice, we demonstrate that a modest decrease in Ramp2 expression causes severe reproductive defects characterized by fetal growth restriction, fetal demise, and postnatal lethality that is independent of the genotype and gender of the offspring. Ramp2(+/-) female mice also exhibit hyperprolactinemia during pregnancy and in basal conditions. Consistent with hyperprolactinemia, Ramp2(+/-) female mice have enlarged pituitary glands, accelerated mammary gland development, and skeletal abnormalities including delayed bone development and decreased bone mineral density. Because RAMP2 has been shown to associate with numerous GPCR, it is likely that signaling of one or more of these GPCR is compromised in Ramp2(+/-) mice, yet the precise identification of these receptors remains to be elucidated. Taken together, this work reveals an essential role for RAMP2 in endocrine physiology and provides the first in vivo evidence for a physiological role of RAMP2 beyond that of AM/CLR signaling.

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y13 receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y13 receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild‐type (WT) and P2Y13 receptor knockout (P2Y13R−/−) mice were subject to non‐invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro‐computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y13R−/− mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y13R−/− mice compared with controls. In vitro, primary P2Y13R−/− osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y13R−/− osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto‐enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y13 receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX‐induced bone loss after P2Y13 receptor depletion as previously described, suggests a potential role for P2Y13 receptor antagonist‐based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.

Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation and supports differentiation

Human megakaryocytes release glutamate and express glutamate-gated Ca(2+)-permeable N-methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca(2+) responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca(2+) flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca(2+) entry into Meg-01 cells, amplified Ca(2+) responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5-100μM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100μM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC50 20μM and 36μM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation. In conclusion, NMDARs provide a novel pathway for Ca(2+) entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts.

Role of Receptor Activity Modifying Protein 1 in Function of the Calcium Sensing Receptor in the Human TT Thyroid Carcinoma Cell Line

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca2+ and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred.