Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

Integrins and signaling in osteoclast function

Integrins are heterodimeric adhesion receptors that mediate cell–matrix and cell–cell interactions. Osteoclasts highly express the αvβ3 integrin, which binds to a variety of extracellular matrix proteins including vitronectin, osteopontin and bone sialoprotein. RGD-containing peptides, RGD-mimetics and αvβ3 blocking antibodies inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in osteoclast function. RGD-containing peptides were shown to raise cytosolic calcium in osteoclasts. Furthermore, several signaling and adaptor molecules were found to be involved in αvβ3 integrin-dependent signaling pathways, including phosphatidylinositol 3-kinase, c-Src, PYK2 and p130^cas. In addition, cytoskeletal molecules such as paxillin, vinculin, gelsolin and F-actin are recruited to adhesion contacts upon integrin activation. Many of these molecules signaling and cytoskeletal localize to the sealing zone of actively resorbing osteoclasts, suggesting that they play a role in linking the adhesion of osteoclasts to the bone matrix with the cytoskeletal organization and the polarization and activation of these cells for bone resorption.     

Glutamatergic regulation of bone resorption

There has been increasing evidence during the last years that glutamate (Glu), the major neuromediator of the nervous system, contributes to the local regulation of bone cell functions. Several classes of Glu receptors and transporters, as well as molecules involved in glutamate signal transduction in neuronal tissue, were identified in bone. While recent findings suggest that Glu may participate in mechanisms underlying bone formation, several studies indicate that Glu may also control bone resorption. Ionotropic NMDA and metabotropic Glu receptors are expressed by osteoclasts and electrophysiological studies have demonstrated that NMDA receptors (NMDAR) are functional on these cells. In vitro studies have shown that NMDAR are important for osteoclast function since several specific antagonists of NMDAR which block the current induced by Glu in these cells also inhibit bone resorption. Preliminary studies investigating the mechanisms of action of NMDAR antagonists on bone resorption are reviewed in this paper. There is also growing evidence that NMDAR are expressed throughout the osteoclastic differentiation sequence and that antagonists of NMDAR affect osteoclastogenesis. Very few in vivo studies have however investigated the role of Glu in skeletal metabolism and bone resorption and clearly further work is required to demonstrate the relevance of glutamate signaling in the physiology of bone resorption in vivo.     

Osteoblastic glutamate receptor function regulates bone formation and resorption

Previous studies showed that a variety of bone cells express protein components necessary for neuronal-like glutamatergic signaling and implicated glutamate as having a role in mechanically induced bone remodeling. Initial functional studies concentrated on the role of glutamate signaling in bone resorption and provided compelling evidence to suggest that glutamate signaling through functional NMDA type ionotropic glutamate receptors (iGluRs) is a prerequisite for in vitro osteoclastogenesis. Originally, effects of iGluR antagonists seen in co-cultures were attributed to antagonists acting directly on osteoclast precursors. However, in the light of recent osteoblast studies it now seems likely that the observed effects on osteoclastogenesis are an indirect effect of modulating the function of pre-osteoblast present within these cultures. The presence of iGluRs in osteoblasts suggests a role for them in bone formation and this paper reviews and discusses the emerging data relating to the role of glutamate signaling in osteoblasts. A number of recently published studies have shown that osteoblasts not only express a wide number of 'pre-synaptic' glutamatergic proteins but also possess the ability to both regulate glutamate release and actively recycle extracellular glutamate. The functionality of osteoblastic 'post-synaptic' glutamatergic components has also been shown as both primary and clonal osteoblasts express electrophysiologically active iGluRs, metabotropic type glutamate receptors (mGluRs) along with a variety of glutamate receptor associated signaling proteins. There is, however, little published data regarding the actual role of glutamatergic signaling in osteoblastic bone formation. In vivo and in vitro studies performed provide evidence that glutamatergic signaling is a necessity for normal osteoblast function. In a number of different models of in vitro bone formation, the addition of non-competitive antagonists of iGluRs prevents the formation of mineralized bone, moreover antagonizing some sub-types of iGluR mediates the differentiation of pre-osteoblasts. iGluR antagonists modulate osteoblast function in a manner that correlates with the previously reported data regarding in vitro osteoclastogenesis. Interestingly iGluR mediated glutamate signaling appears to function differently in osteoblasts derived from flat and long bones. This implies the components of osteoblastic glutamatergic signaling may be adapted in vivo possibly to reflect the differential function of osteoblasts in those regions of the skeleton.     

The BMP signaling and in vivo bone formation

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smads 1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and their signaling molecules have shown that BMP signaling plays critical roles in bone and cartilage development and postnatal bone formation. BMP activities are regulated at different molecular levels. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue.     

Role of Slit/Robo Signaling pathway in Bone Metabolism

Slit/Robo signals were initially found to play an essential role in nerve development as axonal guidance molecules. In recent years, with in-depth study, the role of Slit/Robo in other life activities, such as tumor development, angiogenesis, cell migration, and bone homeostasis, has gradually been revealed. Bone is an organ with an active metabolism. Bone resorption and bone formation are closely related through precise spatiotemporal coordination. There is much evidence that slit, as a new bone coupling factor, can regulate bone formation and resorption. For example, Slit3 can promote bone formation and inhibit bone resorption through Robo receptors, which has excellent therapeutic potential in metabolic bone diseases. Although the conclusions of some studies are contradictory, they all affirm the vital role of Slit/Robo signaling in regulating bone metabolism. This paper reviews the research progress of Slit/Robo signaling in bone metabolism, briefly discusses the contradictions in the existing research, and puts forward the research direction of Slit/Robo in the field of bone metabolism in the future.     

Wnt signaling pathway in rheumatoid arthritis,with special emphasis on the different roles in synovial inflammation and bone remodeling

Rheumatoid arthritis (RA) is a chronic symmetrical autoimmune disease of unknown etiology that affects primarily the diarthrodial joints. Characteristic features of RA pathogenesis are synovial inflammation and proliferation accompanied by cartilage erosion and bone loss. Fibroblast-like synoviocytes (FLS) display an important role in the pathogenesis of RA. Several lines of evidence show that the Wnt signaling pathway significantly participates in the RA pathogenesis. The Wnt proteins are glycoproteins that bind to the Fz receptors on the cell surface, which leads to several important biological functions, such as cell differentiation, embryonic development, limb development and joint formation. Accumulated evidence has suggested that this signaling pathway plays a key role in the FLS activation, bone resorption and joint destruction during RA development. Greater knowledge of the role of the Wnt signaling pathway in RA could improve understanding of the RA pathogenesis and the differences in RA clinical presentation and prognosis. In this review, new advances of the Wnt signaling pathway in RA pathogenesis are discussed, with special emphasis on its different roles in synovial inflammation and bone remodeling. Further studies are needed to reveal the important role of the members of the Wnt signaling pathway in the RA pathogenesis and treatment.     

The endocannabinoid signaling system: a marriage of PUFA and musculoskeletal health

The role of diet in health and diseases related to muscle and bone has been an area of active study. Recently, endocannabinoids (EC), endogenous derivatives of arachidonic acid, an omega-6 (n-6) polyunsaturated fatty acid (PUFA), have been discovered to play regulatory roles in bone mass and muscle energy metabolism. This signaling system consists of the G-protein coupled cannabinoid receptors, CB1 and CB2, expressed in central and peripheral tissues and cells, which are variably activated by the production and on demand release of endogenous and synthetic agonists and antagonists. We propose that the balance between omega-6 and omega-3 (n-3) PUFA is an important modifier for the activation and suppression of endocannabinoid receptors and therefore, downstream signaling actions in cells. The potential of dietary PUFA to regulate this signaling system to influence the metabolic and physiological outcomes favorable to musculoskeletal health is the purpose of this review. The important role of n-3 PUFA in metabolic and physiological processes that attenuate muscle and bone loss under conditions of disease and stress is one aspect described herein. In this review, we first introduce the EC agonists (ligands) and their receptors (CB1 and CB2) and the general actions of EC signaling in various organs and systems. Second, we describe EC signaling in bone and muscle and how dietary PUFA influence the levels of endogenous agonists. Third, we discuss the potential implications of how dietary PUFA impact this system to minimize muscle atrophy and osteopenia and support healthy muscle development and bone modeling.     

FGF signaling in the developing endochondral skeleton

Mutations in fibroblast growth factor receptors (Fgfrs) are the etiology of many craniosynostosis and chondrodysplasia syndromes in humans. The phenotypes associated with these human syndromes and the phenotypes resulting from targeted mutagenesis in the mouse have defined essential roles for FGF signaling in both endochondral and intramembranous bone development. In this review, I will focus on the role of FGF signaling in chondrocytes and osteoblasts and how FGFs regulate the growth and development of endochondral bone.     

The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells

Lactoferrin induces osteoblast proliferation and survival in vitro and is anabolic to bone in vivo. The molecular mechanisms by which lactoferrin exerts these biological actions are not known, but lactoferrin is known to bind to two members of the low-density lipoprotein receptor family, low- density lipoprotein receptor-related proteins 1 (LRP1) and 2 (LRP2). We have examined the role(s) of these receptors in the actions of lactoferrin on osteoblasts. We show that lactoferrin binds to cultured osteoblastic cells, and that LRP1 and LRP2 are expressed in several osteoblastic cell types. In primary rat osteoblastic cells, the LRP1/2 inhibitor receptor associated protein blocks endocytosis of lactoferrin and abrogates lactoferrin-induced p42/44 MAPK signaling and mitogenesis. Lactoferrin-induced mitogenesis is also inhibited by an antibody to LRP1. Lactoferrin also induces receptor associated protein-sensitive activation of p42/44 MAPK signaling and proliferation in osteoblastic human SaOS-2 cells, which express LRP1 but not LRP2. The mitogenic response of LRP1-null fibroblastic cells to lactoferrin is substantially reduced compared with that of cells expressing wild-type LRP1. The endocytic and signaling functions of LRP1 are independent of each other, because lactoferrin can activate mitogenic signaling in conditions in which endocytosis is inhibited. Taken together, these results 1) suggest that mitogenic signaling through LRP1 to p42/44 MAPKs contributes to the anabolic skeletal actions of lactoferrin; 2) demonstrate growth-promoting actions of a third LRP family member in osteoblasts; and 3) provide further evidence that LRP1 functions as a signaling receptor in addition to its recognized role in ligand endocytosis.     

Modulation of cancer cell signaling by long noncoding RNAs

Cellular signaling pathways play a very important role in almost all molecular processes in the cell, and are generally composed of a complex set of cascades in which enzymes and proteins play a key role. These signaling pathways include different types of cellular signaling classified based on their receptors and effector proteins such as enzyme-linked receptors, cytokine receptors, and G-protein-coupled receptors each of which is subdivided into different classes. Signaling pathways are tightly controlled by different mechanisms mostly thorough inhibiting/activating their receptors or effector proteins. In the last two decades, our knowledge of molecular biology has changed dramatically and today we know that more than 85% of the human genome expresses noncoding RNAs most of which are crucial in the cellular and molecular mechanisms of cells. One of these noncoding RNAs are long noncoding RNAs (lncRNA) containing more than 200 nucleotides. LncRNAs participate in the progression of cancer growth through several mechanism including signaling pathways. In this review, we summarize some of the most important of lncRNAs and their effect on important signaling pathways.     

Research resource: Haploinsufficiency of receptor activity-modifying protein-2 (RAMP2) causes reduced fertility, hyperprolactinemia, skeletal abnormalities, and endocrine dysfunction in mice

Receptor activity-modifying protein-2 (RAMP2) is a single-pass transmembrane protein that can regulate the trafficking, ligand binding, and signaling of several G protein-coupled receptors (GPCR). The most well-characterized role of RAMP2 is in the regulation of adrenomedullin (AM) binding to calcitonin receptor-like receptor (CLR), and our previous studies using knockout mouse models support this canonical signaling paradigm. For example, Ramp2(-/-) mice die at midgestation with a precise phenocopy of the AM(-/-) and Calcrl(-/-) mice. In contrast, Ramp2(+/-) mice are viable and exhibit an expanded variety of phenotypes that are distinct from those of Calcrl(+/-) mice. Using Ramp2(+/-) female mice, we demonstrate that a modest decrease in Ramp2 expression causes severe reproductive defects characterized by fetal growth restriction, fetal demise, and postnatal lethality that is independent of the genotype and gender of the offspring. Ramp2(+/-) female mice also exhibit hyperprolactinemia during pregnancy and in basal conditions. Consistent with hyperprolactinemia, Ramp2(+/-) female mice have enlarged pituitary glands, accelerated mammary gland development, and skeletal abnormalities including delayed bone development and decreased bone mineral density. Because RAMP2 has been shown to associate with numerous GPCR, it is likely that signaling of one or more of these GPCR is compromised in Ramp2(+/-) mice, yet the precise identification of these receptors remains to be elucidated. Taken together, this work reveals an essential role for RAMP2 in endocrine physiology and provides the first in vivo evidence for a physiological role of RAMP2 beyond that of AM/CLR signaling.     

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm² at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF₁₆₄, play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.     

A role for ligand-gated ion channels in rod photoreceptor development

Neurotransmitter receptors are central to communication at synapses. Many components of the machinery for neurotransmission are present prior to synapse formation, suggesting a developmental role. Here, evidence is presented that signaling through glycine receptor alpha2 (GlyRalpha2) and GABA(A) receptors plays a role in photoreceptor development in the vertebrate retina. The signaling is likely mediated by taurine, which is present at high levels throughout the developing central nervous system (CNS). Taurine potentiates the production of rod photoreceptors, and this induction is inhibited by strychnine, an antagonist of glycine receptors, and bicuculline, an antagonist of GABA receptors. Gain-of-function experiments showed that signaling through GlyRalpha2 induced exit from mitosis and an increase in rod photoreceptors. Furthermore, targeted knockdown of GlyRalpha2 decreased the number of photoreceptors while increasing the number of other retinal cell types. These data support a previously undescribed role for these ligand-gated ion channels during the early stages of CNS development.     

Cytokine properties of prokineticins

Prokineticins are a novel family of secreted peptides with diverse regulatory roles, one of which is their capacity to modulate immunity in humans and in other species. Prokineticins are small peptides of 8 kDa that mediate their biological activities by signaling through two homologous G-protein-coupled receptors (prokineticin receptor 1 and prokineticin receptor 2). This family of peptides is characterized by a completely conserved N-terminal hexapeptide crucial for their bioactivities and a unique structural motif comprising five disulfide bonds. Prokineticins and their receptors are highly expressed in bone marrow, in peripheral circulating leukocytes, in inflamed tissues and in resident organ immune cells. Their structure, size, signaling and biological activities are reminiscent of the chemokine superfamily. In this review, emphasis is placed on the properties of prokineticins as cytokines and their role in the immune system.     

Expression of OB receptor splice variants during prenatal development of the mouse

In adult animals, signaling through the leptin receptor (OB-R) has been shown to play a critical role in fat metabolism. However, it is not known when these receptors are first expressed and what their role may be during embryonic development. To date, at least 6 splice variants of the OB-R have been identified. Although the function of each of these individual splice variants are unknown, only one of them, ob-rL ,encodes a receptor with a long intracellular domain that is implicated in OB-R signaling. In this study we have used in situ hybridization to examine the localization of OB-R splice variants during embryonic development of C57B1/6J mice. Using a probe, ob-r, that recognizes all of the splice variants, ob-r mRNA was found to be distributed in developing bone, mesenchyme, notochord and liver. In addition, epithelial structures including leptomeninges, choroid plexi and hair follicles also expressed ob-r. No ob-r mRNA was detected in the CNS. ob-rL, expression was only detected in notochord, bone and mesenchyme. The differential expression of these two mRNA isoforms suggests that the extracellular and intracellular domains of the OB receptor perform different biological functions.     

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor β (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.     

Osteotropic effects by the neuropeptides calcitonin gene-related peptide, substance P and vasoactive intestinal peptide

Immunohistochemical phenotypic characterization of skeletal nerve fibers has demonstrated the expression of a restricted number of neuropeptides, including calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP). According to the neuro-osteological hypothesis, such neuropeptides can be released and exert paracrine biological effects on bone cells present close to the nerve endings expressing these signaling molecules. The existence of such interplay is most convincingly shown by the hypothalamic control of bone formation, in the case of leptin stimulation of hypothalamic nuclei mediated by the sympathetic nervous system and inhibitory beta-adrenergic receptors on osteoblasts. In addition to these receptors, osteoblasts and osteoclasts express functional receptors for CGRP, SP and VIP, which can regulate both bone formation and bone resorption. The evidence for these observations is summarized in the present paper.