Title Bcl 2 Deficiency Activates FoxO through Akt Inactivation and AcceleratesOsteoblast Differentiation

Osteoblast apoptosis plays an important role in bone development and maintenance, and is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging. Although Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis, the physiological significance of Bcl2 in osteoblast differentiation has not been fully elucidated. To investigate this, we examined Bcl2-deficient (Bcl2) mice. In Bcl2 mice, bromodeoxyuridine (BrdU)positive osteoblasts were reduced in number, while terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive osteoblasts were increased. Unexpectedly, osteoblast differentiation was accelerated in Bcl2 mice as shown by the early appearance of osteocalcin-positive osteoblasts. Osteoblast differentiation was also accelerated in vitro when primary osteoblasts were seeded at a high concentration to minimize the reduction of the cell density by apoptosis during culture. FoxO transcription factors, whose activities are negatively regulated through the phosphorylation by Akt, play important roles in multiple cell events, including proliferation, death, differentiation, longevity, and stress response. Expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated; the expression and activity of FoxOs were enhanced; and the phosphorylation of Akt and that of FoxO1 and FoxO3a by Akt were reduced in Bcl2 calvariae. Further, the levels of p53 mRNA and protein were increased, and the expression of p53-target genes, Pten and Igfbp3 whose proteins inhibit Akt activation, was upregulated in Bcl2 calvariae. However, Pten but not Igfbp3 was upregulated in Bcl2 primary osteoblasts, and p53 induced Pten but not Igfbp3 in vitro. Silencing of either FoxO1 or FoxO3a inhibited and constitutively-active FoxO3a enhanced osteoblast differentiation. These findings suggest that Bcl2 deficiency induces and activates FoxOs through Akt inactivation, at least in part, by upregulating Pten expression through p53 in osteoblasts, and that the enhanced expression and activities of FoxOs may be one of the causes of accelerated osteoblast differentiation in Bcl2 mice. Citation: Moriishi T, Kawai Y, Komori H, Rokutanda S, Eguchi Y, et al. (2014) Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation. PLoS ONE 9(1): e86629. doi:10.1371/journal.pone. 0086629 Editor: Irina V. Lebedeva, Columbia University, United States of America Received October 27, 2011; Accepted December 17, 2013; Published January 20, 2014 Copyright: 2014 Moriishi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Ministry of Education, Science, Sports and Culture and Technology Grant-in-Aid for Scientific Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: komorit@nagasaki-u.ac. jp