In women, estrogen deficiency after menopause frequently accelerates osteoclastic bone resorption, leading to osteoporosis, the most common skeletal disorder. However, mechanisms underlying osteoporosis resulting from estrogen deficiency remain largely unknown. Here we show that in bone-resorbing osteoclasts, estrogendependent destabilization of hypoxia-inducible factor 1 alpha (HIF1α), which is unstable in the presence of oxygen, plays a pivotal role in promoting bone loss in estrogen-deficient conditions. In vitro, HIF1α was destabilized by estrogen treatment even in hypoxic conditions, and estrogen loss in ovariectomized (Ovx) mice stabilized HIF1α in osteoclasts and promoted their activation and subsequent bone loss in vivo. Osteoclast-specific HIF1α inactivation antagonized bone loss in Ovx mice and osteoclast-specific estrogen receptor alpha deficient mice, both models of estrogen-deficient osteoporosis. Oral administration of a HIF1α inhibitor protected Ovx mice from osteoclast activation and bone loss. Thus, HIF1α represents a promising therapeutic target in osteoporosis.B one mass is tightly regulated by a delicate balance between osteoblastic bone formation and osteoclastic bone resorption. Estrogen loss in women after menopause frequently promotes activation of osteoclastic bone resorption, causing osteoporosis. Osteoporotic bone phenotypes are seen in ovariectomized female mice, and estrogen deficiency-induced bone loss in both mouse models and women is ameliorated by estrogen treatment (1, 2). However, estrogen administration reportedly increases risk of cardiovascular events and carcinogenesis of the mammary gland and uterus (3). Bioavailable estrogens including selective estrogen receptor modulators (SERMs) also protect bone from estrogen deficiency-induced osteoporosis (4), and estrogen and SERMs primarily act via estrogen receptors (ER), ERα and ERβ (5, 6). However, how SERMs act on bone remains largely unknown. Thus, understanding of osteoclast activation following estrogen loss is crucial for development of safe therapeutic reagents.Both the endosteal zone of bone marrow cavities and epiphyseal growth plates are hypoxic areas, and the hypoxia-inducible factor (HIF) signaling pathway governs chondrocyte and osteoblast function in these respective areas (7,8). The HIF1 transcription factor consists of an oxygen-regulated alpha subunit, HIF1α, and a constitutively expressed beta subunit, HIF1β. Under normoxia, HIF1α is posttranslationally modified by prolyl hydroxylases, which catalyze hydroxylation of proline residues in the presence of O 2 and Fe 2+ . Recognition of hydroxylated HIF1α by the von Hippel-Lindau tumor suppressor protein recruits an E3 ubiquitin ligase complex targeting HIF1α for proteasomal degradation. Conversely, under hypoxia, proline hydroxylation is inhibited by substrate (O 2 ) deprivation, allowing HIF1α accumulation and formation of an active transcriptional complex with HIF1β (9). Recently, regulation of HIF1α protein levels by factors other than O 2 , including reactive ...

Cell-cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC-STAMP), and analyzed its function. OC-STAMP-deficient mice exhibited a complete lack of cell-cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage-lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC-STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC-STAMP-overexpressing transgenic mice with OC-STAMP-deficient mice restored inhibited osteoclast and FBGC cell-cell fusion seen in OC-STAMP-deficient mice. Thus, fusogenic mechanisms in macrophage-lineage cells are regulated via OC-STAMP and DC-STAMP. ß

Osteoporosis is a complex disease with various causes, such as estrogen loss, genetics, and aging. Here we show that a dominantnegative form of aldehyde dehydrogenase 2 (ALDH2) protein, ALDH2Ã 2, which is produced by a single nucleotide polymorphism (rs671), promotes osteoporosis due to impaired osteoblastogenesis. Aldh2 plays a role in alcohol-detoxification by acetaldehyde-detoxification; however, transgenic mice expressing Aldh2 Ã 2 (Aldh2 Ã 2 Tg) exhibited severe osteoporosis with increased levels of blood acetaldehyde without alcohol consumption, indicating that Aldh2 regulates physiological bone homeostasis. Wild-type osteoblast differentiation was severely inhibited by exogenous acetaldehyde, and osteoblastic markers such as osteocalcin, runx2, and osterix expression, or phosphorylation of Smad1,5,8 induced by bone morphogenetic protein 2 (BMP2) was strongly altered by acetaldehyde. Acetaldehyde treatment also inhibits proliferation and induces apoptosis in osteoblasts. The Aldh2 Ã 2 transgene or acetaldehyde treatment induced accumulation of the lipid-oxidant 4-hydroxy-2-nonenal (4HNE) and expression of peroxisome proliferator-activated receptor gamma (PPARg), a transcription factor that promotes adipogenesis and inhibits osteoblastogenesis. Antioxidant treatment inhibited acetaldehyde-induced proliferation-loss, apoptosis, and PPARg expression and restored osteoblastogenesis inhibited by acetaldehyde. Treatment with a PPARg inhibitor also restored acetaldehyde-mediated osteoblastogenesis inhibition. These results provide new insight into regulation of osteoporosis in a subset of individuals with ALDH2 Ã 2 and in alcoholic patients and suggest a novel strategy to promote bone formation in such osteopenic diseases. ß

Chronic inflammation is frequently associated with tumorigenesis in elderly people. By contrast, young people without chronic inflammation often develop tumors considered independent of chronic inflammation but driven instead by mutations. Thus, whether inflammation has a significant role in tumor progression in tumors driven by mutations remains largely unknown. Here we show that TNFa is required for the tumorigenesis of osteosarcoma, the most common tumor in children and adolescents. We show that transplantation of AX osteosarcoma cells, which harbor mutations driving c-Myc overexpression and Ink4a-deficiency, in wildtype mice promotes lethal tumorigenesis accompanied by ectopic bone formation and multiple metastases, phenotypes seen in osteosarcoma patients. Such tumorigenesis was completely abrogated in TNFa-deficient mice. AX cells have the capacity to undergo osteoblastic differentiation; however, that activity was significantly inhibited by TNFa treatment, suggesting that TNFa maintains AX cells in an undifferentiated state. TNFa inhibition of AX cell osteoblastic differentiation occurred through ERK activation, and a pharmacological TNFa inhibitor effectively inhibited both AX cell tumorigenesis and increased osteoblastic gene expression and increased survival of tumor-bearing mice. Lethal tumorigenesis of AX cells was also abrogated in IL-1a/IL-1b doubly deficient mice. We found that both TNFa and IL-1 maintained AX cells in an undifferentiated state via ERK activation. Thus, inflammatory cytokines are required to promote tumorigenesis even in mutation-induced tumors, and TNFa/IL-1 and ERK may represent therapeutic targets for osteosarcoma.

Retrospective comparative study, Level III.