Both aging and loss of sex steroids have adverse effects on skeletal homeostasis, but whether and how they may influence each others negative impact on bone remains unknown. We report herein that both female and male C57BL/6 mice progressively lost strength (as determined by load-to-failure measurements) and bone mineral density in the spine and femur between the ages of 4 and 31 months. These changes were temporally associated with decreased rate of remodeling as evidenced by decreased osteoblast and osteoclast numbers and decreased bone formation rate; as well as increased osteoblast and osteocyte apoptosis, increased reactive oxygen species levels, and decreased glutathione reductase activity and a corresponding increase in the phosphorylation of p53 and p66 shc , two key components of a signaling cascade that are activated by reactive oxygen species and influences apoptosis and lifespan. Exactly the same changes in oxidative stress were acutely reproduced by gonadectomy in 5-month-old females or males and reversed by estrogens or androgens in vivo as well as in vitro. We conclude that the oxidative stress that underlies physiologic organismal aging in mice may be a pivotal pathogenetic mechanism of the age-related bone loss and strength. Loss of estrogens or androgens accelerates the effects of aging on bone by decreasing defense against oxidative stress.Age-related loss of bone mass and strength is an invariable feature of human biology, affecting women and men alike. Moreover, population-based studies demonstrate that substantial bone loss begins as early as the 20s in young adult women and men, long before any hormonal changes (1).3 The extent to which estrogen deficiency contributes to age-related bone loss and the slower rate of decline of bone mass and strength during the late postmenopausal years, and the molecular and cellular mechanisms of such putative interactions, are unknown.The universality of age-associated bone loss irrespective of sex steroid status notwithstanding, age is by far a more critical determinant of fracture risk than bone mass in humans indicating that age-related increase in fracture risk reflects a loss of bone strength that is only partly accounted for by loss of bone mass (2). Whereas an increased propensity to fall due to agerelated decline in neuromuscular function is a factor, there are also age-related changes in the bone itself. Such changes include disrupted architecture, altered composition of the bone mineral and matrix, delayed repair of fatigue microdamage, excessive turnover, and inadequate bone size (3-7). The most recently appreciated qualitative factor is loss of osteocytes (8, 9), former osteoblasts entombed into the mineralized matrix. Osteocyte death may influence the signals necessary for mechanical adaptation and repair and also lead to long term changes in bone hydration. The anti-apoptotic effect of sex steroids on osteocytes, which has been well documented in mice, rats, and humans (10 -12), may contribute to their anti-fracture efficacy independently of...
Estrogens and androgens influence the growth and maintenance of the mammalian skeleton and are responsible for its sexual dimorphism. Estrogen deficiency at menopause or loss of both estrogens and androgens in elderly men contribute to the development of osteoporosis, one of the most common and impactful metabolic diseases of old age. In the last 20 years, basic and clinical research advances, genetic insights from humans and rodents, and newer imaging technologies have changed considerably the landscape of our understanding of bone biology as well as the relationship between sex steroids and the physiology and pathophysiology of bone metabolism. Together with the appreciation of the side effects of estrogen-related therapies on breast cancer and cardiovascular diseases, these advances have also drastically altered the treatment of osteoporosis. In this article, we provide a comprehensive review of the molecular and cellular mechanisms of action of estrogens and androgens on bone, their influences on skeletal homeostasis during growth and adulthood, the pathogenetic mechanisms of the adverse effects of their deficiency on the female and male skeleton, as well as the role of natural and synthetic estrogenic or androgenic compounds in the pharmacotherapy of osteoporosis. We highlight latest advances on the crosstalk between hormonal and mechanical signals, the relevance of the antioxidant properties of estrogens and androgens, the difference of their cellular targets in different bone envelopes, the role of estrogen deficiency in male osteoporosis, and the contribution of estrogen or androgen deficiency to the monomorphic effects of aging on skeletal involution.
We have elucidated that oxidative stress is a pivotal pathogenetic factor of age-related bone loss and strength in mice, leading to, among other changes, a decrease in osteoblast number and bone formation. To gain insight into the molecular mechanism by which oxidative stress exerts such adverse effects, we have tested the hypothesis that induction of the Forkhead box O (FoxO) transcription factors by reactive oxygen species may antagonize Wnt signaling, an essential stimulus for osteoblastogenesis. In support of this hypothesis, we report herein that the expression of FoxO target genes increases, whereas the expression of Wnt target genes decreases, with increasing age in C57BL/6 mice. Moreover, we show that in osteoblastic cell models, oxidative stress (exemplified by H 2 O 2 ) promotes the association of FoxOs with ␤-catenin, ␤-catenin is required for the stimulation of FoxO target genes by H 2 O 2 , and H 2 O 2 promotes FoxO-mediated transcription at the expense of Wnt-/T-cell factor-mediated transcription and osteoblast differentiation. Furthermore, ␤-catenin overexpression is sufficient to prevent FoxO-mediated suppression of T-cell factor transcription. These results demonstrate that diversion of the limited pool of ␤-catenin from T-cell factor-to FoxO-mediated transcription in osteoblastic cells may account, at least in part, for the attenuation of osteoblastogenesis and bone formation by the age-dependent increase in oxidative stress.
Mouse models with cell-specific deletion of the estrogen receptor (ER) α, the androgen receptor (AR) or the receptor activator of nuclear factor κB ligand (RANKL), as well as cascade-selective estrogenic compounds have provided novel insights into the function and signalling of ERα and AR. The studies reveal that the effects of estrogens on trabecular versus cortical bone mass are mediated by direct effects on osteoclasts and osteoblasts, respectively. The protection of cortical bone mass by estrogens is mediated via ERα, using a non-nucleus-initiated mechanism. By contrast, the AR of mature osteoblasts is indispensable for the maintenance of trabecular bone mass in male mammals, but not required for the anabolic effects of androgens on cortical bone. Most unexpectedly, and independently of estrogens, ERα in osteoblast progenitors stimulates Wnt signalling and periosteal bone accrual in response to mechanical strain. RANKL expression in B lymphocytes, but not T lymphocytes, contributes to the loss of trabecular bone caused by estrogen deficiency. In this Review, we summarize this evidence and discuss its implications for understanding the regulation of trabecular and cortical bone mass; the integration of hormonal and mechanical signals; the relative importance of estrogens versus androgens in the male skeleton; and, finally, the pathogenesis and treatment of osteoporosis.
Genetic studies in humans and mice have revealed an important role of the Wnt signaling pathway in the regulation of bone mass, resulting from potent effects on the control of osteoblast progenitor proliferation, commitment, differentiation, and perhaps osteoblast apoptosis. To establish the linkage between Wnts and osteoblast survival and to elucidate the molecular pathways that link the two, we have utilized three cell models: the uncommitted bipotential C2C12 cells, the preosteoblastic cell line MC3T3-E1, and bone marrow-derived OB-6 osteoblasts. Serum withdrawal-induced apoptosis was prevented by the canonical Wnts (Wnt3a and Wnt1) and the noncanonical Wnt5a in all cell types. Wnt3a induced LRP5-independent transient phosphorylation and nuclear accumulation of ERKs and phosphorylation of Src and Akt. The anti-apoptotic effect of Wnt3a was abrogated by inhibitors of canonical Wnt signaling, as well as by inhibitors of MEK, Src, phosphatidylinositol 3-kinase (PI3K), or Akt kinases, or by the addition of cycloheximide to the culture medium. Wnt3a-induced phosphorylation of GSK-3␤ and downstream activation of ␤-catenin-mediated transcription required ERK, PI3K, and Akt signaling. Wnt3a increased the expression of the anti-apoptotic protein Bcl-2 in an ERK-dependent manner. ␤-Catenin-mediated transcription was permissive for the anti-apoptotic actions of Wnt1 and Wnt3a but was dispensable for the anti-apoptotic action of Wnt5a. However, Src, ERKs, PI3K, and Akt kinases were required for the anti-apoptotic effects of Wnt5a. These results demonstrate for the first time that Wnt proteins, irrespective of their ability to stimulate canonical Wnt signaling, prolong the survival of osteoblasts and uncommitted osteoblast progenitors via activation of the Src/ERK and PI3K/Akt signaling cascades.Wnts are secreted lipid-modified signaling proteins that influence cell proliferation, differentiation, and survival (1-3). Wnt proteins are divided into two classes. The Wnt1 class activates the canonical Wnt signaling pathway, which involves the formation of a complex between Wnt proteins, Frizzled, and LRP5 2 or LRP6 receptors (4, 5). This complex in turn leads to phosphorylation and inactivation of GSK-3␤, inhibition of ␤-catenin degradation, and subsequent accumulation of ␤-catenin in the nucleus (6, 7). Nuclear ␤-catenin binds the TCF/LEF family of transcription factors and induces target gene expression (8).The noncanonical Wnt5a class binds Frizzled proteins, activates heterotrimeric G proteins, and increases intracellular calcium via protein kinase C-dependent mechanisms or induces Rho-or c-Jun N-terminal kinase (JNK)-dependent changes in the actin cytoskeleton (9). Genetic studies in humans and mice have determined that LRP5 (LRP6)/Wnt signaling plays a major role in the control of bone mass. Mutations in LRP5 or LRP6 lead to disorders associated with either low (10) or high bone mass (11-13). In agreement with observations in humans, LRP5-deficient mice show decreased bone formation and osteoblast proliferation (14), wh...
Summary Aging increases oxidative stress and osteoblast apoptosis and decreases bone mass, whereas forkhead box O (FoxO) transcription factors defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis. Conditional deletion of FoxO1, 3 and 4 in three month-old mice resulted in an increase in oxidative stress in bone and osteoblast apoptosis and a decrease in the number of osteoblasts, the rate of bone formation, and bone mass at cancellous and cortical sites. The effect of the deletion on osteoblast apoptosis was cell autonomous and resulted from oxidative stress. Conversely, overexpression of a FoxO3 transgene in mature osteoblasts decreased oxidative stress and osteoblast apoptosis, and increased osteoblast number, bone formation rate and vertebral bone mass. We conclude that FoxO-dependent oxidative defense provides a mechanism to handle the oxygen free radicals constantly generated by the aerobic metabolism of osteoblasts and is thereby indispensable for bone mass homeostasis.
The Wnt/beta-catenin signaling pathway affects several biological processes ranging from embryonic development, patterning, and postembryonic stem cell fate, to bone formation and insulin secretion in adulthood. beta-Catenin mediates canonical Wnt signaling by binding to and activating members of the T-cell factor (TCF) transcription factor family. Similar to the Wnt/beta-catenin pathway, oxidative stress influences fundamental cellular processes including stem cell fate and has been linked to aging and the development of age-related diseases. However, the molecular details of the pathogenetic effects of oxidative stress on the homeostasis of many different tissues remain unclear. beta-Catenin has been recently implicated as a pivotal molecule in defense against oxidative stress by serving as a cofactor of the forkhead box O (FOXO) transcription factors. In addition, it has been shown that oxidative stress is a pivotal pathogenetic factor of age-related bone loss and strength in mice, leading to, among other changes, a decrease in osteoblast number and bone formation. These particular cellular changes evidently result from diversion of the limited pool of beta-catenin from TCF- to FOXO-mediated transcription in osteoblastic cells. Fascinatingly, attenuation of Wnt-mediated transcription, resulting from an autosomal-dominant missense mutation in LRP6, a coreceptor for the Wnt-signaling pathway, has been linked recently genetically not only to premature osteoporosis, but also to coronary artery disease as well as several features of the metabolic syndrome including hyperlipidemia, hypertension, and diabetes, but not obesity. In this minireview, we highlight evidence linking the age-associated oxidative stress with FOXOs, Wnt/beta-catenin signaling, osteoblastogenesis, adipogenesis, osteoporosis, and several features of the metabolic syndrome. We hypothesize that antagonism of Wnt signaling by oxidative stress with increasing age may be a common molecular mechanism contributing to the development not only of involutional osteoporosis, but several pathologies such as atherosclerosis, insulin resistance, and hyperlipidemia, all of which become more prevalent with advancing age.
The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues. IntroductionThe volume of bone mass is determined by the balance between two opposing processes, bone removal (resorption) by osteoclasts and bone formation by osteoblasts. Under physiologic circumstances, formation compensates for the effect of resorption by adding bone either in the same anatomical site from which it was previously resorbed, in a process called remodeling, or in a different site, as in the case of the sculpting of bones during growth, in a process called modeling (1).After birth, long bones in both sexes increase in length. In parallel with linear growth, females and males experience an enlargement and thickening of the bone cortex, because bone apposition at the periosteal (outer) envelope exceeds the widening of the medullar cavity by endocortical resorption. Importantly, with the onset of puberty, estrogens inhibit, while androgens stimulate, periosteal bone formation, thereby contributing to sexual dimorphism and the greater bone mass in the male (2). At the same time, endocortical resorption is attenuated by either estrogens or androgens, producing increased cortical thickness at the end of puberty in both sexes. In line with the inhibitory effect of estrogens on periosteal bone formation, estrogen deficiency in female rodents and postmenopausal women increases periosteal apposition in an attempt to compensate for the endocortical loss of bone. At the end of puberty, estrogens are essential for the closure of the epiphyses and the cessation of linear growth (3).Estrogens also slow the rate of bone remodeling and help to maintain a balance between bone formation and resorption by attenuating the birth rate of osteoclast and osteoblast progenitors in the bone marrow and exerting a proapop...
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