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...

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.

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.