The PHCCC site increase of FoxOs mRNA in Bcl22/2 mice still remains to be clarified. p53 has been shown to inhibit osteoblast differentiation,. However, it is evident in vitro but not in vivo, because the calvarial bone volume is mildly reduced in p532/2 mice compared with wild-type mice. Since the deletion of p53 enhances proliferation and inhibits apoptosis, p53 deletion should increase the cell density in culture, leading to the acceleration of osteoblast differentiation in vitro, because osteoblast differentiation is dependent on the cell density in vitro. Similarly, the increase in osteoblast number due to increased proliferation and reduced apoptosis should also lead to an increase in bone formation in p532/2 mice as previously reported. Therefore, the function of p53 in osteoblast differentiation needs to be further investigated. Although osteoblast proliferation was not examined in vivo in previously reported Bcl22/2 mice,, we showed that the number of proliferating osteoblasts was reduced in Bcl22/2 mice. Further, we observed a 15481974 reduction in the number of Bcl22/2 primary osteoblasts in the MTT assay, suggesting that Bcl2 enhances osteoblast proliferation. However, it could also have been caused by increased apoptosis during culture. Previous reports showed that Bcl2 inhibits cell proliferation by facilitating G0 arrest and delaying G0 to S phase transition in hematopoietic cells and fibroblasts, and various groups showed that p27 as well as p130 was elevated in Bcl2overexpressing cells during arrest,,,, although overNT-157 expression of Bcl2 in myocytes promoted proliferation. Thus, it is possible that the decrease in proliferating osteoblasts in Bcl22/2 mice was mostly a reflection of enhanced osteoblast differentiation, although the activation of FoxOs should have affected both proliferation and differentiation of osteoblasts in Bcl22/2 mice. In summary, osteoblast differentiation was enhanced in Bcl22/2 mice, at least in part, through FoxOs. FoxOs were Osteoblast Differentiation in Bcl22/2 Mice 8 Osteoblast Differentiation in Bcl22/2 Mice and 10mM b-glycerophosphate were added at day 1, and mRNA was extracted at day 4. The expression of p53, Pten, and Igfbp3 was examined by real-time RT-PCR. Similar results were obtained in two independent experiments and representative data are shown. n = 12213. vs. empty retrovirus. P,0.01, p,0.001. Schematic presentation of the signaling pathway for FoxO activation. p53 induces Pten mRNA and Igfbp3 mRNA. Pten and Igfbp3 inhibit Akt activation. Akt inhibits the activation of FoxOs. Activation of JNK and Mst1 activate FoxOs. p53 failed to induce Igfbp3 in vitro. Dotted arrows indicate that the activation did not occur in Bcl22/2 mice. doi:10.1371/journal.pone.0086629.g004 activated through the suppression of Akt, at least in part, by upregulation of Pten through p53. Although osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid 12926553 excess, and aging, our findings suggest that the 9 Osteoblast Differentiation in Bcl22/2 Mice stresses toward apoptosis may have a positive effect on osteoblast differentiation. Acknowledgments We thank N. Motoyama and T. Sakai for Gadd45a promoter construct, K. Ito for p53-expressing retrovirus, and C. Fukuda for secretarial assistance. Supporting Information Author Contributions Conceived and designed the experiments: TK TM. Performed the experiments: TM YK SR. Analyzed the data: TM. Contributed reagents/materials/analysis tools: HK Y.The increase of FoxOs mRNA in Bcl22/2 mice still remains to be clarified. p53 has been shown to inhibit osteoblast differentiation,. However, it is evident in vitro but not in vivo, because the calvarial bone volume is mildly reduced in p532/2 mice compared with wild-type mice. Since the deletion of p53 enhances proliferation and inhibits apoptosis, p53 deletion should increase the cell density in culture, leading to the acceleration of osteoblast differentiation in vitro, because osteoblast differentiation is dependent on the cell density in vitro. Similarly, the increase in osteoblast number due to increased proliferation and reduced apoptosis should also lead to an increase in bone formation in p532/2 mice as previously reported. Therefore, the function of p53 in osteoblast differentiation needs to be further investigated. Although osteoblast proliferation was not examined in vivo in previously reported Bcl22/2 mice,, we showed that the number of proliferating osteoblasts was reduced in Bcl22/2 mice. Further, we observed a 15481974 reduction in the number of Bcl22/2 primary osteoblasts in the MTT assay, suggesting that Bcl2 enhances osteoblast proliferation. However, it could also have been caused by increased apoptosis during culture. Previous reports showed that Bcl2 inhibits cell proliferation by facilitating G0 arrest and delaying G0 to S phase transition in hematopoietic cells and fibroblasts, and various groups showed that p27 as well as p130 was elevated in Bcl2overexpressing cells during arrest,,,, although overexpression of Bcl2 in myocytes promoted proliferation. Thus, it is possible that the decrease in proliferating osteoblasts in Bcl22/2 mice was mostly a reflection of enhanced osteoblast differentiation, although the activation of FoxOs should have affected both proliferation and differentiation of osteoblasts in Bcl22/2 mice. In summary, osteoblast differentiation was enhanced in Bcl22/2 mice, at least in part, through FoxOs. FoxOs were Osteoblast Differentiation in Bcl22/2 Mice 8 Osteoblast Differentiation in Bcl22/2 Mice and 10mM b-glycerophosphate were added at day 1, and mRNA was extracted at day 4. The expression of p53, Pten, and Igfbp3 was examined by real-time RT-PCR. Similar results were obtained in two independent experiments and representative data are shown. n = 12213. vs. empty retrovirus. P,0.01, p,0.001. Schematic presentation of the signaling pathway for FoxO activation. p53 induces Pten mRNA and Igfbp3 mRNA. Pten and Igfbp3 inhibit Akt activation. Akt inhibits the activation of FoxOs. Activation of JNK and Mst1 activate FoxOs. p53 failed to induce Igfbp3 in vitro. Dotted arrows indicate that the activation did not occur in Bcl22/2 mice. doi:10.1371/journal.pone.0086629.g004 activated through the suppression of Akt, at least in part, by upregulation of Pten through p53. Although osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid 12926553 excess, and aging, our findings suggest that the 9 Osteoblast Differentiation in Bcl22/2 Mice stresses toward apoptosis may have a positive effect on osteoblast differentiation. Acknowledgments We thank N. Motoyama and T. Sakai for Gadd45a promoter construct, K. Ito for p53-expressing retrovirus, and C. Fukuda for secretarial assistance. Supporting Information Author Contributions Conceived and designed the experiments: TK TM. Performed the experiments: TM YK SR. Analyzed the data: TM. Contributed reagents/materials/analysis tools: HK Y.
