FOXOs attenuate bone formation by suppressing Wnt signaling
Srividhya Iyer, Elena Ambrogini, Shoshana M. Bartell, Li Han, Paula K. Roberson, Rafael de Cabo, Robert L. Jilka, Robert S. Weinstein, Charles A. O’Brien, Stavros C. Manolagas, Maria Almeida
Srividhya Iyer, Elena Ambrogini, Shoshana M. Bartell, Li Han, Paula K. Roberson, Rafael de Cabo, Robert L. Jilka, Robert S. Weinstein, Charles A. O’Brien, Stavros C. Manolagas, Maria Almeida
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Research Article Bone biology

FOXOs attenuate bone formation by suppressing Wnt signaling

Abstract

Wnt/β-catenin/TCF signaling stimulates bone formation and suppresses adipogenesis. The hallmarks of skeletal involution with age, on the other hand, are decreased bone formation and increased bone marrow adiposity. These changes are associated with increased oxidative stress and decreased growth factor production, which activate members of the FOXO family of transcription factors. FOXOs in turn attenuate Wnt/β-catenin signaling by diverting β-catenin from TCF- to FOXO-mediated transcription. We show herein that mice lacking Foxo1, -3, and -4 in bipotential progenitors of osteoblast and adipocytes (expressing Osterix1) exhibited increased osteoblast number and high bone mass that was maintained in old age as well as decreased adiposity in the aged bone marrow. The increased bone mass in the Foxo-deficient mice was accounted for by increased proliferation of osteoprogenitor cells and bone formation resulting from upregulation of Wnt/β-catenin signaling and cyclin D1 expression, but not changes in redox balance. Consistent with this mechanism, β-catenin deletion in Foxo null cells abrogated both the increased cyclin D1 expression and proliferation. The elucidation of a restraining effect of FOXOs on Wnt signaling in bipotential progenitors suggests that FOXO activation by accumulation of age-associated cellular stressors may be a seminal pathogenetic mechanism in the development of involutional osteoporosis.

Authors

Srividhya Iyer, Elena Ambrogini, Shoshana M. Bartell, Li Han, Paula K. Roberson, Rafael de Cabo, Robert L. Jilka, Robert S. Weinstein, Charles A. O’Brien, Stavros C. Manolagas, Maria Almeida

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Figure 5

Wnt/TCF signaling is responsible for increased osteoprogenitor proliferation.

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Wnt/TCF signaling is responsible for increased osteoprogenitor prolifera...
(A) Wnt-target genes determined by microarray analysis in Osx1-GFP calvaria cells. (B) mRNA levels by qRT-PCR in calvaria cells treated with vehicle (veh) or 50 ng/ml Wnt3a overnight (triplicates). (C) mRNA levels by qRT-PCR in femoral shafts from 3-month-old mice (n = 8–9/group), and in periosteal and bone marrow osteoblastic cell cultures described in Figure 4, C and G. (D) mRNA by qRT-PCR (left, triplicates) and protein levels by Western blot (right) in calvaria-derived cells infected with Adeno-Cre to induce Foxo deletion. (E) BrdU incorporation in cells, described in D, treated with veh (–) or 50 ng/ml Wnt3a (triplicates). (F) Lysates from nuclear extracts of calvaria cells, described in D, treated with vehicle or 50 ng/ml Wnt3a, immunoprecipitated with an anti–TCF-4 or anti-IgG antibody, and probed with an anti–β-catenin antibody. (G) mRNA levels by qRT-PCR and (H) BrdU incorporation in calvaria cells infected with Adeno-Cre and with lentivirus expressing shRNA directed against β-catenin or nontargeted shRNA followed by treatment with vehicle or 50 ng/ml Wnt3a overnight (triplicates). #P < 0.05 by Student’s t test; *P < 0.05 by ANOVA with Bonferroni’s test. Bars represent mean + SD.