Conditional disruption of calcineurin B1 in osteoblasts increases bone formation and reduces bone resorption

We recently reported that the pharmacological inhibition of calcineurin (Cn) by low concentrations of cyclosporin A increases osteoblast differentiation in vitro and bone mass in vivo. To determine whether Cn exerts direct actions in osteoblasts, we generated mice lacking Cnb1 (Cn regulatory subunit) in osteoblasts (DeltaCnb1(OB)) using Cre-mediated recombination methods. Transgenic mice expressing Cre recombinase, driven by the human osteocalcin promoter, were crossed with homozygous mice that express loxP-flanked Cnb1 (Cnb1(f/f)). Microcomputed tomography analysis of tibiae at 3 months showed that DeltaCnb1(OB) mice had dramatic increases in bone mass compared with controls. Histomorphometric analyses showed significant increases in mineral apposition rate (67%), bone volume (32%), trabecular thickness (29%), and osteoblast numbers (68%) as well as a 40% decrease in osteoclast numbers as compared with the values from control mice. To delete Cnb1 in vitro, primary calvarial osteoblasts, harvested from Cnb1(f/f) mice, were infected with adenovirus expressing the Cre recombinase. Cre-expressing osteoblasts had a complete inhibition of Cnb1 protein levels but differentiated and mineralized more rapidly than control, green fluorescent protein-expressing cells. Deletion of Cnb1 increased expression of osteoprotegerin and decreased expression of RANKL. Co-culturing Cnb1-deficient osteoblasts with wild type osteoclasts demonstrated that osteoblasts lacking Cnb1 failed to support osteoclast differentiation in vitro. Taken together, our findings demonstrate that the inhibition of Cnb1 in osteoblasts increases bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis.     

Lipopolysaccharide-induced bone resorption is mediated by prostaglandins

In organ cultures of new-born mouse calvaria both bovine parathyroid hormone and a lipopolysaccharide preparation from E. coli promoted bone resorption. Dead bone cultures did not respond to either of these agents. When bone prostaglandin synthesis was inhibited by indomethacin the lipopolysaccharide no longer promoted bone resorption. Similar treatment did not affect the ability of parathyroid hormone to cause bone dissolution.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Osteoblast-osteoclast relationships in bone resorption: osteoblasts enhance osteoclast activity in a serum-free co-culture system.

Osteoblast-osteoclast relationships in bone resorption are unclear. We investigated whether osteoblasts constitutively influence osteoclast activity. We employed a serum-free co-culture system in which chicken osteoclasts and chick calvaria or, alternatively, isolated chick osteoblasts were cultured in two different compartments separated by a 0.45 micron porous membrane permeable to soluble molecules. Osteoclastic bone resorption, evaluated by release of 3H-proline from prelabeled bone fragments, was significantly enhanced by bone cells resident in the calvaria, as well as by isolated osteoblasts. Stimulation was specific, since periosteal cells, or skin fibroblasts, failed to mimic osteoblast activity. Conditioned medium from osteoblast cultures stimulated osteoclast function in a similar manner, indicating that paracrine signals, capable of crossing the porous membrane separating the two compartments, are released by the bone forming cells.     

Echistatin is a potent inhibitor of bone resorption in culture

The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone resorption, that this RGD-binding integrin is at adhesion structures, and that it colocalizes with vinculin and has an alpha-like subunit.     

T-cell protein tyrosine phosphatase regulates bone resorption and whole-body insulin sensitivity through its expression in osteoblasts

Insulin signaling in osteoblasts contributes to whole-body glucose homeostasis in the mouse and in humans by increasing the activity of osteocalcin. The osteoblast insulin signaling cascade is negatively regulated by ESP, a tyrosine phosphatase dephosphorylating the insulin receptor. Esp is one of many tyrosine phosphatases expressed in osteoblasts, and this observation suggests that other protein tyrosine phosphatases (PTPs) may contribute to the attenuation of insulin receptor phosphorylation in this cell type. In this study, we sought to identify an additional PTP(s) that, like ESP, would function in the osteoblast to regulate insulin signaling and thus affect activity of the insulin-sensitizing hormone osteocalcin. For that purpose, we used as criteria expression in osteoblasts, regulation by isoproterenol, and ability to trap the insulin receptor in a substrate-trapping assay. Here we show that the T-cell protein tyrosine phosphatase (TC-PTP) regulates insulin receptor phosphorylation in the osteoblast, thus compromising bone resorption and bioactivity of osteocalcin. Accordingly, osteoblast-specific deletion of TC-PTP promotes insulin sensitivity in an osteocalcin-dependent manner. This study increases the number of genes involved in the bone regulation of glucose homeostasis.     

Effects of rat fetuin on stimulation of bone resorption in the presence of parathyroid hormone.

Rat fetuin, which is the rat counterpart of human alpha 2-HS glycoprotein and bovine fetuin, is only detectable in calcified tissues such as bone matrices and dentin, and bone cells such as osteoblasts and osteocytes immunohistochemically. The effect of this protein on bone resorption was examined to study its physiological role in bone metabolism. Rat fetuin increased bone resorption in the presence of low concentrations of parathyroid hormone (PTH), but it had no activity on bone resorption without PTH. The increase in bone resorption by PTH and PTH plus rat fetuin was inhibited by the addition of chymostatin, an inhibitor for cathepsin L. Moreover, we found that when type I collagen from rat was preincubated with rat fetuin, the digestion of rat type I collagen by cathepsin L was increased. These findings suggest that rat fetuin present in bone matrix is important in bone resorption.     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.     

The effect of resorption cavities on bone stiffness is site dependent

Resorption cavities formed during the bone remodelling cycle change the structure and thus the mechanical properties of trabecular bone. We tested the hypotheses that bone stiffness loss due to resorption cavities depends on anatomical location, and that for identical eroded bone volumes, cavities would cause more stiffness loss than homogeneous erosion. For this purpose, we used beam–shell finite element models. This new approach was validated against voxel-based FE models. We found an excellent agreement for the elastic stiffness behaviour of individual trabeculae in axial compression (R² = 1.00) and in bending (R²>0.98), as well as for entire trabecular bone samples to which resorption cavities were digitally added (R² = 0.96, RMSE = 5.2%). After validation, this new method was used to model discrete cavities, with dimensions taken from a statistical distribution, on a dataset of 120 trabecular bone samples from three anatomical sites (4th lumbar vertebra, femoral head, iliac crest). Resorption cavities led to significant reductions in bone stiffness. The largest stiffness loss was found for samples from the 4th lumbar vertebra, the lowest for femoral head samples. For all anatomical sites, resorption cavities caused significantly more stiffness loss than homogeneous erosion did. This novel technique can be used further to evaluate the impact of resorption cavities, which are known to change in several metabolic bone diseases and due to treatment, on bone competence.     

In vitro bone resorption is dependent on physiological concentrations of zinc

The addition of physiological concentrations of zinc (25-200 (Μg/dL) to Dulbecco’s Modified Eagle’s Medium containing tibiae from 19-d chick embryos resulted in a concentration-dependent increase in tibial content of tartrate-resistant acid phosphatase (TRAP) and an increase in bone resorption, as measured by tibial calcium release. This increase in bone resorption was additive to the resorptive effect resulting from the addition of 10^-9-10^-7 M parathyroid hormone (PTH), but was not additive to similar effects produced by the addition of 10^-9-10^-7 M prostaglandin E₂ (PGE₂). An inhibitor of prostaglandin synthesis, flurbiprofen (10^-6 M), did not influence the effect of zinc on bone resorption. However, the addition of 2,6-pyridinedicarboxylic acid (10^-3 M, 2,6-PDCA), a chelator of zinc, did attenuate the effects of zinc, as did the addition of an inhibitor of DNA replication (hydroxyurea, 10^-3 M). Hydroxyurea also attenuated the bone resorptive response to PGE², but had no influence on the effects of PTH. These results indicate that physiological concentrations of zinc alter bone resorptive rates in vitro by a mechanism that is dependent on DNA replication.     

Role of cytokines in bone resorption

Osteoclastic bone resorption is modulated in humans by powerful osteotropic factors which are generated in the immediate vicinity of bone resorbing surfaces. These factors are released from marrow mononuclear cells and from some bone cells, and some are actually incorporated into the noncollagenous bone matrix from where they are released when bone is resorbed. They are likely important not only in the control of normal bone remodeling, but also in a number of disease states associated with disordered remodeling. In this review, current concepts of the effects of these factors on cells in the osteoclast lineage will be discussed.     

Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars

Lactobacillus rhamnosus GG is an industrially significant probiotic strain with proven health benefits. In this study, the effect of glucose on L. rhamnosus GG survival was analyzed in simulated gastric juice at pH 2.0. It was found that the presence of 19.4 mM glucose resulted in up to 6-log10-enhanced survival following 90 min of exposure. Further work with dilute HCl confirmed that glucose was the sole component responsible. Comparative analysis with other Lactobacillus strains revealed that enhanced survival was apparent in all strains, but at different pH values. The presence of glucose at concentrations from 1 to 19.4 mM enhanced L. rhamnosus GG survival from 6.4 to 8 log10 CFU ml(-1) in simulated gastric juice. The mechanisms behind the protective effect of glucose were investigated. Addition of N',N'-dicyclohexylcarbodiimide to simulated gastric juice caused survival to collapse, which was indicative of a prominent role in inhibition of F0F1-ATPase. Further work with neomycin-resistant mutants that exhibited 38% to 48% of the F0F1-ATPase activity of the parent confirmed this, as the survival in the presence of glucose of these mutants decreased 3 x 10(6)-fold compared with the survival of the wild type (which had a viability of 8.02 log10 CFU ml(-1)). L. rhamnosus GG survival in acidic conditions occurred only in the presence of sugars that it could metabolize efficiently. To confirm the involvement of glycolysis in the glucose effect, iodoacetic acid was used to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. The reduction in GAPDH activity caused survival to decrease by 8.30 log10 CFU ml(-1) in the presence of glucose. The data indicate that glucose provides ATP to F0F1-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit.     

Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorption

The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.     

Neuropeptidergic regulation of bone resorption and bone formation

Immunohistochemical studies have revealed an extensive network of nerve fibers in the vicinity and within the skeleton, not only in the periosteum of bone but also in cortical and trabecular bone as well as in the bone marrow. Phenotyping of the skeletal nerve fibers have demonstrated the expression of a restrictive panel of different signalling molecules including neuropeptides, neurotransmitters and neurotrophins. In this review, the presence of receptors for the neuropeptides vasoactive intestinal peptide, calcitonin gene-related peptide and substance P on osteoblasts and osteoclasts and the capacity of these receptors to regulate bone formation, osteoclast formation and activity are described. These findings, together with data obtained by chemically and surgically targeted nerve deletion and observations made in paraplegic patients, strongly suggest that neuro-osteogenic interactions play an important role in skeletal function.