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The bone organ integrates the activity of bone tissue, bone marrow, and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP+/+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (×4) and vascular volume (×7) than BSP+/+. Vessel numbers in the shaft of ablated BSP+/+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin- (×3) as well as LSK (Lin- IL-7Rα- Sca-1hi c-Kithi , ×2) and hematopoietic stem cells (HSC: Flt3- LSK, ×2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin- was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants. In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. J. Cell. Physiol. 232: 2528-2537, 2017. © 2016 Wiley Periodicals, Inc.

Matrix proteins of the SIBLING family interact with bone cells, extracellular matrix and mineral and are thus in a key position to regulate the microenvironment of the bone tissue, including its hematopoietic component. In this respect, osteopontin (OPN) has been implicated in the hematopoietic stem cell (HSC) niche as negative regulator of the HSC function. We investigated the impact on hematopoietic regulation of the absence of the cognate bone sialoprotein (BSP). BSP knockout (−/−) mice display increased bone marrow cellularity, and an altered commitment of hematopoietic precursors to myeloid lineages, leading in particular to an increased frequency of monocyte/macrophage cells. The B cell pool is increased in −/− bone marrow, and its composition is shifted toward more mature lymphocyte stages. BSP-null mice display a decreased HSC fraction among LSK cells and a higher percentage of more committed progenitors as compared to +/+. The fraction of proliferating LSK progenitors is higher in −/− mice, and after PTH treatment the mutant HSC pool is lower than in +/+. Strikingly, circulating levels of OPN as well as its expression in the bone tissue are much higher in the −/−. Thus, a BSP-null bone microenvironment affects the hematopoietic system both quantitatively and qualitatively, in a manner in part opposite to the OPN knockout, suggesting that the effects might in part reflect the higher OPN expression in the absence of BSP. J. Cell. Physiol. 230: 1342–1351, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1–84 (iPTH) over the calvaria of BSP knockout (−/−) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and −/− mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from −/− mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5× higher in −/− versus +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in −/− cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP +/+ calvaria, while it blunted iPTH effects in −/− mice, reduced to a modest increase in periosteum thickness. In −/− (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins. J. Cell. Physiol. 230: 568–577, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Bone vessel functions during bone remodeling are poorly understood. They depend on both vessel network structure and vasomotor regulation. Parathyroid hormone (PTH) is a systemic vasodilator that may modulate microvascularization. Moreover, although intermittent PTH is anti-osteoporotic, continuous PTH administration can be catabolic for bone. Finally, ovariectomy (OVX) reduces bone perfusion and vessel density in mice. We reasoned that the effects of PTH on bone vascularization might depend on its administration regimen and be impacted by ovariectomy. A 100-µg/kg PTH 1-84 daily dose was administered for 15 days to 4-month-old female C57BL/6 mice, either as daily sc injection (iPTH) or continuously (cPTH; ALZET minipump). Blood pressure (BP) and tibia bone perfusion were measured in vivo with a laser Doppler device. Histomorphometry of bone and barium-contrasted vascular network were performed on the same tibia. Compared with untreated controls, both iPTH and cPTH increased bone formation but had opposite effects on resorption. Both iPTH and cPTH were slightly angiogenic. Intermittent PTH increased microvessel size (+48%, p

Intermittent parathyroid hormone (PTH) is anabolic for bone. Our aims were to determine (1) whether PTH stimulates bone angiogenesis and (2) whether vascular endothelial growth factor (VEGF A) mediates PTH-induced bone accrual. Male Wistar rats were given PTH(1–84) daily, and trabecular bone mass increased 150% and 92% after 30 and 15 days, respectively. The vascular system was contrasted to image and quantify bone vessels with synchrotron radiation microtomography and histology. Surprisingly, bone vessel number was reduced by approximately 25% and approximately 40% on days 30 and 15, respectively. PTH redistributed the smaller vessels closer to bone-formation sites. VEGF A mRNA expression in bone was increased 2 and 6 hours after a single dose of PTH and returned to baseline by 24 hours. Moreover, anti-VEGF antibody administration (1) blunted the PTH-induced increase in bone mass and remodeling parameters, (2) prevented the relocation of bone vessels closer to bone-forming sites, and (3) inhibited the PTH-induced increase in mRNA of neuropilin 1 and 2, two VEGF coreceptors associated with vascular development and function. In conclusion, PTH(1–84) is osteoanabolic through VEGF-related mechanism(s). Further, PTH spatially relocates blood vessels closer to sites of new bone formation, which may provide a microenvironment favorable for growth. © 2011 American Society for Bone and Mineral Research

Excess thyroid hormone (TH) in adults causes osteoporosis and increases fracture risk. However, the mechanisms by which TH affects bone turnover are not elucidated. In particular, the roles of thyroid hormone receptor (TR) isotypes in the mediation of TH effects on osteoblast-mediated bone formation and osteoclast-mediated bone resorption are not established. In this study we have induced experimental hypothyroidism or hyperthyroidism in adult wild-type, TRα- or TRβ-deficient mice and analyzed the effects of TH status on the structure and remodeling parameters of trabecular bone. In wild-type mice, excess TH decreased bone volume and mineralization. High TH concentrations were associated with a high bone-resorption activity, assessed by increased osteoclast surfaces and elevated concentrations of serum bone-resorption markers. Serum markers of bone formation also were higher in TH-treated mice. TRα deficiency did not prevent TH action on bone volume, bone mineralization, bone formation, or bone resorption. In contrast, TRβ deficiency blocked all the early effects of excess TH observed in wild-type mice. However, prolonged exposure to low or high TH concentrations of TRβ-deficient mice induced mild modifications of bone structure and remodeling parameters. Together our data suggest that TRβ receptors mediate the acute effects produced by transient changes of TH concentrations on bone remodeling, whereas TRα receptors mediate long-term effects of chronic alterations of TH metabolism. These data shed new light on the respective roles of TRs in the control of bone metabolism by TH.

Serum bone GLA-protein (BGP), a sensitive and specific marker of bone formation, was measured in 54 normal children and in 50 children with growth disorders. In normal children, the pattern of variations of serum BGP with age was similar to the pattern of variations of the growth velocity. Mean serum BGP was very high during the first year of life (25.3 +/- 8.5 ng/ml), decreased to 14.8 +/- 2.2 ng/ml from 2 to 6 years, increased to 18.4 +/- 4.1 ng/ml from 7 to 10 years and to 18.8 +/- 6.5 ng/ml from 11 to 14 years. After puberty, mean sBGP decreased to 12.9 +/- 5.4 ng/ml from 15 to 18 years and to 6.5 +/- 1.4 ng/ml in young adults. In 32 patients with untreated growth hormone (GH) deficiency, mean sBGP was markedly lower than in age matched controls (6.8 +/- 4.4 ng/ml vs. 17.5 +/- 4.9 ng/ml, p less than .001). In 19 patients with GH deficiency who were undergoing treatment with human GH, sBGP was higher than in untreated patients (20.5 +/- 9.3 ng/ml vs. 6.8 +/- 4.4 ng/ml, p less than .001) and was not different from controls. Repeated measurements performed in 14 GH-deficient patients under chronic GH therapy showed that serum BGP: (1) increased in most patients during treatment (p less than .005); (2) was correlated with the duration of treatment (p less than .001); (3) decreased to pretreatment values after discontinuing therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

We previously showed that thrombospondin, a major alpha-granule glycoprotein of human platelets, forms a specific complex with osteonectin, a phosphoglycoprotein originally described in bone that is also present in human platelets. The storage organelles and the function of osteonectin in platelets are still unknown. In this study, using electron microscopy in combination with immunogold staining, the major storage organelle for platelet-secreted proteins, the alpha-granules. Furthermore, osteonectin was qualitatively and quantitatively assessed by studying normal platelets and the platelets from a patient with gray platelet syndrome. Gray platelet syndrome is a rare congenital bleeding disorder characterized by a selective deficiency in morphologically recognizable platelet alpha-granules and in the alpha-granule secretory proteins. Binding of an iodinated antiosteonectin monoclonal antibody to gray platelet proteins transferred to nitrocellulose from SDS-polyacrylamide gels showed no band corresponding to osteonectin compared to control platelets. Using a polyclonal antiosteonectin antibody-based radioimmunoassay, gray platelets contained 0.2 +/- 0.03 ng osteonectin per 10(6) platelets, which is only 20% of the normal platelet content of osteonectin (0.93 +/- 0.16 ng per 10(6) platelets). Study of the localization of osteonectin to the surface of human platelets demonstrated that a radioiodinated antiosteonectin polyclonal antibody bound specifically to thrombin-stimulated platelets but not to resting platelets. Binding was concentration-dependent, saturable (1710 +/- 453 binding sites per platelet, Kd = 1 microM), and inhibited by an excess of cold antiosteonectin polyclonal antibody. No binding was observed on the surface of thrombin-stimulated gray platelets. To gain further insights into the role of osteonectin released from activated platelets, the effect of an antiosteonectin polyclonal antibody was tested on the aggregation of washed platelets. F(ab')2 fragments from the antiosteonectin polyclonal antibody inhibited in a dose-dependent manner the aggregation of collagen-stimulated, washed human platelets without affecting collagen-induced platelet serotonin release. To characterize the mechanism through which antiosteonectin F(ab')2 fragments inhibit platelet aggregation, the expression of endogenous thrombospondin (TSP) on the surface of thrombin-activated platelets was studied using 125I-labeled anti-TSP monoclonal antibody P10. The endogenous surface expression of TSP to thrombin-stimulated platelets was significantly inhibited in the presence of antiosteonectin F(ab')2 fragments (6286 +/- 2065 molecules of P10 per platelet) compared to 11,230 +/- 766 molecules of P10 per platelet in the presence of nonimmune F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

International audience; The objective of this study was to develop and validate a technique for both 3D imaging and quantification of the vascular network of bone tissue in the rat. Five month-old male Wistar rats were divided into tail-suspension (21 days) and control groups. Sixty percent barium sulfate solution was infused into the vena cava. The tibiae were evaluated in 2D and 3D before and after decalcification, using conventional microcompu-terized tomography (lCT) at 10 and 5 lm resolution and synchrotron radiation (SR) lCT. The perfusion technique and tomography exhibited excellent bone vasculature imaging. Significant positive correlations were observed between 2D histomorphometric and 3D lCT vascular parameters (P < 0.05). 3DlCT discriminated significant changes of vessel structures in unloading condition: vessel number decreased by 25%, (P < 0.005), vessel separation increased by 27%, P < 0.01. SRlCT could image sinusoid clusters in bone. lCT is an accurate and reproducible technique for 3D quantitative evaluation of long bone vascularisation in the rat. Anat Rec, 293:215-224

Although the responses of bone to increased loading or exercise have been studied in detail, our understanding of the effects of decreased usage of the skeleton has been limited by the scarcity of suitable models. Such models should ideally not affect bone innervation, which appears to be a mediator of physiological responses of bone to unloading. MyoD-/-/Myf5-/- (dd/ff) mice lack skeletal muscle, so the fetuses develop without any active movement in utero and die soon after birth. We used micro-compter tomography and histology to analyse their bone development and structure during endochondral ossification in parallel with the establishment of bone innervation. Long bones from mutant mice were found to be profoundly different from controls, with shorter mineralized zones and less mineralization. They lacked many characteristics of adult bones - curvatures, changes in shaft diameter and traction epiphyses where muscles originate or insert - that were evident in the controls. Histologically, dd/ff mice showed the same degree of endochondral development as wild-type animals, but presented many more osteoclasts in the newly layed bone. Innervation and the expression pattern of semaphorin-3A signalling molecules were not disturbed in the mutants. Overall, we have found no evidence for a major defect of development in dd/ff mice, and specifically no alteration or delay in endochondral ossification and bone innervation. The altered morphological features of dd/ff mice and the increased bone resorption show the role of muscle activity in bone shaping and the consequences of bone unloading.

Leukemia inhibitory factor (LIF) is a cytokine produced by multiple cell types including osteoblasts and which is active on bone metabolism. We have previously shown that in a bone nodule forming in vitro model of osteogenesis, the fetal rat calvaria (RC) cell model, LIF inhibits osteoblast differentiation, acting on late osteoprogenitors and/or early osteoblasts. These results are in contrast to in vivo experiments, in which LIF has been found to increase bone formation. To resolve this discrepancy, we have tested the effect of LIF on rat bone marrow (RBM) stromal cell cultures, an in vitro model encompassing earlier osteoprogenitor stages. LIF inhibited cell growth in early, proliferating RBM cultures, but increased the culture saturation density. The effect of LIF on bone nodule formation in this model was cell density dependent and biphasic. Continuous treatment with LIF reduced the number of bone nodules present in confluent, more mature cultures, and the inhibitory effect was strongest when cells were plated at higher cell density than lower. In contrast, during the early stages of RBM culture, nodule numbers were higher in LIF-treated dishes than in controls, and this effect was greater in lower density cultures. Acute LIF treatment restricted to early time points increased the final number of bone nodules formed in mature RBM cell cultures, but not in RC cell cultures. Our results indicate that LIF exerts complex, stage-specific effects on osteoprogenitor recruitment, differentiation, and bone formation, and that the effects are cell nonautonomous, in the rat bone marrow stromal cell model. J. Cell. Biochem. Suppl. 36: 63-70, 2001.

Cytokines of the interleukin 6 (IL-6) subfamily are a group of factors produced by osteoblasts and acting through the same transducing element, membrane protein gp130. We have previously shown that exogenous (added to the culture medium) leukemia inhibitory factor (LIF) inhibits bone nodule formation and expression of osteoblast-associated genes in fetal rat calvaria (RC) cell cultures and that dexamethasone (Dex) increases the ID50 of LIF. To investigate the respective roles of IL-6-related cytokines and receptors in osteprogenitor differentiation, and their regulatory interplay with Dex, we used reverse transcribed polymerase chain reaction, bioassay, and blocking antibody techniques to assess the time courses of LIF, IL-6, LIF transmembrane receptor, IL-6 receptor, and gp130 expression in RC cell cultures grown with and without Dex. The levels of the mRNAs for IL-6, LIF, and gp130 decreased concomitantly with the formation of bone nodules. Dex treatment, which stimulates bone nodule formation, reduced the expression of LIF and IL-6 mRNAs and IL-6 bioactivity in the culture medium. LIF treatment strongly stimulated the expression of IL-6. Incubation with anti-LIF antibodies increased the number of nodules, while an antibody blocking IL-6 activity had little or no effect on nodule numbers and did not antagonize the action of exogenous LIF, indicating that IL-6 does not mediate the action of LIF in this system. Moreover, although exogenously added IL-6 was active in the cultures as noted by a reduction of nodule mineralization, it had no effect on nodule numbers, i.e., on osteoprogenitor differentiation, in the presence or absence of Dex. In conclusion, IL-6, LIF, and their receptors are expressed throughout the time-course of osteogenesis in RC cell cultures. However, only LIF, but not IL-6, appears to play a significant role in autocrine regulation of osteoblastic differentiation in this system. The antagonist action of Dex on the effects of exogenously added LIF, as well as the bone-promoting action of Dex in RC cell cultures, could be exerted partly through the down-regulation of the expression of endogenous LIF.

Skin decorin (DCN) is an antiadhesive dermatan sulfate-rich proteoglycan that interacts with thrombospondin-1 (TSP) and inhibits fibroblast adhesion to TSP [Winnemöller et al., 1992]. Molecular mechanisms by which DCN interacts with TSP and inhibits cell adhesion to TSP are unknown. In the present study, we showed that skin DCN and bone DCN (chondroitin sulfate-rich proteoglycan) were quantitatively identical with respect to their ability to interact with TSP. Using a series of fusion proteins corresponding to the different structural domains of TSP, binding of [125I]DCN to TSP was found to be dependent of the N-terminal domain and, to a lesser extent, of the type 1 repeats and the C-terminal domain of TSP. In addition, heparan sulfate drastically inhibited [125I]DCN binding to solid-phase adsorbed TSP (80% inhibition), suggesting that DCN could bind to the N-terminal domain of TSP through interaction with heparin-binding sequences. To address this question, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), KKTR (residues 80-83) and RLRIAKGGVNDN (residues 178-189), were synthesized and tested for their ability to interact with DCN. [125I]DCN interacted only with peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS containing the heparin-binding consensus sequence KKTR. These peptides contained glycosaminoglycan-dependent and -independent binding sites because [125I]DCN binding to VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS was partially reduced upon removal of the glycosaminoglycan chain (65% and 46% inhibition, respectively). [125I]DCN poorly bound to subpeptide MKKTRG and did not bind at all to subpeptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS, suggesting that heparin-binding sequence MKKTRG constituted a DCN binding site when flanked with peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS. The sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS constitutes a cell adhesive active site in the N-terminal domain of TSP [Clezardin et al., 1997], and DCN inhibited the attachment of fibroblastic and osteoblastic cells to peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS by about 50 and 80%, respectively. Although fibroblastic cells also attached to type 3 repeats and the C-terminal domain of TSP, DCN only inhibited cell attachment to the C-terminal domain. Overall, these data indicate that modulation by steric exclusion of cell adhesion to a KKTR-dependent cell adhesive site present within the N-terminal domain of TSP could explain the antiadhesive properties of DCN.

Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, beta-glycerophosphate and 10(-8) M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317-330; Aubin et al., 1990, J. Bone Miner. Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of osteoblastic markers. Immunocytochemistry was combined with Northern blotting, enzymatic assay, and radioimmunoassay to analyze the expression of bone-related proteins during the growth and differentiation sequence. By mRNA levels, protein production and/or enzymatic activity, expression of osteocalcin, bone sialoprotein, and alkaline phosphatase increased concomitantly with the development of bone nodules, while osteopontin mRNA levels decreased and those of SPARC/osteonectin did not change significantly. In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteocalcin, declined. Immunolabeling revealed that cells in early cultures stained poorly for SPARC/osteonectin and strongly for thrombospondin. Later, SPARC/osteonectin staining increased in most cells, while thrombospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity. At all time points studied, osteoblasts within bone nodules stained homogeneously for thrombospondin and alkaline phosphatase, and with marked heterogeneity of intensity amongst cells for SPARC/osteonectin and osteocalcin. Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cells which intensely labels osteoblasts and osteocytes (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), co-localized with osteocalcin. Alkaline phosphatase activity and the amount of osteocalcin determined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules. These studies indicate that the bone marrow stromal cell system is a useful model to study the temporal and spatial expression of bone-related proteins during osteogenesis and formation, mineralization, and maturation of bone nodules. Further, immunolabelling at the individual cell and single bone nodule level allowed discrimination of marked variability of expression of osteoblast markers during the differentiation sequence.

Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (Kd) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (Kd = 0.86 nM). The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81% inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calcium-dependent as shown by a 50-80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A--Sepharose 4B. Elution of the anti-thrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred.