The reduced osteogenic potential of Nf1-deficient osteoprogenitors is EGFR-independent.

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations in the NF1 gene. Recalcitrant bone healing following fracture (i.e. pseudarthrosis) is one of the most problematic skeletal complications associated with NF1. The etiology of this condition is still unclear; thus, pharmacological options for clinical management are limited. Multiple studies have shown the reduced osteogenic potential of Nf1-deficient osteoprogenitors. A recent transcriptome profiling investigation revealed that EREG and EGFR, encoding epiregulin and its receptor Epidermal Growth Factor Receptor 1, respectively, were among the top over-expressed genes in cells of the NF1 pseudarthrosis site. Because EGFR stimulation is known to inhibit osteogenic differentiation, we hypothesized that increased EREG and EGFR expression in NF1-deficient skeletal progenitors may contribute to their reduced osteogenic differentiation potential. In this study, we first confirmed via single-cell mRNA sequencing that EREG over-expression was associated with NF1 second hit somatic mutations in human bone cells, whereas Transforming Growth Factor beta 1 (TGFß1) expression was unchanged. Second, using ex-vivo recombined Nf1-deficient mouse bone marrow stromal cells (mBMSCs), we show that this molecular signature is conserved between mice and humans, and that epiregulin generated by these cells is overexpressed and active, whereas soluble TGFß1 expression and activity are not affected. However, blocking either epiregulin function or EGFR signaling by EGFR1 or pan EGFR inhibition (using AG-1478 and Poziotinib respectively) did not correct the differentiation defect of Nf1-deficient mBMSCs, as measured by the expression of Alpl, Ibsp and alkaline phosphatase activity. These results suggest that clinically available drugs aimed at inhibiting EGFR signaling are unlikely to have a significant benefit for the management of bone non-union in children with NF1 PA.

Medication therapy for attention deficit/hyperactivity disorder is associated with lower risk of fracture: a retrospective cohort study.

UNLABELLED: The impact of pharmacotherapy for attention deficit/hyperactivity disorder on fracture risk has not been well studied. In this retrospective cohort study, medication therapy was associated with lower fracture incidence. Further studies are needed to better characterize the short-term and long-term effects of these medications on bone health and fracture risk. INTRODUCTION: Attention deficit/hyperactivity disorder (ADHD) is associated with increased risk of bone fractures. The impact of pharmacotherapy with either stimulant or non-stimulant medications on fracture risk has not been well characterized. We performed a study to compare fracture incidence in ADHD patients treated with stimulant or non-stimulant medications vs. no pharmacotherapy. METHODS: In this retrospective cohort study, data were extracted from a large electronic medical record. A total of 10,066 subjects with ADHD, 40 years or younger, were included. We extracted data regarding stimulant and non-stimulant ADHD medications, corticosteroids, fracture data, demographic data, and diabetes history. RESULTS: A total of 1015 patients (10 %) sustained fractures. Multivariable Cox proportional hazard analysis indicated that compared to those with two or more prescriptions for an ADHD medication, individuals without documented medication therapy had a significantly increased hazard of fracture (hazard ratio [HR] 3.9, 95 % confidence interval [CI] 2.6-5.9). However, the hazard ratio for stimulant vs. non-stimulant medication (HR 0.92, 95 % CI 0.60-1.4) was not statistically significant. CONCLUSIONS: Three times as many patients with no documented ADHD medication prescriptions suffer a fracture compared to patients with a history of two or more prescriptions for an ADHD medication. Treatment and adherence are thus important to prevent fracture in this population.

The effect of an inhibitor of gut serotonin (LP533401) during the induction of periodontal disease.

BACKGROUND AND OBJECTIVE: LP533401 is an inhibitor of tryptophan hydroxylase 1, which regulates serotonin production in the gut. Previous work indicates that LP533401 has an anabolic effect in bone. Thus, we hypothesized that inhibition of gut serotonin production may modulate the host response in periodontal disease. In this study, we aimed to analyze the effects of LP533401 in a rat periodontitis model to evaluate the role of gut serotonin in periodontitis pathophysiology. MATERIAL AND METHODS: Twenty-four rats were divided into three groups: treated group (T: ligature-induced periodontal disease and LP533401, 25 mg/kg/d) by gavage; ligature group (L: ligature-induced periodontal disease only); and control group (C: without ligature-induced periodontal disease). After 28 d, radiographic alveolar bone support was measured on digital radiographs, and alveolar bone volume fraction, tissue mineral density and trabeculae characteristics were quantified by microcomputed tomography in the right hemi-mandible. Left hemi-mandibles were decalcified and alveolar bone loss, attachment loss and area of collagen in the gingiva were histologically analyzed. RESULTS: Significant difference between the L and C groups was found, confirming that periodontal disease was induced. We observed no difference between the T and L groups regarding alveolar bone destruction and area of collagen. CONCLUSION: LP533401 (25 mg/kg/d) for 28 d does not prevent bone loss and does not modulate host response in a rat model of induced periodontal disease.

TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity. We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification. This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

The Vestibular System: A Newly Identified Regulator of Bone Homeostasis Acting Through the Sympathetic Nervous System.

The vestibular system is a small bilateral structure located in the inner ear, known as the organ of balance and spatial orientation. It senses head orientation and motion, as well as body motion in the three dimensions of our environment. It is also involved in non-motor functions such as postural control of blood pressure. These regulations are mediated via anatomical projections from vestibular nuclei to brainstem autonomic centers and are involved in the maintenance of cardiovascular function via sympathetic nerves. Age-associated dysfunction of the vestibular organ contributes to an increased incidence of falls, whereas muscle atrophy, reduced physical activity, cellular aging, and gonadal deficiency contribute to bone loss. Recent studies in rodents suggest that vestibular dysfunction might also alter bone remodeling and mass more directly, by affecting the outflow of sympathetic nervous signals to the skeleton and other tissues. This review will summarize the findings supporting the influence of vestibular signals on bone homeostasis, and the potential clinical relevance of these findings.

The new field of neuroskeletal biology.

The fields of neuroscience and bone biology have recently converged following the discovery that bone remodeling is directly regulated by the brain. This work has defined bone remodeling as one of the cardinal physiological functions of the body, subject to homeostatic regulation and integrated with the other major physiological functions by the hypothalamus. Central to this discovery was the definition of the adipocyte-derived hormone leptin as a regulator of both arms of bone remodeling, formation and resorption, through its action on the ventromedial hypothalamus and subsequently via the sympathetic nervous system to osteoblasts. The characterization of the sympathetic nervous system as a regulator of bone remodeling has led to several large clinical studies demonstrating a substantial protective effect of beta-blockers, particularly beta1-blockers, on fracture risk. Studies in model organisms have reinforced the role of the central nervous system in the regulation of bone remodeling in vivo by the identification of several additional genes, namely cocaine and amphetamine regulated transcript (Cart), melanocortin 4 receptor (Mc4R), neuropeptide Y (NPY), Y2 receptor, cannabinoid receptor CB1 (Cnbr1), and the genes of the circadian clock. These genes have several common features, including high levels of expression in the hypothalamus and the ability to regulate other major physiological functions in addition to bone remodeling including energy homeostasis, body weight, and reproduction. We review the major pathways that define the new field of neuroskeletal biology and identify further avenues of inquiry.

Neuronal signaling and the regulation of bone remodeling.

An increasing number of studies suggest that nerve-derived signals play an important role in the regulation of bone remodeling. Neuropeptides and receptors/transporters of adrenergic, glutaminergic, serotoninergic, dopaminergic and sensory nature have been described in osteoblasts in vitro. Downstream signaling pathways and targets genes have been identified, but the in vivo relevance of these findings remained controversial until more recent gene gain and loss of function studies confirmed the role of CGRP and beta2-adrenergic receptor signaling in osteoblasts. Tissue and time-conditional mutant mice originally generated for studies unrelated to bone are now available tools to determine the role of neuronal signaling in bone and to dissociate the central and peripheral role of these signals. Lastly, understanding how the central nervous system integrates homeostatic signals with the regulation of bone homeostasis will be the next exciting subject of research in the field.

[Bone mass regulation by leptin: a hypothalamic control of bone formation]. / Régulation de la masse osseuse par la leptine: un contrôle hypothalamique de la formation osseuse.

Bone mass is maintained constant between puberty and menopause by the balance between osteoblasts and osteoclasts activity. The existence of a hormonal control of osteoblast activity has been speculated for years by analogy to osteoclast biology. Through the search for such humoral signal(s) regulating bone formation, leptin has been identified as a powerful inhibitor of bone formation. Furthermore, by means of intracerebroventricular infusion of leptin, it has been shown that the effect of this adipocyte-derived hormone on bone is mediated via a brain relay, like all its other functions. Subsequent studies have led to the identification of hypothalamic neurons involved in leptin's antiosteogenic function. In addition, it has been shown that those neurons or neuronal pathways are distinct from neurons responsible for the regulation of energy metabolism. Finally, the peripheral mediator of leptin's antiosteogenic function has been identified as being the sympathetic nervous system. Catecholamine-deficient mice have a high bone mass and sympathomimetics administered to mice decreased bone formation and bone mass. Conversely, beta-blockers increased bone formation and bone mass and blunt the bone loss induced by ovariectomy.

Serum leptin level is a regulator of bone mass.

Leptin is a powerful inhibitor of bone formation in vivo. This antiosteogenic function involves leptin binding to its receptors on ventromedial hypothalamic neurons, the autonomous nervous system and beta-adrenergic receptors on osteoblasts. However, the mechanisms whereby leptin controls the function of ventromedial hypothalamic antiosteogenic neurons remain unclear. In this study, we compared the ability of leptin to regulate body weight and bone mass and show that leptin antiosteogenic and anorexigenic functions are affected by similar amounts of leptin. Using a knock-in of LacZ in the leptin locus, we failed to detect any leptin synthesis in the central nervous system. However, increasing serum leptin level, even dramatically, reduced bone mass. Conversely, reducing serum-free leptin level by overexpressing a soluble receptor for leptin increased bone mass. Congruent with these results, the high bone mass of lipodystrophic mice could be corrected by restoring serum leptin level, suggesting that leptin is an adipocyte product both necessary and sufficient to control bone mass. Consistent with the high bone mass phenotype of lipodystrophic mice, we observed an advanced bone age, an indirect reflection of premature bone formation, in lipodystrophic patients. Taken together, these results indicate that adipocyte-derived circulating leptin is a determinant of bone formation and suggests that leptin antiosteogenic function is conserved in vertebrates.

Binding of tenascin-X to decorin.

Tenascin-X (TN-X) is an extracellular matrix protein whose absence results in an alteration of the mechanical properties of connective tissue. To understand the mechanisms of integration of TN-X in the extracellular matrix, overlay blot assays were performed on skin extracts. A 100 kDa molecule interacting with TN-X was identified by this method and this interaction was abolished when the extract was digested by chondroitinase. By solid-phase assays, we showed that dermatan sulfate chains of decorin bind to the heparin-binding site included within the fibronectin-type III domains 10 and 11 of TN-X. We thus postulate that the association of TN-X with collagen fibrils is mediated by decorin and contributes to the integrity of the extracellular network.

Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X.

Tenascin-X is known as a heparin-binding molecule, but the localization of the heparin-binding site has not been investigated until now. We show here that, unlike tenascin-C, the recombinant fibrinogen-like domain of tenascin-X is not involved in heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at physiological pH, hence a set of recombinant proteins comprising these domains was tested for interaction with heparin. Using solid phase assays and affinity chromatography, we found that interaction with heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis. In this way, we identified the basic residues within each domain that are crucial for this interaction. Blocking experiments using antibodies against domain 10 were performed to test the efficiency of this site within intact tenascin-X. Binding was significantly reduced, arguing for the activity of a heparin-binding site involving domains 10 and 11 in the whole molecule. Finally, the biological significance of this site was tested by cell adhesion studies. Heparan sulfate cell surface receptors are able to interact with proteins bearing domains 10 and 11, suggesting that tenascin-X may activate different signals to regulate cell behavior.

Cell adhesion to tenascin-X mapping of cell adhesion sites and identification of integrin receptors.

Adhesive properties of tenascin-X (TN-X) were investigated using TN-X purified from bovine skin and recombinant proteins encompassing the RGD sequence located within the tenth fibronectin type-III domain, and the fibrinogen-like domain. Osteosarcoma (MG63) and bladder carcinoma cells (ECV304) cells were shown to adhere to purified TN-X, but did not spread and did not assemble actin stress fibers. Both cell types adhered to recombinant proteins harboring the contiguous fibronectin type-III domains 9 and 10 (FNX 9-10) but not to the FNX 10 domain alone. This adhesion to FNX 9-10 was shown to be mediated by alphavbeta3 integrin, was inhibited by RGD peptides and was strongly reduced in proteins mutated within the RGD site. As antibodies against alphavbeta3 integrin had no effects on cell adhesion to purified TN-X, we suggest that the RGD sequence is masked in intact TN-X. Cell attachment to the recombinant TN-X fibrinogen domain (FbgX) and to purified TN-X was greater for MG63 than for ECV304 cells. A beta1-containing integrin was shown to be involved in MG63 cell attachment to FbgX and to purified TN-X. Although the existence of other cell interaction sites is likely in this huge molecule, these similar patterns of adhesion and inhibition suggest that the fibrinogen domain might be a dominant site in the whole molecule.

Characterization of the bovine tenascin-X.

The primary structure of flexilin, an extracellular matrix glycoprotein previously identified in bovine tissues (Lethias, C., Descollonges, Y., Boutillon, M.-M., and Garrone, R. (1996) Matrix Biol. 15, 11-19) was determined by cDNA cloning. The deduced amino acid sequence (4135 residues) reveals that this protein is composed of a succession of peptide motifs characteristic of the tenascin family: an amino-terminal domain containing cysteine residues and heptads of hydrophobic amino acids, 18.5 epidermal growth factor-like repeats, 30 fibronectin type III-like (FNIII) domains, and a carboxyl-terminal fibrinogen-like motif. Sequence analysis indicated that this protein is the bovine orthologue of human tenascin-X. By rotary shadowing, bovine tenascin-X was identified as monomers with a flexible aspect, which are ended by a globule. More FNIII motifs were characterized in the bovine protein than in human tenascin-X. The main difference between the human and bovine tenascin-X is found in the arrangement of the three classes of highly similar FNIII repeat types in the central region of tenascin-X. The bovine FNIII motif b10 exhibits an RGD putative cell attachment site. The functional role of this sequence is corroborated by cell adhesion on purified tenascin-X, which is inhibited by RGD peptides. Moreover, we demonstrate that this RGD site is conserved at the same location in the human molecule.