Cell-specific paracrine actions of IL-6 family cytokines from bone,marrow and muscle that control bone formation and resorption

Bone renews itself and changes shape throughout life to account for the changing needs of the body; this requires co-ordinated activities of bone resorbing cells (osteoclasts), bone forming cells (osteoblasts) and bone’s internal cellular network (osteocytes). This review focuses on paracrine signaling by the IL-6 family of cytokines between bone cells, bone marrow, and skeletal muscle in normal physiology and in pathological states where their levels may be locally or systemically elevated. These functions include the support of osteoclast formation by osteoblast lineage cells in response to interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM) and cardiotrophin 1 (CT-1). In addition it will discuss how bone-resorbing osteoclasts promote osteoblast activity by secreting CT-1, which acts as a “coupling factor” on osteocytes, osteoblasts, and their precursors to promote bone formation. OSM, produced by osteoblast lineage cells and macrophages, stimulates bone formation via osteocytes. IL-6 family cytokines also mediate actions of other bone formation stimuli like parathyroid hormone (PTH) and mechanical loading. CT-1, OSM and LIF suppress marrow adipogenesis by shifting commitment of pluripotent precursors towards osteoblast differentiation. Ciliary neurotrophic factor (CNTF) is released as a myokine from skeletal muscle and suppresses osteoblast differentiation and bone formation on the periosteum (outer bone surface in apposition to muscle). Finally, IL-6 acts directly on marrow-derived osteoclasts to stimulate release of “osteotransmitters” that act through the cortical osteocyte network to stimulate bone formation on the periosteum. Each will be discussed as illustrations of how the extended family of IL-6 cytokines acts within the skeleton in physiology and may be altered in pathological conditions or by targeted therapies.     

Oxytocin deficiency impairs maternal skeletal remodeling

We have reported that the posterior pituitary hormone, oxytocin (OT), known for its effects in inducing parturition, lactation and social bonding, is also a skeletal hormone. Here, we demonstrate that OT plays a key role in enabling maternal skeletal mobilization during pregnancy by enhancing the formation of bone resorbing osteoclasts. Osteoclast formation ex vivo is thus diminished in pregnant mothers with genetic OT-deficiency. OT^−/− pups at day E20 also show a defect in trabecular bone. μCT measurements reveal normal bone volume, but increased trabecular numbers, suggesting that trabeculae in OT^−/− pups are hypomineralized. We suggest that OT facilitates intergenerational transfer of calcium ions from a pregnant mother to the pups.     

Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice

The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.     

The cytokine interleukin-11 crucially links bone formation,remodeling and resorption

Bone development is a complex process that requires the activity of several different signaling pathways and cell types. It involves the coordinated action of osteoclasts (cells that are capable of resorbing bone), osteoblasts (cells that are able to form bone), osteocytes (cells that form a syncytial network within the bone), skeletal muscle cells and the bone marrow. In recent years, the cytokine interleukin-11 (IL-11), a member of the IL-6 family of cytokines, has emerged as an important regulatory protein for bone formation, remodeling and resorption. Furthermore, coding missense mutations in the IL11RA gene, which encodes the IL-11 receptor (IL-11R), have recently been linked to craniosynostosis, a human disease in which the sutures that line the head bones close prematurely. This review summarizes current knowledge about IL-11 and highlights its role in bone development and homeostasis. It further discusses the specificity and redundancy provided by the other members of the IL-6 cytokine family and how they facilitate signaling and cross-talk between skeletal muscle cells, bone cells and the bone marrow. We describe their actions in physiological and in pathological states and discuss how this knowledge could be translated into therapy.     

Influence of RGD-containing oligopeptide-coated surface on bone formation in vitro and in vivo

New bone formation on an RGD-containing oligopeptide-coated surface in vitro and in vivo was investigated. The surface showed two-fold higher osteoblastic cell adhesion and differentiation in vitro, and revealed statistically significant in vivo bone formation compared with the control (P < 0.05).     

Extracellular matrix vesicles in rat bone after parathyroidectomy

Summary The enzymatic activity of bone matrix vesicles from parathyroidectomized rats was determined and compared to the activity of vesicles from sham operated and normal animals. The vesicles were isolated from the alveolar bone by collagenase digestion and differential centrifugation and further purified on a discontinuous sucrose density gradient. The amount of extractable protein and the activity of alkaline phosphatase, acid phosphatase, and ATPase in the vesicle fractions thus obtained did not differ significantly from the values characteristic of preparations from control rats. It may therefore be suggested that parathyroid hormone depletion and the associated hypocalcemia have no significant effect on the occurrence and phosphatase activity of bone matrix vesicles.     

Death of osteocytes turns off the inhibition of osteoclasts and triggers local bone resorption

Osteocytes have been suggested to play a role in the regulation of bone resorption, although their effect on bone turnover has remained controversial. In order to study this open question, we developed an organ culture system based on isolated rat calvaria, where the osteocyte viability and its effect on osteoclastic bone resorption can be monitored. Our results suggest that osteocytes are constitutively negative regulators of osteoclastic activity. Osteoclasts, which were cultured on calvarial slices with living osteocytes inside, failed to form actin rings which are the hallmarks of resorbing cells. A similar inhibitory effect was also achieved by the conditioned medium obtained from calvarial organ culture, suggesting that living osteocytes produce yet unrecognized osteoclast inhibitors. On the contrary, when osteocyte apoptosis was induced, this inhibitory effect disappeared and strong osteoclastic bone resorption activity was observed. Thus, local apoptosis of osteocytes may play a major role in triggering local bone remodeling.     

Selenoproteins and selenium status in bone physiology and pathology

Background

Emerging evidence supports the view that selenoproteins are essential for maintaining bone health.

Scope of review

The current state of knowledge concerning selenoproteins and Se status in bone physiology and pathology is summarized.

Major conclusions

Antioxidant selenoproteins including glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), as a whole, play a pivotal role in maintaining bone homeostasis and protecting against bone loss. GPx1, a major antioxidant enzyme in osteoclasts, is up-regulated by estrogen, an endogenous inhibitor of osteoclastogenesis. TrxR1 is an immediate early gene in response to 1α,25-dihydroxyvitamin D3, an osteoblastic differentiation agent. The combination of 1α,25-dihydroxyvitamin D3 and Se generates a synergistic elevation of TrxR activity in Se-deficient osteoblasts. Of particular concern, pleiotropic TrxR1 is implicated in promoting NFκB activation. Coincidentally, TrxR inhibitors such as curcumin and gold compounds exhibit potent osteoclastogenesis inhibitory activity. Studies in patients with the mutations of selenocysteine insertion sequence-binding protein 2, a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins have clearly established a causal link of selenoproteins in bone development. Se transport to bone relies on selenoprotein P. Plasma selenoprotein P concentrations have been found to be positively correlated with bone mineral density in elderly women.

General significance

A full understanding of the role and function of selenoproteins and Se status on bone physiology and pathology may lead to effectively prevent against or modify bone diseases by using Se.     

Mammalian collagenase predisposes bone surfaces to osteoclastic resorption

Summary The cell-free endocranial surface of young adult rat parietal bones was used as a substrate for bone cell-derived mammalian collagenase. Incubation of parietal bones in a concentration of enzyme comparable to that secreted by osteoblastic cells in vitro caused destruction of surface osteoid, and resulted in exposure of mineral onto the bone surface. Bones so pre-treated were considerably more susceptible to osteoclastic resorption than bones preincubated in the absence of collagenase. These results are consistent with the view that the osteoid layer which covers bone surfaces acts as a barrier to osteoclastic contact with underlying, resorption — stimulating bone mineral; and that cells of the osteoblastic lineage induce osteoclastic resorption through collagenase secretion which, by digestion of the surface osteoid, exposes bone mineral to osteoclastic contact.     

The role of calcitonin and alpha-calcitonin gene-related peptide in bone formation

The Calca gene encodes two polypeptides, calcitonin (CT) and α-calcitonin gene-related peptide (α-CGRP), generated through alternative splicing. While CT, a hormone mainly produced by thyroidal C cells, has been described as a major regulator of bone resorption, α-CGRP, a neuropeptide expressed in the cells of the central and peripheral nervous system, is mostly known as a regulator of vascular tone. Surprisingly, the generation and skeletal analyses of two mouse deficiency models has recently uncovered a physiological function for both peptides in the regulation of bone formation. In the first model, where the replacement of exons 2-5 of the Calca gene resulted in the combined deficiency of CT and α-CGRP, an increased bone formation rate (BFR) was observed, whereas decreased BFR was found in the second model, where the introduction of a translational termination codon into exon 5 of the Calca gene resulted in the specific absence of α-CGRP.     

Bone and bone marrow: The same organ

Interplays between bone and bone marrow are not limited to merely anatomic and histological connections, but include a tight functional correlation. Bone marrow resides within the medullary cavity of the bones and the process of hematopoiesis is regulated, at least in part, by bone cells. Moreover, osteoclasts and osteoblasts derive from precursors of hematopoietic and mesenchymal origin, respectively, both residing within the bone marrow. Alterations in one of these components typically cause impairment in the other, so diseases of the bone marrow compartment often affect the bone and vice versa. All these findings could make us to speculate that bone and bone marrow are not two separate districts, but can be considered as the two elements of the same unique functional unit, the bone-bone marrow organ. Here we will describe histological and functional interplays between bone and bone marrow, and will illustrate some diseases in which this tight correlation is evident.     

A molecular analysis of matrix remodeling and angiogenesis during long bone development

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a ‘molecular map’ of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.     

Osteoclast and its roles in calcium metabolism and bone development and remodeling

Osteoclasts are multinucleated cells responsible for bone resorption and play important roles in normal skeletal development, in the maintenance of its integrity throughout life, and in calcium metabolism. During bone resorption, the cytoskeleton of osteoclasts undergoes extensive reorganization, with polarization and formation of ruffled borders to secrete acid and formation of sealing zone to prevent leakage. The differentiation and function of osteoclasts are in turn regulated by osteoblasts, stromal cells, and bone. They are also subjected to negative feedback regulation by extracellular and intracellular calcium concentrations.     

Regulation of bone remodeling by the central and peripheral nervous system

The homeostatic nature of bone remodeling has become a notion further supported lately by the demonstration that neuropeptides and their receptors regulate osteoblast and osteoclast function in vivo. Following initial studies reporting the presence of nerves and nerve-derived products within the bone microenvironment and the expression of receptors for these neuropeptides in bone cells, new experimental and mechanistic evidence based on in vivo murine genetic and pharmacologic models recently demonstrated that inputs from the central and peripheral nervous system feed into the already complex regulatory machinery controlling bone remodeling. The function of a number of “osteo-neuromediators” has been characterized, including norepinephrine and the beta2-adrenergic receptor, Neuropeptide Y and the Y1 and Y2 receptors, endocannabinoids and the CB1 and CB2 receptors, as well as dopamine, serotonin and their receptors and transporters, Calcitonin gene-related peptide, and neuronal NOS. This new body of evidence suggests that neurons in the central nervous system integrate clues from the internal and external milieux, such as energy homeostasis, glycemia or reproductive signals, with the regulation of bone remodeling. The next major tasks in this new area of bone biology will be to understand, at the molecular level, the mechanisms by which common central neural systems regulate and integrate these major physiological functions, the relative importance of the central and peripheral actions of neuropeptides present in both compartments and their relationship, and how bone cells signal back to central centers, because the definition of a homeostatic function implies the existence of feedback signals. Together, these findings shed a new light on the complexity of the mechanisms regulating bone remodeling and uncovered new potential therapeutic strategies for the design of bone anabolic treatments. This review summarizes the latest advances in this area, focusing on investigations based on in vivo animal studies.     

Pleiotropic activity of lysophosphatidic acid in bone metastasis

Bone is a common metastatic site for solid cancers. Bone homeostasis is tightly regulated by intimate cross-talks between osteoblast (bone forming cells) and osteoclasts (bone resorbing cells). Once in the bone microenvironment, metastatic cells do not alter bone directly but instead perturb the physiological balance of the bone remodeling process controlled by bone cells. Tumor cells produce growth factors and cytokines stimulating either osteoclast activity leading to osteolytic lesions or osteoblast function resulting in osteoblastic metastases. Growth factors, released from the resorbed bone matrix or throughout osteoblastic bone formation, sustain tumor growth. Therefore, bone metastases are the sites of vicious cycles wherein tumor growth and bone metabolism sustain each other. Lysophosphatidic acid (LPA) promotes the growth of primary tumors and metastatic dissemination of cancer cells. We have shown that by acting on cancer cells via the contribution of blood platelets and the LPA-producing enzyme Autotaxin (ATX), LPA promotes the progression of osteolytic bone metastases in animal models. In the light of recent reports it would appear that the role of LPA in the context of bone metastases is complex involving multiple sources of lipid combined with direct and indirect effects on target cells. This review will present our current knowledge on the LPA/ATX axis involvement in osteolytic and osteoblastic skeletal metastases and will discuss the potential activity of LPA upstream and downstream metastasis seeding of cancer cells to bone as well as its implication in cancer induced bone pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Apigenin inhibits osteoblastogenesis and osteoclastogenesis and prevents bone loss in ovariectomized mice

Polyphenol have been reported to have physiological effects with respect to alleviating diseases such as osteoporosis and osteopetrosis. We recently reported that the olive polyphenol hydroxytyrosol accelerates bone formation both in vivo and in vitro. The present study was designed to evaluate the in vivo and in vitro effects of apigenin (4′,5,7-trihydroxyflavone), one of the major polyphenols in olives and parsley, on bone formation by using cultured osteoblasts and osteoclasts and ovariectomized (OVX) mice, respectively. Apigenin markedly inhibited cell proliferation and indices of osteoblast differentiation, such as collagen production, alkaline phosphatase activity, and calcium deposition in osteoblastic MC3T3-E1 cells at concentrations of 1–10 μM. At 10 μM, apigenin completely inhibited the formation of multinucleated osteoclasts from mouse splenic cells. Moreover, injection of apigenin at 10 mg kg⁻¹ body weight significantly suppressed trabecular bone loss in the femurs of OVX mice. Our findings indicate that apigenin may have critical effects on bone maintenance in vivo.     

Recombinant expression of mouse osteocalcin protein in Escherichia coli

Osteocalcin is the most abundant non-collagenous protein of bone. Recombinant mouse osteocalcin protein (mOC) that includes the highly conserved central domain for binding to hydroxyapatite (HA), a mineral component of bone, was expressed in Escherichia coli. Purified mOC protein exhibited a significant increase in HA adhesion and differentiation in osteoblast cells as well as binding to HA with high affinity.     

The response of bone to mechanical loading and disuse: fundamental principles and influences on osteoblast/osteocyte homeostasis

Bone’s response to increased or reduced loading/disuse is a feature of many clinical circumstances, and our daily life, as habitual activities change. However, there are several misconceptions regarding what constitutes loading or disuse and why the skeleton gains or loses bone. The main purpose of this article is to discuss the fundamentals of the need for bone to experience the effects of loading and disuse, why bone loss due to disuse occurs, and how it is the target of skeletal physiology which drives pathological bone loss in conditions that may not be seen as being primarily due to disuse. Fundamentally, if we accept that hypertrophy of bone in response to increased loading is a desirable occurrence, then disuse is not a pathological process, but simply the corollary of adaptation to increased loads. If adaptive processes occur to increase bone mass in response to increased load, then the loss of bone in disuse is the only way that adaptation can fully tune the skeleton to prevailing functional demands when loading is reduced. The mechanisms by which loading and disuse cause bone formation or resorption are the same, although the direction of any changes is different. The osteocyte and osteoblast are the key cells involved in sensing and communicating the need for changes in mass or architecture as a result of changes in experienced loading. However, as those cells are affected by numerous other influences, the responses of bone to loading or disuse are not simple, and alter under different circumstances. Understanding the principles of disuse and loading and the mechanisms underlying them therefore represents an important feature of bone physiology and the search for targets for anabolic therapies for skeletal pathology.     

An electron-microscopic study of the bone-remodeling sequence in the rat

Summary A detailed chronological electron-microscopic study of the bone remodeling sequence has been performed in the rat based on a previously described model (Tran Van et al. 1982) in which the remodeling activity is synchronized. This allowed the observation of the cellular and extracellular events during the bone remodeling process, including the activation of the sequential process and the reversal phase, intermediate between osteoclastic resorption and osteoblastic formation. Most important is the fact that throughout the whole process cells with the morphological characteristics of mononuclear phagocytes have been observed in proximity or in contact with the bone surface and/or the various bone cells. Coated pits (receptor-mediated endocytosis) are frequently observed in close apposition to bone spicules and gap junctions are frequent between the cells. These observations suggest that, besides being likely candidates as osteoclast precursors, mononuclear phagocytes may play an important role in bone remodeling.     

Extracellular matrix in ovarian follicular development and disease

The ovarian follicle contains several different cell types and separate compartments and undergoes substantial development during its growth and maturation. Extracellular matrix (ECM) could be expected to play a major role in these processes. Most research on ECM in follicles has focused on the follicular basal lamina and its changing composition during folliculogenesis and on the specialised matrix formed at ovulation by the cumulus cells surrounding the oocyte and the zona pellucida. We review these aspects. Few naturally occurring gene mutations have identified unique roles for ECM molecules in follicular function. Presumably, any mutations leading to reduced fertility are eliminated quickly by natural selection and, when mutations are not eliminated, considerable redundancy occurs to ensure successful reproduction. In mice, in which the genome can be easily manipulated, the modification of matrix components associated with cumulus and oocytes has often resulted in partial infertility, suggesting redundancy. We provide an update of basal lamina components focusing on newer discoveries. In addition, we review matrix associated with the occyte and cumulus cells (excluding the zona pellucida) and other components of ECM. Where possible, we examine evidence for the role of the ECM in follicular development and diseases.Research in the authors' laboratories has been supported by the National Health and Medical Research Council of Australia, The University of Adelaide, Wellcome Foundation, and the Clive and Vera Ramaciotti Foundation.