Summary Osteoporosis, a low bone mass disease, is associated with decreased osteoblast numbers and increased levels of oxidative stress in these cells. Since the FoxO family of transcription factors, confers stress resistance, we investigated their potential impact on skeletal integrity. We show here through cell-specific deletion and molecular analyses that, among the 3 FoxO proteins, only FoxO1 is required for proliferation and redox balance in osteoblasts, and as a result controls bone formation. FoxO1 regulation of osteoblast proliferation occurs because of its interaction with ATF4, a transcription factor regulating amino acid import; and of its regulation of a stress-dependent pathway influencing p53 signaling. Accordingly, decreasing oxidative stress levels or increasing protein intake normalizes bone formation and bone mass in mice lacking FoxO1 in osteoblasts only. These results identify FoxO1 as a crucial regulator of osteoblast physiology and provide a direct mechanistic link between oxidative stress and the regulation of bone remodeling.

Background: The obesity epidemic, recognized in developed nations for decades, is now a worldwide phenomenon. All age groups are affected, including women of childbearing age, fueling concern that maternal obesity before and during pregnancy and lactation impairs developmental establishment of body weight regulatory mechanisms in the fetus or infant, causing transgenerational amplification of obesity prevalence and severity. The biological mechanisms underlying such processes remain unknown. Methods: We used agouti viable yellow (A vy ) mice to test the hypothesis that maternal obesity induces transgenerational amplification of obesity. We passed the A vy allele through three generations of A vy /a females and assessed cumulative effects on coat color and body weight. By studying two separate but contemporaneous populations of mice, one provided a standard diet and the other a methyl-supplemented diet that induces DNA hypermethylation during development, we tested whether potential transgenerational effects on body weight might be mediated by alterations in epigenetic mechanisms including DNA methylation. Results: The genetic tendency for obesity in A vy mice was progressively exacerbated when the A vy allele was passed through successive generations of obese A vy females. This transgenerational amplification of body weight was prevented by a promethylation dietary supplement. Importantly, the effect of methyl supplementation on body weight was independent of epigenetic changes at the A vy locus, indicating this model may have direct relevance to human transgenerational obesity. Conclusion: Our results show that in a population with a genetic tendency for obesity, effects of maternal obesity accumulate over successive generations to shift the population distribution toward increased adult body weight, and suggest that epigenetic mechanisms are involved in this process. /a females were mated with a/a males at P56, and their (F2) offspring were treated identically to the F1 generation. In the F3 generation, both females and males were weaned onto the standard mouse diet, and females were not mated. Body weight of all A vy /a mice (F0, F1, F2 and F3) was measured at P180. The digital photographs enabled A vy /a coat color phenotype to be rated by a single observer (RAW) blind to diet group. Coat color was rated as yellow (o5% brown), slightly mottled (X5% but less than half brown), mottled ( approximately half brown), heavily mottled (more than half but p95% brown) or pseudoagouti (495% brown).15 Body composition was measured by dual-energy X-ray absorptiometry (Piximus Series Densitometer, GE Medical Systems, Madison, WI, USA), as per the manufacturers' instructions. We certify that all applicable institutional and governmental regulations concerning the ethical use of animals were followed during this research. The protocol was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. Statistical approachesAll data were entered independently into two Excel spreadsheets, which were...

Osteoblasts have recently been found to play a role in regulating glucose metabolism through secretion of osteocalcin. It is unknown, however, how this osteoblast function is regulated transcriptionally. As FoxO1 is a forkhead family transcription factor known to regulate several key aspects of glucose homeostasis, we investigated whether its expression in osteoblasts may contribute to its metabolic functions. Here we show that mice lacking Foxo1 only in osteoblasts had increased pancreatic β cell proliferation, insulin secretion, and insulin sensitivity. The ability of osteoblast-specific FoxO1 deficiency to affect metabolic homeostasis was due to increased osteocalcin expression and decreased expression of Esp, a gene that encodes a protein responsible for decreasing the bioactivity of osteocalcin. These results indicate that FoxO1 expression in osteoblasts contributes to FoxO1 control of glucose homeostasis and identify FoxO1 as a key modulator of the ability of the skeleton to function as an endocrine organ regulating glucose metabolism.

The perichondrium, a structure made of undifferentiated mesenchymal cells surrounding growth plate cartilage, regulates chondrocyte maturation through poorly understood mechanisms. Analyses of loss-and gain-of-function models show that Twist-1, whose expression in cartilage is restricted to perichondrium, favors chondrocyte maturation in a Runx2-dependent manner. Runx2, in turn, enhances perichondrial expression of Fgf18, a regulator of chondrocyte maturation. Accordingly, compound heterozygous embryos for Runx2 and Fgf18 deletion display the same chondrocyte maturation phenotype as Fgf18-null embryos. This study identifies a transcriptional basis for the inhibition of chondrocyte maturation by perichondrium and reveals that Runx2 fulfills antagonistic functions during chondrogenesis.Supplemental material is available at http://www.genesdev.org.Received August 16, 2006; revised version accepted September 11, 2006. Endochondral bone formation is a complex process that involves several cell types with distinct patterns of gene expression. The initial step in this process is characterized by the aggregation of undifferentiated mesenchymal cells expressing ␣1(I) and ␣1(III) Collagen into condensations at the location and with the overall shape of future skeletal elements. Subsequently, cells within these condensations differentiate into chondrocytes that do not express any more ␣1(I) Collagen but instead, and among other genes, ␣1(II) b Collagen. Cartilage anlagen then enlarge through proliferation of chondrocytes that elongate to form prehypertrophic chondrocytes that will eventually exit the cell cycle to become bona fide hypertrophic chondrocytes. Genetically, hypertrophic chondrocytes differ from proliferating chondrocytes as they express ␣1(X) Collagen but not ␣1(II) Collagen (Kronenberg 2003). Throughout skeletogenesis, layers of ␣1(I) Collagen-expressing undifferentiated mesenchymal cells persist and surround cartilage anlagen to form a structure called the perichondrium (Kronenberg 2003).The transcriptional control of cell differentiation during skeletogenesis has been a topic of intense studies in the last 10 yr, leading to the identification of several key genes. For instance, Sox9 along with Sox5 and Sox6 are seen as the main transcription factors triggering mesenchymal condensations and initiating chondrocyte differentiation (Lefebvre et al. 1998;Bi et al. 1999;Smits et al. 2001). On the other hand, Runx2 appears to be the earliest transcriptional determinant of osteoblast differentiation (Ducy et al. 1997;Komori et al. 1997;Otto et al. 1997). Runx2 has broader functions during skeletogenesis since it is, along with Runx3, an inducer of chondrocyte hypertrophy (Takeda et al. 2001;Ueta et al. 2001;Yoshida et al. 2004). This latter role of Runx2 is explained by its transient expression in prehypertrophic chondrocytes.Runx2 is also expressed at high levels and throughout skeletogenesis in cells of the perichondrium, suggesting that it may have additional roles during chondrogenesis (Ducy et al. 1997). T...

The periosteum is now widely recognized as a homeostatic and therapeutic target for actions of sex steroids and intermittent PTH administration. The mechanisms by which estrogens suppress but PTH promotes periosteal expansion are not known. In this report, we show that intermittent PTH(1-34) promotes differentiation of periosteal osteoblast precursors as evidenced by the stimulation of the expression or activity of alkaline phosphatase as well as of targets of the bone morphogenetic protein 2 (BMP-2) and Wnt pathways. In contrast, 17beta-estradiol (E2) had no effect by itself. However, it attenuated PTH- or BMP-2-induced differentiation of primary periosteal osteoblast progenitors. Administration of intermittent PTH to ovariectomized mice induced rapid phosphorylation of the BMP-2 target Smad1/5/8 in the periosteum. A replacement dose of E2 had no effect by itself but suppressed PTH-induced phosphorylation of Smad1/5/8. In contrast to its effects to stimulate periosteal osteoblast differentiation, PTH promoted and subsequently suppressed proliferation of periosteal osteoblast progenitors in vitro and in vivo. E2 promoted proliferation and attenuated the antiproliferative effect of PTH. Both hormones protected periosteal osteoblasts from apoptosis induced by various proapoptotic agents. These observations suggest that the different effects of PTH and estrogens on the periosteum result from opposing actions on the recruitment of early periosteal osteoblast progenitors. Intermittent PTH promotes osteoblast differentiation from periosteum-derived mesenchymal progenitors through ERK-, BMP-, and Wnt-dependent signaling pathways. Estrogens promote proliferation of early osteoblast progenitors but inhibit their differentiation by osteogenic agents such as PTH or BMP-2.