Uptake of horseradish peroxidase by bone cells during endochondral bone development

Summary To investigate the mechanisms whereby bone cells absorb organic bone-matrix components during endochondral bone development, rat humeri were examined, employing horseradish peroxidase as a soluble protein tracer.Intravenously-injected peroxidase filled the osteoid layer and penetrated into the osteocyte lacunae and canaliculi, but did not enter the mineralized bone matrix. Whereas osteocytes rarely took up exogenous peroxidase, osteoblasts and osteoclasts actively endocytosed peroxidase in pinocytotic coated vesicles, tubular structures, and vacuoles. They also formed endocytotic vacuoles containing peroxidase in the Golgi area. The Golgi apparatus and dense bodies of these bone cells were, however, free of reaction products. Osteoclast ruffled borders were responsible for peroxidase absorption. In the osteoblast, osteocyte and osteoclast, endogenous peroxidatic reaction was detected only in mitochondria and not in other membrane-bounded vesicles and bodies. These results strongly suggest that both osteoblasts and osteoclasts participate in the resorption of bone-matrix organic components during bone remodelling.     

Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblastic cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

Differential actions of VEGF-A isoforms on perichondrial angiogenesis during endochondral bone formation

During endochondral bone formation, vascular invasion initiates the replacement of avascular cartilage by bone. We demonstrate herein that the cartilage-specific overexpression of VEGF-A₁₆₄ in mice results in the hypervascularization of soft connective tissues away from cartilage. Unexpectedly, perichondrial tissue remained avascular in addition to cartilage. Hypervascularization of tissues similarly occurred when various VEGF-A isoforms were overexpressed in the chick forelimb, but also in this case perichondrial tissue and cartilage were completely devoid of vasculature. However, following bony collar formation, anti-angiogenic properties in perichondrial tissue were lost and perichondrial angiogenesis was accelerated by VEGF-A₁₄₆, VEGF-A₁₆₆, or VEGF-A₁₉₀. Once the perichondrium was vascularized, osteoclast precursors were recruited from the circulation and the induction of MMP9 and MMP13 can be observed in parallel with the activation of TGF-β signaling. Neither perichondrial angiogenesis nor the subsequent cartilage vascularization was found to be accelerated by the non-heparin-binding VEGF-A₁₂₂ or by the VEGF-A₁₆₆ΔE₁₆₂-R₁₆₆ mutant lacking a neuropilin-binding motif. Hence, perichondrial angiogenesis is a prerequisite for subsequent cartilage vascularization and is differentially regulated by VEGF-A isoforms.     

Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.     

The role of bone morphogenetic proteins in endochondral bone formation

Bone morphogenetic proteins (BMPs) were originally identified as proteins capable of inducing endochondral bone formation when implanted at extraskeletal sites. BMPs have diverse biological activities during early embryogenesis and various aspects of organogenesis. BMPs bind to BMP receptors on the cell surface, and these signals are transduced intracellularly by Smad proteins. BMP signal pathways can be inhibited by both extra- and intracellular mechanisms. As for skeletal development, genetic studies suggest that BMPs are skeletal mesoderm inducers. Recent studies of tissue-specific activation and inactivation of BMP signals have revealed that BMP signals control proliferation and differentiation of chondrocytes, differentiation of osteoblasts and bone quality. These findings may contribute not only to understanding of bone biology and pathology, but also to improvement of the clinical efficacy of BMPs.     

Influence of electromagnetic fields on endochondral bone formation

Endochondral ossification is a basic physiological process in limb development and is central to bone repair and linear growth. Factors which regulate endochondral ossification include several biophysical and biochemical agents and are of interest from clinical and biological perspectives. One of these agents, electric stimulation, has been shown to result in enhanced synthesis of extracellular matrix, calcification, and bone formation in a number of experimental systems and is the subject of this review. The effects of electric stimulation have been studied in embryonic limb rudiments, growth plates, and experimental endochondral ossification induced with decalcified bone matrix and, in all these models, endochondral ossification has been enhanced. It is not known definitively whether electric fields stimulate cell differentiation or modulate an increased number of molecules synthesized by committed cell population and this is a fertile area of current study.     

Basal activities and hormone responsiveness of osteoclast-like and osteoblast-like bone cells are regulated by glucocorticoids.

Isolated bone cells demonstrate cell-type specific responses to glucocorticoids. Osteoclast-like (OC) cells exhibit a large decrease in basal hyaluronate synthesis at physiological doses of glucocorticoids and resistance to further inhibition by pharmacological doses up to 10(-4) M. This effect is not accompanied by decreases in protein synthesis. In contrast, osteoblast-like (OB) cell metabolism is not inhibited by physiological doses of glucocorticoids. However, in OB cells both citrate decarboxylation and collagen synthesis are decreased at pharmacological doses of glucocorticoids and these effects are accompanied by a decrease in general protein synthesis. In addition to these effects on basal and general cell activities, physiological doses of glucocorticoids modulate the hormonal sensitivity of OC and OB bone cells such that lower concentrations of bovine parathyroid hormone (PTH) are necessary to elicit measurable biochemical changes. As a result, the presence of glucocorticoids permits significant responses to PTH to be detected at doses as low as 2 x 10(-13) M in OC and OB bone cells.     

Germ cells in Hydra oligactis males. I. Isolation of a subpopulation of interstitial cells that is developmentally restricted to sperm production

Single clones of interstitial cells were generated and analyzed to determine if one interstitial cell has the capacity to differentiate both somatic and germ cells. Such clones were produced by using hydroxyurea to selectively eliminate interstitial cells from normal Hydra oligactis males. The number of animals devoid of interstitial cells within the population was determined by staining whole animals with toluidine blue which renders the interstitial cells visible. The number of animals containing single clones of interstitial cells was then estimated using single hit Poisson statistics. In treatments which rendered 60-80% of the population devoid of interstitial cells, the majority of the animals containing interstitial cells lost the ability to produce somatic cells, including nerves and nematocytes, but retained the capacity to produce sperm. This result strongly suggests the presence of a separate germ line in hydra.     

Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues.     

Melanocortin-3 receptors are involved in adaptation to restricted feeding

The central nervous melanocortin system forms a neural network that maintains energy homeostasis. Actions involving neural melanocortin-3 receptors (MC3Rs) regulate the expression rhythms in ingestive behaviors and metabolism anticipating nutrient intake. Here, we characterized the response of Mc3r knockout (Mc3r(-/-)) and wild type (WT) mice to a restricted feeding (RF) schedule where food access was limited to a 4-h period mid light cycle using a mechanical barrier. Mc3r(-/-) mice adapted poorly to the food restriction schedule. Anticipatory activity and the initial bout of intense feeding activity associated with granting food access were attenuated in Mc3r(-/-) mice, resulting in increased weight loss relative to controls. To investigate whether activity in specific hypothalamic nuclei contribute to the Mc3r(-/-) phenotype observed, we assessed hypothalamic FOS-immunoreactivity (FOS-IR) associated with food restriction. Food access markedly increased FOS-IR in the dorsomedial hypothalamus (DMH), but not in the suprachiasmatic or ventromedial hypothalamic nuclei (SCN and VMN, respectively) compared to ad libitum fed mice. Mc3r(-/-) mice displayed a significant reduction in FOS-IR in the DMH during feeding. Analysis of MC3R signaling in vitro indicated dose-dependent stimulation of the extracellular signal-regulated kinase (ERK) pathway by the MC3R agonist d-Trp(8)-γMSH. Treatment of WT mice with d-Trp(8)-γMSH administered intracerebroventricularly increased the number of pERK neurons 1.7-fold in the DMH. These observations provide further support for the involvement of the MC3Rs in regulating adaptation to food restriction. Moreover, MC3Rs may modulate the activity of neurons in the DMH, a region previously linked to the expression of the anticipatory response to RF.     

Bacterial persisters are a stochastically formed subpopulation of low-energy cells

Persisters represent a small subpopulation of non- or slow-growing bacterial cells that are tolerant to killing by antibiotics. Despite their prominent role in the recalcitrance of chronic infections to antibiotic therapy, the mechanism of their formation has remained elusive. We show that sorted cells of Escherichia coli with low levels of energy-generating enzymes are better able to survive antibiotic killing. Using microfluidics time-lapse microscopy and a fluorescent reporter for in vivo ATP measurements, we find that a subpopulation of cells with a low level of ATP survives killing by ampicillin. We propose that these low ATP cells are formed stochastically as a result of fluctuations in the abundance of energy-generating components. These findings point to a general “low energy” mechanism of persister formation.

Persisters represent a small subpopulation of non- or slow-growing bacterial cells that are tolerant to killing by antibiotics, but the mechanism of their formation has remained elusive. This study of E. coli shows that stochastic heterogeneity in levels of energy-generating enzymes results in a subpopulation of cells with low ATP that are tolerant to antibiotics.     

Ovotransferrin and ovotransferrin receptor expression during chondrogenesis and endochondral bone formation in developing chick embryo

Ovotransferrin expression during chick embryo tibia development has been investigated in vivo by immunocytochemistry and in situ hybridization. Ovotransferrin was first observed in the 7 day cartilaginous rudiment. At later stages, the factor was localized in the articular zone of the bone epiphysis and in the bone diaphysis where it was concentrated in hypertrophic cartilage, in zones of cartilage erosion and in the osteoid at the chondro-bone junction. When the localization of the ovotransferrin receptors was investigated, it was observed that chondrocytes at all stages of differentiation express a low level of the oviduct (tissue) specific receptor. Interestingly, high levels of the receptor were detectable in the 13-d old tibia in the diaphysis collar of stacked-osteoprogenitor cells and in the layer of derived osteoblasts. High levels of oviduct receptor were also observed in the primordia of the menisci. Metabolic labeling of proteins secreted by cultured chondrocytes and osteoblasts and Northern blot analysis of RNA extracted from the same cells confirmed and completed the above information. Ovotransferrin was expressed by in vitro differentiating chondrocytes in the early phase of the culture and, at least when culture conditions allowed extracellular matrix assembly, also by hypertrophic chondrocytes and derived osteoblast-like cells. Osteoblasts directly obtained from bone chips produced ovotransferrin only at the time of culture mineralization. By Western blot analysis, oviduct receptor proteins were detected at a very low level in extract from differentiating and hypertrophic chondrocytes and at a higher level in extract from hypertrophic chondrocytes undergoing differentiation to osteoblast-like cells and from mineralizing osteoblasts. Based on these results, the existence of autocrine and paracrine loops involving ovotransferrin and its receptor during chondrogenesis and endochondral bone formation is discussed.     

Isomer-specific effects of conjugated linoleic acid on mineralized bone nodule formation from human osteoblast-like cells

Mixed isomers of conjugated linoleic acid (CLA) have been shown to have variable effects on bone formation and resorption in animals. The variable effects of CLA on bone physiology may be due to the different isomers present in common commercial preparations of CLA, and the effects of the predominant individual isomers (9cis,11trans and 10trans,12cis CLA) are not clear. The objective of this study was to determine the effects of individual and mixed isomers of CLA on mineralized bone nodule formation and alkaline phosphatase (ALP) activity in vitro using long-term cultures of SaOS-2 cells. Mineralized bone nodules were stained using the von Kossa method, and ALP activity in cell lysates was measured as a marker of early osteoblast differentiation. The 9cis,11trans isomer increased the number (~4- to 11-fold) and size (~2- to 5-fold) of mineralized bone nodules from 25 to 100 microM, but the 10trans,12cis isomer did not. The increase in mineralized bone nodule formation by 9cis,11trans CLA was accompanied by a variable increase in ALP activity. These results show that the 9cis,11trans isomer of CLA increases the formation of mineralized bone nodules using bone cells of human origin, and provide evidence for isomer-specific effects of CLA on bone health.