New insights into the epigenetics of osteoporosis

Osteoporosis is characterized by reduced bone formation and accumulation of adipocytes in the bone marrow compartment. The decrease in bone mass results from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The deficiency of bone cells to replace the resorpted bone can be due to a preferential differentiation of bone marrow stromal cells into adipocytes at the expense of osteoblasts. Consequently, the processes that control the differentiation of osteoclasts, osteoblasts and adipocytes play a crucial role in bone metabolism. It is known that epigenetic mechanisms are critical regulator of the differentiation programs for cell fate and moreover are subject to changes during aging. Here, we summarize recent findings on the role of epigenetics in the modulation of mechanisms that may be associated with osteoporosis. In particular, we focus on disturbances in the bone remodeling process described in human studies that address the epigenetic regulation of the osteoblast/adipocyte balance.     

Pathobiology and prevention of cancer chemotherapy-induced bone growth arrest, bone loss, and osteonecrosis

Cancer chemotherapy has been recognized as one severe risk factor that influences bone growth and bone mass accumulation during childhood and adolescence. This article reviews on the importance of this clinical issue, current understanding of the underlying mechanisms for the skeletal defects and potential preventative strategies. Both clinical and basic studies that appeared from 1990 to 2010 were reviewed for bone defects (growth arrest, bone loss, osteonecrosis, and/or fractures) caused by paediatric cancer chemotherapy. As chemotherapy has become more intensive and achieved greater success in treating paediatric malignancies, skeletal complications such as bone growth arrest, low bone mass, osteonecrosis, and fractures during and/or after chemotherapy have become a problem for some cancer patients and survivors particularly those that have received high dose glucocorticoids and methotrexate. While chemotherapy-induced skeletal defects are likely multi-factorial, recent studies suggest that different chemotherapeutic agents can directly impair the activity of the growth plate and metaphysis (the two major components of the bone growth unit) through different mechanisms, and can alter bone modeling/remodeling processes via their actions on bone formation cells (osteoblasts), bone resorption cells (osteoclasts) and bone "maintenance" cells (osteocytes). Intensive use of multi-agent chemotherapy can cause growth arrest, low bone mass, fractures, and/or osteonecrosis in some paediatric patients. While there are currently no specific strategies for protecting bone growth during childhood cancer chemotherapy, regular BMD monitoring and exercise are have been recommended, and possible adjuvant treatments could include calcium/vitamin D, antioxidants, bisphosphonates, resveratrol, and/or folinic acid.     

Semaphorin 3A: A new player in bone remodeling

Semaphorin 3A (Sema3A) is a protein identified originally as a diffusible axonal chemorepellent. Sema3A has multifunctional roles in embryonic development, immune regulation, vascularization, and oncogenesis. Bone remodeling consists of two phases: the removal of mineralized bone by osteoclasts and the formation of new bone by osteoblasts, and plays an essential role in skeletal diseases such as osteoporosis. Recent studies have shown that Sema3A is implicated in the regulation of osteoblastgenesis and osteoclastgenesis. Moreover, low bone mass in mice with specific knockout of Sema3A in the neurons indicates that Sema3A regulates bone remodeling indirectly. This review highlights recent advances on our understanding of the role of sema3A as a new player in the regulation of bone remodeling and proposes the potential of sema3A in the diagnosis and therapy of bone diseases.     

Regulation of adult bone turnover by sex steroids

Recent reports reveal increasing complexity of mechanisms underlying the bone sparing effects of sex steroids. This review focuses on mechanisms by which sex steroids attenuate endocortical and trabecular adult bone turnover, perhaps their most important property as bone mass regulators. Clearly, estrogen withdrawal increases osteoclast number and bone resorption; however, important open questions are the extent to which osteoblasts and their precursors are involved, and the relative contributions of the RANK/RANKL/OPG system, Fas ligand and Runx2. In addition to reviewing these aspects of estrogen action, we also discuss proskeletal effects of androgens on the adult male skeleton, including aromatization to estrogens and male‐specific mechanisms. Detailed understanding of skeletal site‐ and gender‐dependent mechanisms by which sex steroids protect the adult skeleton will provide the foundation for improved risk assessment, prevention and management of osteoporosis. J. Cell. Physiol. 224: 305–310, 2010. © 2010 Wiley‐Liss, Inc.     

Lactoferrin promotes bone growth

We have demonstrated bovine or human lactoferrin to be an anabolic factor in skeletal tissue. In vitro, lactoferrin stimulates the proliferation of bone forming cells, osteoblasts, and cartilage cells at physiological concentrations (above 0.1 microg/ml). The magnitude of this effect exceeds that observed in response to other skeletal growth factors such as IGF-1 and TGFbeta. DNA synthesis is also stimulated in a bone organ culture system likely reflecting the proliferation of cells of the osteoblast lineage. Lactoferrin is also a potent osteoblast survival factor. In TUNEL and DNA fragmentation assays, lactoferrin decreased apoptosis, induced by serum withdrawal, by up to 70%. In addition, lactoferrin has powerful effects on bone resorbing cells, osteoclasts, decreasing osteoclast development at concentrations > 1 microg/ml in a murine bone marrow culture system. However, lactoferrin did not alter bone resorption in calvarial organ culture, suggesting that it does not influence mature osteoclast function. In vivo, local injection of lactoferrin in adult mice resulted in increased calvarial bone growth, with significant increases in bone area and dynamic histomorphometric indices of bone formation after only 5 injections. Taken together, these data demonstrate that the naturally-occurring glycoprotein lactoferrin is anabolic to bone in vivo, an effect which is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis. Lactoferrin may therefore have a physiological role in bone growth, and a potential therapeutic role in osteoporosis.     

Skeletal effects of androgen withdrawal

Hypogonadism is considered to be one of the major risk factors for osteoporosis in men. Therefore, it is an important goal for skeletal research to improve our understanding of the skeletal effects of androgens. Androgen deficiency during growth is associated with a failure to acquire normal peak bone mass, and there is good evidence that the effects of androgens on skeletal growth and the development of a male skeletal phenotype are mediated through the androgen receptor. In adult men, acute withdrawal of androgens by surgical or chemical castration induces high turnover bone loss. Similarly, orchidectomy of aged, non-growing male rats is associated with a pronounced and sustained increase in bone turnover and with true loss of cancellous and cortical bone. Interestingly, the changes in bone turnover induced by orchidectomy are paralleled by a concomitant increase in B lymphopoiesis in bone marrow of rats and mice. Although there is firm evidence that male bone metabolism can be influenced by androgens and estrogen, a variety of clinical and animal experimental data have strongly suggested that, under physiological circumstances, the maintenance of cancellous bone mass in males involves the skeletal action of estrogen derived from aromatization of androgens. Aged male rats appear to closely mimic the conditions induced by androgen withdrawal in adult humans, and this animal model may be used 1) to elucidate further the role of muscle as a mediator of the actions of androgens on bone, 2) to explore the regulatory functions of androgens and estrogens in the male skeleton and the immune system, and 3) to find new treatment strategies for the prevention and treatment of osteoporosis in men.     

Central control of bone remodeling by neuromedin U

Bone remodeling, the function affected in osteoporosis, the most common of bone diseases, comprises two phases: bone formation by matrix-producing osteoblasts and bone resorption by osteoclasts. The demonstration that the anorexigenic hormone leptin inhibits bone formation through a hypothalamic relay suggests that other molecules that affect energy metabolism in the hypothalamus could also modulate bone mass. Neuromedin U (NMU) is an anorexigenic neuropeptide that acts independently of leptin through poorly defined mechanisms. Here we show that Nmu-deficient (Nmu-/-) mice have high bone mass owing to an increase in bone formation; this is more prominent in male mice than female mice. Physiological and cell-based assays indicate that NMU acts in the central nervous system, rather than directly on bone cells, to regulate bone remodeling. Notably, leptin- or sympathetic nervous system-mediated inhibition of bone formation was abolished in Nmu-/- mice, which show an altered bone expression of molecular clock genes (mediators of the inhibition of bone formation by leptin). Moreover, treatment of wild-type mice with a natural agonist for the NMU receptor decreased bone mass. Collectively, these results suggest that NMU may be the first central mediator of leptin-dependent regulation of bone mass identified to date. Given the existence of inhibitors and activators of NMU action, our results may influence the treatment of diseases involving low bone mass, such as osteoporosis.     

IL-33 is expressed in human osteoblasts, but has no direct effect on bone remodeling

The aim of the present study was to investigate the potential role of the recently discovered IL-1 family member IL-33 in bone remodeling. Our results indicate that IL-33 mRNA is expressed in osteocytes in non-inflammatory human bone. Moreover, IL-33 levels are increased by TNF-α and IL-1β in human bone marrow stromal cells, osteoblasts and adipocytes obtained from three healthy donors. Experiments with the inhibitor GW-9662 suggested that expression of IL-33, in contrast to that of IL-1β, is not repressed by PPARγ likely explaining why IL-33, but not IL-1β, is expressed in adipocytes. The IL-33 receptor ST2L is not constitutively expressed in human bone marrow stromal cells, osteoblasts or CD14-positive monocytes, and IL-33 has no effect on these cells. In addition, although ST2L mRNA is induced by TNF-α and IL-1β in bone marrow stromal cells, IL-33 has the same effects as TNF-α and IL-1β, and, therefore, the biological activity of IL-33 may be redundant in this system. In agreement with this hypothesis, MC3T3-E1 osteoblast-like cells constitutively express ST2L mRNA, and IL-33 and TNF-α/IL-1β similarly decrease osteocalcin RNA levels in these cells. In conclusion, our results suggest that IL-33 has no direct effects on normal bone remodeling.     

Osteoblast recruitment to sites of bone formation in skeletal development,homeostasis, and regeneration

During endochondral bone development, bone‐forming osteoblasts have to colonize the regions of cartilage that will be replaced by bone. In adulthood, bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, as a prerequisite for skeletal health. A failure of osteoblasts to reach the sites in need of bone formation may contribute to impaired fracture repair. Conversely, stimulation of osteogenic cell recruitment may be a promising osteo‐anabolic strategy to improve bone formation in low bone mass disorders such as osteoporosis and in bone regeneration applications. Yet, still relatively little is known about the cellular and molecular mechanisms controlling osteogenic cell recruitment to sites of bone formation. In vitro, several secreted growth factors have been shown to induce osteogenic cell migration. Recent studies have started to shed light on the role of such chemotactic signals in the regulation of osteoblast recruitment during bone remodeling. Moreover, trafficking of osteogenic cells during endochondral bone development and repair was visualized in vivo by lineage tracing, revealing that the capacity of osteoblast lineage cells to move into new bone centers is largely confined to undifferentiated osteoprogenitors, and coupled to angiogenic invasion of the bone‐modeling cartilage intermediate. It is well known that the presence of blood vessels is absolutely required for bone formation, and that a close spatial and temporal relationship exists between osteogenesis and angiogenesis. Studies using genetically modified mouse models have identified some of the molecular constituents of this osteogenic–angiogenic coupling. This article reviews the current knowledge on the process of osteoblast lineage cell recruitment to sites of active bone formation in skeletal development, remodeling, and repair, considering the role of chemo‐attractants for osteogenic cells and the interplay between osteogenesis and angiogenesis in the control of bone formation. Birth Defects Research (Part C) 99:170–191, 2013. © 2013 Wiley Periodicals, Inc.     

MicroRNA-218-5p relieves postmenopausal osteoporosis through promoting the osteoblast differentiation of bone marrow mesenchymal stem cells

MicroRNAs (miRs) are short noncoding RNAs that play key regulatory roles in osteoblast differentiation. In this study, the specific regulatory roles of miR-218-5p on postmenopausal osteoporosis (PMOP) were investigated. The mouse model of PMOP was established by bilateral ovariectomy, and the injection of miR-218-5p mimics significantly relieved PMOP degree. Then, bone marrow mesenchymal stem cells (BMMSCs) isolated from PMOP mice were induced into osteoblasts. When compared with normal BMMSCs , PMOP BMMSCs exhibited significantly lower alkaline phosphatase (ALP) activity and less mineralized nodules, as well as downregulated miR-218-5p, Runx2, Osterix, COL1A1, and OCN after induction (P < .05). The transfection of miR-218-5p mimics, and inhibitor significantly promoted, inhibited the osteoblast differentiation of PMOP BMMSCs, respectively. In addition, COL1A1 was a target of miR-218-5p. The transfection of miR-218-5p mimics into PMOP BMMSCs significantly upregulated COL1A1 at 14th and 21st day post-induction, but not at 7th day. Our findings suggest miR-218-5p may relieve PMOP through promoting the osteoblast differentiation of BMMSCs.     

JunD suppresses bone formation and contributes to low bone mass induced by estrogen depletion

JunD is an activator protein-1 (AP-1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD-deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency-induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD-deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy-induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c-jun, fra-1, and fra-2 in bone in conjunction with elevated expression levels of runx2, type I collagen, and osteocalcin. Thus, JunD is involved in estrogen depletion-induced osteopenia via its action to suppress bone formation and to enhance bone resorption.     

Injections of leptin into rat ventromedial hypothalamus increase adipocyte apoptosis in peripheral fat and in bone marrow

The accumulation of fat cells (adipocytes) in bone marrow is now thought to be a factor contributing to age-related bone loss. Women with osteoporosis have higher numbers of marrow adipocytes than women with healthy bone, and bone formation rate is inversely correlated with adipocyte number in bone tissue biopsies from both men and women. Adipogenic differentiation of bone marrow stromal cells increases with age, but the factors regulating populations of mature adipocytes are not well understood. Leptin is thought to regulate adipose tissue mass via its receptors in the ventromedial hypothalamus (VMH). We have therefore tested the hypothesis that stimulation of leptin receptors in the VMH regulates adipocyte number in bone marrow. Results indicate that unilateral twice-daily injections of leptin into the rat VMH for only 4 or 5 days cause a significant reduction in the number of adipocytes in peripheral fat pads and bone marrow and indeed eliminate adipocytes almost entirely from bone marrow of the proximal tibia. Osteoblast surface is not affected with leptin treatment. Apoptosis assays performed on bone marrow samples from control and treated rats have revealed a significant increase in protein concentration of the apoptosis marker caspase-3 with leptin treatment. We conclude that stimulation of leptin receptors in the VMH significantly decreases the adipocyte population in bone marrow, primarily through apoptosis of marrow adipocytes. Elimination of marrow adipocytes via this central pathway may represent a useful strategy for the treatment and prevention of osteoporosis.     

Sex steroids and stem cell function

Gender dimorphisms exist in the pathogenesis of a variety of cardiovascular, cardiopulmonary, neurodegenerative, and endocrine disorders. Estrogens exert immense influence on myocardial remodeling following ischemic insult, partially through paracrine growth hormone production by bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells. Estrogens also facilitate the mobilization of endothelial progenitor cells to the ischemic myocardium and enhance neovascularization at the ischemic border zone. Moreover, estrogens limit pathological myocardial remodeling through the inhibitory effects on the proliferation of the cardiac fibroblasts. Androgens also may stimulate endothelial progenitor cell migration from the bone marrow, yet the larger role of androgens in disease pathogenesis is not well characterized. The beneficial effects of sex steroids include alteration of lipid metabolism in preadipocytes, modulation of bone metabolism and skeletal maturation, and prevention of osteoporosis through their effects on osteogenic precursors. In an example of sex steroid-specific effects, neural stem cells exhibit enhanced proliferation in response to estrogens, whereas androgens mediate inhibitory effects on their proliferation. Although stem cells can offer significant therapeutic benefits in various cardiovascular, neurodegenerative, endocrine disorders, and disorders of bone metabolism, a greater understanding of sex hormones on diverse stem cell populations is required to improve their ultimate clinical efficacy. In this review, we focus on the effects of estrogen and testosterone on various stem and progenitor cell types, and their relevant intracellular mechanisms.     

Low bone mass may not be the only cause of skeletal fragility in osteoporosis

Skeletal fragility in postmenopausal osteoporosis is not due solely to reduction in bone mass. This fact explains some of the overlap in bone mineral measurements observed between patients who are fracturing and age- and sex-matched normals who are not. Changes in skeletal architecture and bone remodeling occur with age which can account for some of the fragility. These changes are exaggerated in patients with postmenopausal osteoporosis who are suffering spine fractures. Three abnormalities have been described by histomorphometric methods which can account for skeletal fragility out of proportion to the degree of bone loss. They are: (i) loss of trabecular connectivity such that vertical weight-bearing bars lose their cross-attachments with each other, thus becoming susceptible to buckling; (ii) inefficient and prolonged microdamage repair due to periods of pause in the formation phase of remodeling; and (iii) accumulation of unrepaired microdamage in unremodeled bone tissue in the central part of trabeculae due to reduced osteon wall thickness coupled with maintenance of trabecular thickness. Recognition of these abnormalities should broaden our approach to the study of skeletal fragility in the syndrome of postmenopausal osteoporosis.     

Overview of osteoporosis.

Osteoporosis is a common age-related disorder manifested clinically by skeletal fractures, especially fractures of the vertebrae, hip, and distal forearm. The major cause of these fractures is low bone mass, although an increase in trauma due to falls in the elderly also contributes. There are multiple causes for the low bone mass which, in any given individual, may contribute differently to the development of the osteopenia. The most important groups of causes are failure to achieve adequate peak bone mass, slow bone loss due to processes relating to aging, the menopause in women, and a variety of sporadic behavioral, nutritional, and environmental factors that affect bone mass in some but not in other individuals. The most important approach is prevention. Drugs and behavioral factors known to cause bone loss should be eliminated and perimenopausal women should be evaluated for possible preventive administration of estrogen. For patients with fractures due to established osteoporosis, the only drugs approved by the Food and Drug Administration are the antiresorptive agents calcium, estrogen, and calcitonin. Formation-stimulating regimens, however, are being developed and may be available for clinical use in the foreseeable future. These regimens may be capable of increasing bone mass to above the fracture threshold, thereby resulting in a clinical cure of the osteoporosis.