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Pathological bone loss is caused by an imbalance between bone formation and resorption. The bone microenvironments are hypoxic, and hypoxia-inducible factor (HIF) is known to play notable roles in bone remodeling. However, the relevant functions of HIF-2α are not well understood. Here, we have shown that HIF-2α deficiency in mice enhances bone mass through its effects on the differentiation of osteoblasts and osteoclasts. In vitro analyses revealed that HIF-2α inhibits osteoblast differentiation by targeting Twist2 and stimulates RANKL-induced osteoclastogenesis via regulation of Traf6 . In addition, HIF-2α appears to contribute to the crosstalk between osteoblasts and osteoclasts by directly targeting RANKL in osteoprogenitor cells. Experiments performed with osteoblast- and osteoclast-specific conditional knockout mice supported a role of HIF-2α in this crosstalk. HIF-2α deficiency alleviated ovariectomy-induced bone loss in mice, and specific inhibition of HIF-2α with ZINC04179524 significantly blocked RANKL-mediated osteoclastogenesis. Collectively, our results suggest that HIF-2α functions as a catabolic regulator in bone remodeling, which is critical for the maintenance of bone homeostasis.

Although a second stem cell transplantation (SCT) can be used as salvage therapy in patients with relapsing leukemia after SCT, most of these patients have a poor outcome. We tried clinical vaccination using monocyte-derived dendritic cells (DCs) pulsed with leukemic lysates to treat relapsing acute myeloid leukemia (AML) after autologous SCT. To generate DCs, CD14+ cells isolated from peripheral blood stem cell products were cultured in AIM-V in the presence of GM-CSF and IL-4. Adding TNF-alpha on day 6 induced maturation of the DCs, which were harvested on day 8 or 9. The DCs were incubated with tumor lysate and KLH for 2 hr at 37 degrees C. After certifying the absence of microorganisms and endotoxins, the patients received four DC vaccinations at two- to three-week intervals. Two patients received four DC vaccinations with means of 7.8 x 10(6) and 9 x 10(6) DCs at two- to three-week intervals. The DC vaccinations were well tolerated with no apparent side effects. After the vaccinations, the patients showed immunological responses with positive delayed-type hypersensitivity skin reaction and increasing autologous T cells stimulatory capacity to the DCs; however, the BM blast percentage of the patients did not improve. The results suggest that DCs are a feasible cellular therapy for relapsing AML after autologous SCT.

Aging is associated with cellular senescence followed by bone loss leading to bone fragility in humans. However, the regulators associated with cellular senescence in aged bones need to be identified. Hypoxia-inducible factor (HIF)−2α regulates bone remodeling via the differentiation of osteoblasts and osteoclasts. Here, we report that HIF-2α expression was highly upregulated in aged bones. HIF-2α depletion in male mice reversed age-induced bone loss, as evidenced by an increase in the number of osteoblasts and a decrease in the number of osteoclasts. In an in vitro model of doxorubicin-mediated senescence, the expression of Hif-2α and p21, a senescence marker gene, was enhanced, and osteoblastic differentiation of primary mouse calvarial preosteoblast cells was inhibited. Inhibition of senescence-induced upregulation of HIF-2α expression during matrix maturation, but not during the proliferation stage of osteoblast differentiation, reversed the age-related decrease in Runx2 and Ocn expression. However, HIF-2α knockdown did not affect p21 expression or senescence progression, indicating that HIF-2α expression upregulation in senescent osteoblasts may be a result of aging rather than a cause of cellular senescence. Osteoclasts are known to induce a senescent phenotype during in vitro osteoclastogenesis. Consistent with increased HIF-2α expression, the expression of p16 and p21 was upregulated during osteoclastogenesis of bone marrow macrophages. ChIP following overexpression or knockdown of HIF-2α using adenovirus revealed that p16 and p21 are direct targets of HIF-2α in osteoclasts. Osteoblast-specific (Hif-2αfl/fl;Col1a1-Cre) or osteoclast-specific (Hif-2αfl/fl;Ctsk-Cre) conditional knockout of HIF-2α in male mice reversed age-related bone loss. Collectively, our results suggest that HIF-2α acts as a senescence-related intrinsic factor in age-related dysfunction of bone homeostasis.

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