Human primary endothelial cells stimulate human osteoprogenitor cell differentiation.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly endothelial cells that may be pivotal members of a complex interactive communication network in bone. While cell cooperation was previously established between Human OsteoProgenitor cells (HOP) and Human Umbilical Vein Endothelial Cells (HUVEC) the aim of our study was to investigate if this interaction is specific to Human Endothelial cell types (ECs) from different sources. Osteoblastic cell differentiation analysis performed using different co-culture models with direct contact revealed that Alkaline Phosphatase (Al-P) activity was only increased by the direct contact of HOP with human primary vascular endothelial cell types including endothelial precursor cells (EPCs) isolated from blood cord, endothelial cells from Human Saphen Vein (HSV) while a transformed cell line, the Human Bone Marrow Endothelial Cell Line (HBMEC) did not modify osteoblastic differentiation of HOP. Because connexin 43, a specific gap junction protein, seemed to be involved in HUVEC/HOP cell cooperation, expression by RT-PCR and immunocytochemistry of this gap junctional protein was investigated in EPCs, HSV and HBMEC. Both endothelial cells are positive to this protein and the disruption of gap junction communication using 18alpha-glycyrrhetinic acid treatment decreased the positive effect of these endothelial co-cultures on HOP differentiation as was previously demonstrated for HUVEC and HOP co-cultures. These data seem to indicate that this cross talk between HOP and ECs, through gap junction communication constitutes an additional concept in cell differentiation control.     

Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication

Bone development and remodeling depend oncomplex interactions between bone-forming osteoblasts and other cellspresent within the bone microenvironment, particularly vascularendothelial cells that may be pivotal members of a complex interactivecommunication network in bone. Our aim was to investigate theinteraction between human umbilical vein endothelial cells (HUVEC) andhuman bone marrow stromal cells (HBMSC). Cell differentiation analysisperformed with different cell culture models revealed that alkalinephosphatase activity and type I collagen synthesis were increased onlyby the direct contact of HUVEC with HBMSC. This "juxtacrinesignaling" could involve a number of different heterotypic connexionsthat require adhesion molecules or gap junctions. A dyecoupling assay with Lucifer yellow demonstrated a functional couplingbetween HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSCbut also in the endothelial cell network and that these two cell typescan communicate via a gap junctional channel constituted at least byCx43. Moreover, functional inhibition of the gap junction by18-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVECcocultures on HBMSC differentiation. This stimulation could be mediatedby the intercellular diffusion of signaling molecules that permeate thejunctional channel.

     

Osteoclast-osteoblast communication

Cells in osteoclast and osteoblast lineages communicate with each other through cell-cell contact, diffusible paracrine factors and cell-bone matrix interaction. Osteoclast-osteoblast communication occurs in a basic multicellular unit (BMU) at the initiation, transition and termination phases of bone remodeling. At the initiation phase, hematopoietic precursors are recruited to the BMU. These precursors express cell surface receptors including c-Fms, RANK and costimulatory molecules, such as osteoclast-associated receptor (OSCAR), and differentiate into osteoclasts following cell-cell contact with osteoblasts, which express ligands. Subsequently, the transition from bone resorption to formation is mediated by osteoclast-derived ‘coupling factors’, which direct the differentiation and activation of osteoblasts in resorbed lacunae to refill it with new bone. Bidirectional signaling generated by interaction between ephrinB2 on osteoclasts and EphB4 on osteoblast precursors facilitates the transition. Such interaction is likely to occur between osteoclasts and lining cells in the bone remodeling compartment (BRC). At the termination phase, bone remodeling is completed by osteoblastic bone formation and mineralization of bone matrix. Here, we describe molecular communication between osteoclasts and osteoblasts at distinct phases of bone remodeling.     

Asymmetric intercellular communication between bone cells: Propagation of the calcium signaling

Bone functional adaptation by remodeling is achieved by harmonized activities of bone cells in which osteocytes in the bone matrix are believed to play critical roles in sensing mechanical stimuli and transmitting signals to osteoclasts/osteoblasts on the bone surface in order to regulate their bone remodeling activities through the lacuno-canalicular network with many slender osteocytic processes. In this study, we investigated the intercellular communication between bone cells, particularly focusing on its directionality, through in vitro observations of the calcium signaling response to mechanical stimulus and its propagation to neighboring cells (NCs). Direct mechanical stimulus was applied to isolated bone cells from chick calvariae, osteocytes (Ocys) and bone surface cells (BSCs) mainly containing osteoblasts, and the percentage of calcium signaling propagation from the stimulated cell to NCs was analyzed. The results revealed that, regardless of the type of stimulated cell, the signaling propagated to BSCs with a significantly higher percentage, implying that calcium signaling propagation between bone cells strongly depends on the type of receiver cell and not the transmitter cell. In addition, in terms of mutual communication between Ocys and BSCs, the percentage of propagation from Ocys to BSCs is significantly higher than that in the opposite direction, suggesting that the calcium signaling mainly propagates asymmetrically with a bias from Ocys in bone matrix to BSCs on bone surfaces. This asymmetric communication between Ocys and BSCs suggests that osteocytic mechanosensing and cellular communications, which significantly affect bone surface remodeling activities to achieve functional adaptation, seem to be well coordinated and active at the location of biologically suitable and mechanically sensitive regions close to the bone surfaces.     

Angiogenic factors in bone local environment

Angiogenesis plays an important role in physiological bone growth and remodeling, as well as in pathological bone disorders such as fracture repair, osteonecrosis, and tumor metastasis to bone. Vascularization is required for bone remodeling along the endosteal surface of trabecular bone or Haversian canals within the cortical bone, as well as the homeostasis of the cartilage-subchondral bone interface. Angiogenic factors, produced by cells from a basic multicellular unit (BMU) within the bone remodeling compartment (BRC) regulate local endothelial cells and pericytes. In this review, we discuss the expression and function of angiogenic factors produced by osteoclasts, osteoblasts and osteocytes in the BMU and in the cartilage-subchondral bone interface. These include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), BMP7, receptor activator of NF-κB ligand (RANKL) and epidermal growth factor (EGF)-like family members. In addition, the expression of EGFL2, EGFL3, EGFL5, EGFL6, EGFL7, EGFL8 and EGFL9 has been recently identified in the bone local environment, giving important clues to their possible roles in angiogenesis. Understanding the role of angiogenic factors in the bone microenvironment may help to develop novel therapeutic targets and diagnostic biomarkers for bone and joint diseases, such as osteoporosis, osteonecrosis, osteoarthritis, and delayed fracture healing.     

Osteoblast migration in vertebrate bone

Bone formation, for example during bone remodelling or fracture repair, requires mature osteoblasts to deposit bone with remarkable spatial precision. As osteoblast precursors derive either from circulation or resident stem cell pools, they and their progeny are required to migrate within the three‐dimensional bone space and to navigate to their destination, i.e. to the site of bone formation. An understanding of this process is emerging based on in vitro and in vivo studies of several vertebrate species. Receptors on the osteoblast surface mediate cell adhesion and polarization, which induces osteoblast migration. Osteoblast migration is then facilitated along gradients of chemoattractants. The latter are secreted or released proteolytically by several cell types interacting with osteoblasts, including osteoclasts and vascular endothelial cells. The positions of these cellular sources of chemoattractants in relation to the position of the osteoblasts provide the migrating osteoblasts with tracks to their destination, and osteoblasts possess the means to follow a track marked by multiple chemoattractant gradients. In addition to chemotactic cues, osteoblasts sense other classes of signals and utilize them as landmarks for navigation. The composition of the osseous surface guides adhesion and hence migration efficiency and can also provide steering through haptotaxis. Further, it is likely that signals received from surface interactions modulate chemotaxis. Besides the nature of the surface, mechanical signals such as fluid flow may also serve as navigation signals for osteoblasts. Alterations in osteoblast migration and navigation might play a role in metabolic bone diseases such as osteoporosis.     

Bone remodeling: Multiple cellular interactions required for coupling of bone formation and resorption

The dynamic nature of the skeleton is achieved by a process called "remodeling" which involves the co-ordinated actions of osteoclasts, osteoblasts, osteocytes within the bone matrix and osteoblast-derived lining cells that cover the surface of bone. Remodeling commences with signals that initiate osteoclast formation followed by osteoclast-mediated bone resorption, a reversal period, and then a long period of bone matrix formation mediated by osteoblasts, followed by mineralisation of the matrix. This review will discuss each of these steps with particular emphasis on the communication pathways between each cell type involved and the roles of ephrins, sclerostin, RANKL and PTHrP.     

Membrane trafficking in osteoblasts and osteoclasts: new avenues for understanding and treating skeletal diseases

The endocytic and exocytic/secretory pathways are two major intracellular membrane trafficking routes that regulate numerous cellular functions in a variety of cell types. Osteoblasts and osteoclasts, two major bone cells responsible for bone remodeling and homeostasis, are no exceptions. During the past few years, emerging evidence has pinpointed a critical role for endocytic and secretory pathways in osteoblast and osteoclast differentiation and function. The endosomal membrane provides a platform to integrate bone tropic signals of hormones and growth factors in osteoblasts. In osteoclasts, endocytosis, followed by transcytosis, of degraded bone matrix promotes bone resorption. Secretory pathways, especially lysosome secretion, not only participate in bone matrix deposition by osteoblasts and degradation of mineralized bone matrix by osteoclasts; they may also be involved in the coupling of bone resorption and bone formation during bone remodeling. More importantly, mutations in genes encoding regulatory factors within the endocytic and secretory pathways have been identified as causes for bone diseases. Identification of the molecular mechanisms of these genes in bone cells may provide new therapeutic targets for skeletal disorders.     

Cell-to-cell interactions in bone

Bone development (modeling) occurs by migration, aggregation, and condensation of immature osteo/chondroprogenitor cells to form the cartilaginous anlage. This process requires precisely controlled cell-cell interactions. Likewise, bone remodeling in the adult skeleton is a dynamic process that requires coordinated cellular activities among osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. The cooperative nature of both bone modeling and remodeling requires tightly regulated mechanisms of intercellular recognition and communication that permit the cells to sort and migrate, synchronize activity, equalize hormonal responses, and diffuse locally generated signals. Osteoblasts and osteocytes achieve these interactions through cadherin-based adherens junctions as well as by formation of communicating junctions, gap junctions. This review examines the current knowledge of how direct cell-to-cell interactions modulate osteoblast function.     

HMGB1 expression and release by bone cells

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.     

Cell-cell interactions in regulating osteogenesis and osteoblast function

Endochondral bone formation requires an elaborate interplay among autocrine, paracrine, and endocrine signals, positional cues, and cell-cell contacts to mediate the complex three-dimensional architecture and function of the skeleton. Embryonic bone development occurs by migration, aggregation, and condensation of immature mesenchymal progenitor cells to form the cartilaginous anlage. Upon vascular invasion, the cartilaginous scaffold is colonized and subsequently mineralized by osteoblasts. Likewise, bone remodeling in the adult skeleton is a dynamic process that requires coordinated cellular activities among osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. This review examines the role of cell-cell interactions mediated by adherens junctions formed by cadherins and communicative gap junctions formed by connexins in regulating bone development and osteogenic function.     

A supra-cellular model for coupling of bone resorption to formation during remodeling: lessons from two bone resorption inhibitors affecting bone formation differently

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.     

Modeling of bone adaptative behavior based on cells activities

Bone remodeling occurs in an adult’s skeleton to adapt its architecture to external loadings. This involves bone resorption by osteoclasts cells followed by formation of new bone by osteoblasts cells. During bone remodeling, osteoclasts and osteoblasts interact with each other by expressing autocrine and paracrine factors that regulate cells’ population. Therefore, changes in bone density depend on the amount of each acting cell population. The aim of this paper is to propose a model for the bone remodeling process, which takes into account the opposite activity of both types of cells. For this purpose, a system of differential equations, proposed by Komarova et al. (Bone 33:206–215, 2003), is introduced to describe bone cell interactions using parameters which characterize the autocrine and paracrine factors. Such equations allow us to determine how the autocrine and paracrine factors vary in response to an external stimulus. It is assumed that an equilibrium state can be obtained for values of stimulus near to some reference quantity. Far from this value, unbalanced activity of osteoblasts and osteoclasts is observed, which leads to bone apposition or resorption. The proposed model has been implemented into the finite element software ABAQUS to analyze the qualitative response of a bone structure when subjected to certain mechanical loadings. Obtained results are satisfactory and in accordance with the expected bone remodeling behavior.     

The relationship between bone marrow adipose tissue and bone metabolism in postmenopausal osteoporosis

Postmenopausal osteoporosis (PMOP) is a prevalent skeletal disorder associated with menopause-related estrogen withdrawal. PMOP is characterized by low bone mass, deterioration of the skeletal microarchitecture, and subsequent increased susceptibility to fragility fractures, thus contributing to disability and mortality. Accumulating evidence indicates that abnormal expansion of marrow adipose tissue (MAT) plays a crucial role in the onset and progression of PMOP, in part because both bone marrow adipocytes and osteoblasts share a common ancestor lineage. The cohabitation of MAT adipocytes, mesenchymal stromal cells, hematopoietic cells, osteoblasts and osteoclasts in the bone marrow creates a microenvironment that permits adipocytes to act directly on other cell types in the marrow. Furthermore, MAT, which is recognized as an endocrine organ, regulates bone remodeling through the secretion of adipokines and cytokines. Although an enhanced MAT volume is linked to low bone mass and fractures in PMOP, the detailed interactions between MAT and bone metabolism remain largely unknown. In this review, we examine the possible mechanisms of MAT expansion under estrogen withdrawal and further summarize emerging findings regarding the pathological roles of MAT in bone remodeling. We also discuss the current therapies targeting MAT in osteoporosis. A comprehensive understanding of the relationship between MAT expansion and bone metabolism in estrogen deficiency conditions will provide new insights into potential therapeutic targets for PMOP.     

Regulation of apoptosis in osteoclasts and osteoblastic cells

In postnatal life, the skeleton undergoes continuous remodeling in which osteoclasts resorb aged or damaged bone, leaving space for osteoblasts to make new bone. The balance of proliferation, differentiation, and apoptosis of bone cells determines the size of osteoclast or osteoblast populations at any given time. Bone cells constantly receive signals from adjacent cells, hormones, and bone matrix that regulate their proliferation, activity, and survival. Thus, the amount of bone and its microarchitecture before and after the menopause or following therapeutic intervention with drugs, such as sex hormones, glucocorticoids, parathyroid hormone, and bisphosphonates, is determined in part by effects of these on survival of osteoclasts, osteoblasts, and osteocytes. Understanding the mechanisms and regulation of bone cell apoptosis will enhance our knowledge of bone cell function and help us to develop better therapeutics for the management of osteoporosis and other bone diseases.     

Extracellular matrix networks in bone remodeling

Bones are constantly remodeled throughout life to maintain robust structure and function. Dysfunctional remodeling can result in pathological conditions such as osteoporosis (bone loss) or osteosclerosis (bone gain). Bone contains 100s of extracellular matrix (ECM) proteins and the ECM of the various bone tissue compartments plays essential roles directing the remodeling of bone through the coupled activity of osteoclasts (which resorb bone) and osteoblasts (which produce new bone). One important role for the ECM is to serve as a scaffold upon which mineral is deposited. This scaffold is primarily type I collagen, but other ECM components are involved in binding of mineral components. In addition to providing a mineral scaffolding role, the ECM components provide structural flexibility for a tissue that would otherwise be overly rigid. Although primarily secreted by osteoblast-lineage cells, the ECM regulates cells of both the osteoblast-lineage (such as progenitors, mature osteoblasts, and osteocytes) and osteoclast-lineage (including precursors and mature osteoclasts), and it also influences the cross-talk that occurs between these two oppositional cells. ECM influences the differentiation process of mesenchymal stem cells to become osteoblasts by both direct cell-ECM interactions as well as by modulating growth factor activity. Similarly, the ECM can influence the development of osteoclasts from undifferentiated macrophage precursor cells, and influence osteoclast function through direct osteoclast cell binding to matrix components. This comprehensive review will focus on how networks of ECM proteins function to regulate osteoclast- and osteoblast-mediated bone remodeling. The clinical significance of these networks on normal bone and as they relate to pathologies of bone mass and geometry will be considered. A better understanding of the dynamic role of ECM networks in regulating tissue function and cell behavior is essential for the development of new treatment approaches for bone loss.     

Interleukin‐27 expression modifies prostate cancer cell crosstalk with bone and immune cells in vitro

Prostate cancer is frequently associated with bone metastases, where the crosstalk between tumor cells and key cells of the bone microenvironment (osteoblasts, osteoclasts, immune cells) amplifies tumor growth. We have explored the potential of a novel cytokine, interleukin‐27 (IL‐27), for inhibiting this malignant crosstalk, and have examined the effect of autocrine IL‐27 on prostate cancer cell gene expression, as well as the effect of paracrine IL‐27 on gene expression in bone and T cells. In prostate tumor cells, IL‐27 upregulated genes related to its signaling pathway while downregulating malignancy‐related receptors and cytokine genes involved in gp130 signaling, as well as several protease genes. In both undifferentiated and differentiated osteoblasts, IL‐27 modulated upregulation of genes related to its own signaling pathway as well as pro‐osteogenic genes. In osteoclasts, IL‐27 downregulated several genes typically involved in malignancy and also downregulated osteoclastogenesis‐related genes. Furthermore, an osteogenesis‐focused real‐time PCR array revealed a more extensive profile of pro‐osteogenic gene changes in both osteoblasts and osteoclasts. In T‐lymphocyte cells, IL‐27 upregulated several activation‐related genes and also genes related to the IL‐27 signaling pathway and downregulated several genes that could modulate osteoclastogenesis. Overall, our results suggest that IL‐27 may be able to modify interactions between prostate tumor and bone microenvironment cells and thus could be used as a multifunctional therapeutic for restoring bone homeostasis while treating metastatic prostate tumors. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity

The cells of bone are of two lineages, the osteoblasts arising from pluripotential mesenchymal cells and osteoclasts from hemopoietic precursors of the monocyte-macrophage series. Resorption of bone by the multinucleate osteoclast requires the generation of new osteoclastsw and their activation. Many hormones and cytokines are able to promote bone resorption by influencing these processes, but they achieve this without acting directly on osteoclastws. Most evidence indicates that their actions are mediated by cells of the osteoblast lineage. Evidence for hormone-and cytokine-induced activation of osteoclasts requiring the mediation of osteoblasts comes from studies of rsorption by isolated osteoclasts. However, consistent evidence for a spiceific “activating factor” is lacking, and the argument is presented that the isolated osteoclast resorption assays have not been shown convincingly to be assays of osteoclast activation. The view is presented that osteoblast-mediated osteoclast activation is the result of several events in the microenvironment without necessarily requiring the existence of a spicific, essential osteoclast activator. On the other hand, a specific promoter of osteoclast differentiation does seem likely to be a product of cells of the stromal/osteoblast series. Evidence in facour of this comes from studies of osteoclast generation in co-cultures of osteoblast/stromal cells with hemopoietic cells. Conflicting view, maintaining that osteoclasts can develop from hemooietic cells without stromal intervention, might be explaind by varying criteria used in identification of osteoclasts. Osteoblastic and osteoclastic renewal, and the interactions of these lineages, are central to the process of bone remodeling.