Efrat Monsonego-Ornan

Bone Gla Protein Increases HIF-1α–Dependent Glucose Metabolism and Induces Cartilage and Vascular Calcification

Objective— Bone Gla Protein (BGP, osteocalcin) is commonly present in the calcified vasculature and was recently shown as energy metabolism-regulating hormone. This study investigates the role of BGP in cartilage and vasculature mineralization. Methods and Results— We established an in vitro BGP-overexpression model in chondrocytes (ATDC5) and vascular smooth muscle cells (MOVAS). BGP overexpression upregulated markers of chondrogenic differentiation and intensified staining for minerals. BGP overexpression enhanced glucose uptake and increased expression of glucose transporters and glycolysis enzymes while decreasing gluconeogenesis enzymes. Treatment with purified BGP activated insulin signaling pathway and upregulated genes of glucose transport and utilization. Both BGP overexpression and treatment with purified BGP resulted in stabilization of hypoxia-inducible factor 1&agr; (HIF-1&agr;) in chondrocytes and vascular smooth muscle cells, shown essential in mediating the direct metabolic effect of BGP. The in vivo model of 1,25(OH)2D3-induced vascular calcification in rats revealed a correlation between calcification, elevated BGP levels, and increased HIF-1&agr; expression in aortas and bone growth plates. The in vivo introduction of BGP siRNA, coadministered with 1,25(OH)2D3, prevented 1,25(OH)2D3-induced HIF-1&agr; stabilization, and diminished osteochondrogenic differentiation and mineralization of aortas. Conclusion— This study demonstrates novel mechanism by which BGP locally shifts cells toward glycolytic breakdown of glucose, in a HIF-1&agr;–dependent manner, and stimulates calcification of cartilage and vasculature.

Suppressors of cytokine signaling (SOCS) 1 and SOCS3 interact with and modulate fibroblast growth factor receptor signaling

Fibroblast growth factor receptor (FGFR) signaling is transduced by the mitogen-activated protein kinase (MAPK) cascade and the signal transducers and activators of transcription (STATs). Suppressors of cytokine signaling (SOCS) proteins are expressed in response to cytokine-inducible stimulation of STAT phosphorylation, acting in a negative-feedback mechanism to hinder the activities of these receptors. However, there are no data concerning the role of SOCS proteins in the regulation of fibroblast growth factor receptor (FGFR) signaling. In the present study, we show that activation of FGFR in chondrocytes induces the expression of SOCS1 and SOCS3 mRNA, and that these proteins are constitutively associated with FGFR3, as demonstrated by co-immunoprecipitation studies. Transfection of cells with FGFR3-GFP and SOCS1-CFP revealed their colocalization, clustered prominently in the perinuclear cytosolic part of the cell. The effect of the interaction between FGFR3 and SOCS1 on receptor activity was investigated in a chondrocytic cell line overexpressing SOCS1. In these cells, STAT1 phosphorylation is repressed, MAPK phosphorylation is elevated and prolonged, and FGFR3 downregulation is attenuated. Expression of osteopontin (OPN), which is directly upregulated by FGF in chondrocytes, was stimulated by lower levels of FGF in cells expressing SOCS1 compared with parental cells. Blocking of MAPK phosphorylation by PD98059 decreased OPN expression in both cell types, but this decrease was more marked in cells expressing SOCS1. The presented results suggest a novel interaction between the SOCS1 and SOCS3 proteins and the FGFR3 signaling pathway.

Matrix metalloproteinase 9/gelatinase B is required for neural crest cell migration.

This study determined the role of MMP9/gelatinase B during the migration onset of Neural Crest Cells (NCC) in avian embryos. NCC are neuroepithelial progenitors that convert into mesenchyme and migrate along defined paths throughout the embryo. To engage in migration, NCC loose cell contacts, detach from the neural tube and invade the surrounding environment. Multiple signals and transcription factors that regulate these events have been identified. Nevertheless, little is known regarding effectors that act downstream to execute the actual NCC migration. Matrix metalloproteinases (MMPs) compose a large family of enzymes whose principal substrates are basement membranes, adhesion proteins and the extracellular matrix (ECM) components. A major subgroup of MMPs, the gelatinases (MMP9 and 2) are central to many adult physiological and pathological processes, such as tumor metastasis and angiogenesis, in which cell-cell and cell-matrix contacts are degraded to allow migration. As NCC undergo similar processes during development, we hypothesized that MMP9 may also promote the migration of NCC. MMP9 was found to be expressed in delaminating and migrating NCC of both cranial and trunk axial levels. Blocking MMP9 resulted in a dramatic inhibition of NCC delamination and migration, without perturbing specification or survival. This inhibition occurred at regions containing both premigratory and migrating cells, indicative for the central role of MMP9 in executing the detachment of NCC from the neural tube as well as their migration. Conversely, excess MMP9 enhanced mesenchymalization and delamination of NCC and accelerated progenitors to undergo precocious migration. Examination of the mechanistic activity of MMP9 revealed its capability to degrade the adhesion molecule N-cadherin as well as the basement-membrane protein laminin within or around NCC, respectively. Altogether, our study reveals MMP9 as a novel effector which is required for NCC delamination and migration.

Comparison of structural, architectural and mechanical aspects of cellular and acellular bone in two teleost fish

SUMMARY The histological diversity of the skeletal tissues of fishes is impressive compared with that of other vertebrate groups, yet our understanding of the functional consequences of this diversity is limited. In particular, although it has been known since the mid-1800s that a large number of fish species possess acellular bones, the mechanical advantages and consequences of this structural characteristic – and therefore the nature of the evolution of this feature – remain unclear. Although several studies have examined the material properties of fish bone, these have used a variety of techniques and there have been no direct contrasts of acellular and cellular bone. We report on a comparison of the structural and mechanical properties of the ribs and opercula between two freshwater fish – the common carp Cyprinus carpio (a fish with cellular bone) and the tilapia Oreochromis aureus (a fish with acellular bone). We used light microscopy to show that the bones in both fish species exhibit poor blood supply and possess discrete tissue zones, with visible layering suggesting differences in the underlying collagen architecture. We performed identical micromechanical testing protocols on samples of the two bone types to determine the mechanical properties of the bone material of opercula and ribs. Our data support the consensus of literature values, indicating that Young’s moduli of cellular and acellular bones are in the same range, and lower than Young’s moduli of the bones of mammals and birds. Despite these similarities in mechanical properties between the bone tissues of the fish species tested here, cellular bone had significantly lower mineral content than acellular bone; furthermore, the percentage ash content and bone mineral density values (derived from micro-CT scans) show that the bone of these fishes is less mineralized than amniote bone. Although we cannot generalize from our data to the numerous remaining teleost species, the results presented here suggest that while cellular and acellular fish bone may perform similarly from a mechanical standpoint, there are previously unappreciated differences in the structure and composition of these bone types.

Tibial dyschondroplasia – tools, new insights and future prospects

Tibial Dyschondroplasia (TD) is one of the most prevalent skeletal abnormalities observed in avian species; it causes enormous economic losses and is a major animal welfare problem. It is characterized by lesions composed of uncalcified, unvascularized cartilage that can extend from the epiphyseal growth plate into the metaphysis. The disease development and progress have been attributed to dietary, environmental and genetic factors. Irregular cell differentiation of the chondrocytes that populate the growth plate, with consequently aberrant cartilage vascularization and mineralization, has been hypothesized to be involved in the etiology of the disease. Various tools available for the study of TD are described in the present review, and their advantages and limitations are discussed. We describe the morphology of the growth plate, with especial emphasis on the differences between the mammalian and the avian ones. We highlight vascularization as a possible cause of TD, and suggest that matrix metalloproteinases (MMPs) play an important role in cartilage vascularization and TD development. The disparity between broilers and turkeys MMPs suggests that they differ in their regulation of vascularization, so that different strategies may be required in dealing with TD in broilers and in turkeys. The high body mass of the modern meat-type birds has been implicated in the development of TD. A model was established to evaluate the effect of mechanical loading on the bones of young chickens, without any dietary manipulations: increased loading caused enhanced vascularization of the growth plate together with increased MMP-9 expression, without any changes in the incidence of TD, which suggests that the increased loading is not in itself the cause of TD. At present, the cause of TD is not known, but multidisciplinary research at various levels such as a genomic approach based on microarray technology and the chicken genome project, together with cell and organ culture methodology, genetic selection, nutritional manipulation and environmental approaches will provide us with better understanding of the molecular mechanisms underlying TD, and should pave the way for future reduction in its incidence.

The influence of severe prolonged exercise restriction on the mechanical and structural properties of bone in an avian model

Many studies have described the effects of exercise restriction on the mammalian skeleton. In particular, human and animal models have shown that reduction in weight bearing leads to generalised bone loss and deterioration of its mechanical properties. The aim of this study was to assess the effect of prolonged exercise restriction coupled with heavy calcium demands on the micro-structural, compositional and mechanical properties of the avian skeleton. The tibiae and humeri of 2-year-old laying hens housed in conventional caging (CC) and free-range (FR) housing systems were compared by mechanical testing and micro-computed tomography (microCT) scanning. Analyses of cortical, cancellous and medullary bone were performed. Mechanical testing revealed that the tibiae and humeri of birds from the FR group had superior mechanical properties relative to those of the CC group, and microCT scanning indicated larger cortical and lower medullary regions in FR group bones. Cancellous bone analysis revealed higher trabecular thickness and a higher bone volume fraction in the FR group, but no difference in mineral density. The biomechanical superiority of bones from the FR group was primarily due to structural rather than compositional differences, and this was reflected in both the cortical and cancellous components of the bones. The study demonstrated that prolonged exercise restriction in laying hens resulted in major structural and mechanical effects on the bird skeleton.

Acidic peptide hydrogel scaffolds enhance calcium phosphate mineral turnover into bone tissue.

Designed peptides may generate molecular scaffolds in the form of hydrogels to support tissue regeneration. We studied the effect of hydrogels comprising β-sheet-forming peptides rich in aspartic amino acids and of tricalcium phosphate (β-TCP)-loaded hydrogels on calcium adsorption and cell culture in vitro, and on bone regeneration in vivo. The hydrogels were found to act as efficient depots for calcium ions, and to induce osteoblast differentiation in vitro. In vivo studies on bone defect healing in rat distal femurs analyzed by microcomputerized tomography showed that the peptide hydrogel itself induced better bone regeneration in comparison to non-treated defects. A stronger regeneration capacity was obtained in bone defects treated with β-TCP-loaded hydrogels, indicating that the peptide hydrogels and the mineral act synergistically to enhance bone regeneration. In vivo regeneration was found to be better with hydrogels loaded with porous β-TCP than with hydrogels loaded with non-porous mineral. It is concluded that biocompatible and biodegradable matrices, rich in anionic moieties that efficiently adsorb calcium ions while supporting cellular osteogenic activity, may efficiently promote β-TCP turnover into bone mineral.

Exposure to omega-3 fatty acids at early age accelerate bone growth and improve bone quality

Omega-3 fatty acids (FAs) are essential nutritional components that must be obtained from foods. Increasing evidence validate that omega-3 FAs are beneficial for bone health, and several mechanisms have been suggested to mediate their effects on bone, including alterations in calcium absorption and urinary calcium loss, prostaglandin synthesis, lipid oxidation, osteoblast formation and inhibition of osteoclastogenesis. However, to date, there is scant information regarding the effect of omega-3 FAs on the developing skeleton during the rapid growth phase. In this study we aim to evaluate the effect of exposure to high levels of omega-3 FAs on bone development and quality during prenatal and early postnatal period. For this purpose, we used the fat-1 transgenic mice that have the ability to convert omega-6 to omega-3 fatty acids and the ATDC5 chondrogenic cell line as models. We show that exposure to high concentrations of omega-3 FAs at a young age accelerates bone growth through alterations of the growth plate, associated with increased chondrocyte proliferation and differentiation. We further propose that those effects are mediated by the receptors G-protein coupled receptor 120 (GPR120) and hepatic nuclear factor 4α, which are expressed by chondrocytes in culture. Additionally, using a combined study on the structural and mechanical bone parameters, we show that high omega-3 levels contribute to superior trabecular and cortical structure, as well as to stiffer bones and improved bone quality. Most interestingly, the fat-1 model allowed us to demonstrate the role of maternal high omega-3 concentration on bone growth during the gestation and postnatal period.

Interleukin-1β Promotes Proliferation and Inhibits Differentiation of Chondrocytes through a Mechanism Involving Down-Regulation of FGFR-3 and p21

The proinflammatory cytokine IL-1β is elevated in many childhood chronic inflammatory diseases as well as obesity and can be associated with growth retardation. Here we show that IL-1β affects bone growth by directly disturbing the normal sequence of events in the growth plate, resulting in increased proliferation and widening of the proliferative zone, whereas the hypertrophic zone becomes disorganized, with impaired matrix structure and increased apoptosis and osteoclast activity. This was also evident in vitro: IL-1β increased proliferation and caused a G1-to-S phase shift in the cell cycle in ATDC5 chondrocytes, accompanied by a reduction in fibroblast growth factor receptor-3 (FGFR-3) and its downstream gene, the cell-cycle inhibitor p21 and its family member p57, whereas the cell-cycle promoter E2F-2 was increased. The reduction in FGFR-3, p21, and p57 was followed by delayed cell differentiation, manifested by decreases in proteoglycan synthesis, mineralization, alkaline phosphatase activity, and the expression of Sox9, RunX2, collagen type II, collagen type X, and other matrix proteins. Taken together, we suggest that IL-1β alters normal chondrogenesis and bone growth through a mechanism involving down-regulation of FGFR-3 and p21.

1,25(OH)2D3 Alters Growth Plate Maturation and Bone Architecture in Young Rats with Normal Renal Function

Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)2D3, the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)2D3 is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)2D3 treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 for one week, or intermittent 3 µg/kg 1,25(OH)2D3 for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)2D3-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)2D3 increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)2D3 lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)2D3-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)2D3 on intracortical bone formation. This study shows negative effects of 1,25(OH)2D3 on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients.