Ed p53 to p532/2 osteoblast cell line. Pten mRNA but not Igfbp3 mRNA was induced by p53. Further, FoxO3a mRNA was not induced by p53. These findings suggest that Akt phosphorylation was reduced, at least in part, by the induction of Pten through upregulated p53. FoxOs Enhanced Osteoblast Differentiation To examine whether FoxOs are able to enhance osteoblast differentiation, a constitutively active form of FoxO3a Real-time RT-PCR analysis of Bcl2, Runx2, Osterix, Col1a1, get SIS3 osteopontin, and osteocalcin. RNA was directly extracted from newborn calvariae of wild-type and Bcl22/2 mice. The values of wild-type mice were defined as 1, and relative levels are shown. wild-type mice, n = 6; Bcl22/2 mice, n = 15. vs. wild-type mice. P,0.05, P,0.01, P,0.001. In situ hybridization analysis of Col1a1, osteopontin, and osteocalcin. The sections of femurs from Bcl2+/2 mice, Bcl22/2 mice, and wild-type mice at birth and at 2 weeks 22948146 of age were stained with HE or hybridized with Col1a1, osteopontin, and osteocalcin probes. Boxed regions in H, I, R, and S are magnified in J, K, T, and U, respectively. Arrows in I indicate the appearance of osteocalcin-expressing cells in the bone collar. Similar results were obtained in two newborn mice and three 2-week-old mice in each genotype and representative data are shown. In situ hybridization using the sense probes showed no significant signals. Bars: 100 mm; 50 mm. doi:10.1371/journal.pone.0086629.g002 5 Osteoblast Differentiation in Bcl22/2 Mice 6 Osteoblast Differentiation 16574785 in Bcl22/2 Mice expression were examined at day 6, and mineralization was examined at day 17. The value of primary BTZ-043 site osteoblasts from wild-type mice was set as 1 and the relative level is shown. n = 3. Similar results were obtained in three independent experiments and representative data are shown. Frequencies of TUNEL-positive cells during culture. Primary osteoblasts from calvariae of 6 wild-type and 10 Bcl22/2 mice were stained for TUNEL before confluence, at confluence, and at 3 and 9 days after confluence. n = 425. Similar results were obtained in two independent experiments and representative data are shown. Differentiation of primary osteoblasts from calvariae of Bcl22/2 mice. Primary osteoblasts were seeded at a concentration of 26105/cm2, 50 mg/ml ascorbic acid and 10mM b-glycerophosphate were added at day 1, ALP activity was examined at day 2, mineralization was examined at day 9, and the osteoblast marker gene expression was examined at day 2 and 9 by real-time RT-PCR. n = 7 in G; n = 5 in H; n = 10212 in I. Similar results were obtained in three independent experiments and representative data are shown. vs. wild-type primary osteoblasts. P,0.05; , ##P,0.01; p,0.001. doi:10.1371/journal.pone.0086629.g003 TM) was introduced into primary osteoblasts. FoxO3a-TM enhanced ALP activity, mineralization, and the expression of Runx2, Osterix, ALP, and osteocalcin. Further, retroviral introduction of shRNA of either FoxO1 or FoxO3a into MC3T3-E1 cells reduced mineralization. These findings suggest that FoxOs may be involved in the enhanced osteoblast differentiation in Bcl22/2 mice. Discussion The proliferation was reduced and apoptosis was increased, but the differentiation was accelerated in osteoblasts and mature osteoblasts were increased in Bcl22/2 mice. Therefore, our findings indicate that bone mass was increased in Bcl22/2 mice not only due to the decrease in osteoclast number but also due to the acceleration of osteoblast differentiati.Ed p53 to p532/2 osteoblast cell line. Pten mRNA but not Igfbp3 mRNA was induced by p53. Further, FoxO3a mRNA was not induced by p53. These findings suggest that Akt phosphorylation was reduced, at least in part, by the induction of Pten through upregulated p53. FoxOs Enhanced Osteoblast Differentiation To examine whether FoxOs are able to enhance osteoblast differentiation, a constitutively active form of FoxO3a Real-time RT-PCR analysis of Bcl2, Runx2, Osterix, Col1a1, osteopontin, and osteocalcin. RNA was directly extracted from newborn calvariae of wild-type and Bcl22/2 mice. The values of wild-type mice were defined as 1, and relative levels are shown. wild-type mice, n = 6; Bcl22/2 mice, n = 15. vs. wild-type mice. P,0.05, P,0.01, P,0.001. In situ hybridization analysis of Col1a1, osteopontin, and osteocalcin. The sections of femurs from Bcl2+/2 mice, Bcl22/2 mice, and wild-type mice at birth and at 2 weeks 22948146 of age were stained with HE or hybridized with Col1a1, osteopontin, and osteocalcin probes. Boxed regions in H, I, R, and S are magnified in J, K, T, and U, respectively. Arrows in I indicate the appearance of osteocalcin-expressing cells in the bone collar. Similar results were obtained in two newborn mice and three 2-week-old mice in each genotype and representative data are shown. In situ hybridization using the sense probes showed no significant signals. Bars: 100 mm; 50 mm. doi:10.1371/journal.pone.0086629.g002 5 Osteoblast Differentiation in Bcl22/2 Mice 6 Osteoblast Differentiation 16574785 in Bcl22/2 Mice expression were examined at day 6, and mineralization was examined at day 17. The value of primary osteoblasts from wild-type mice was set as 1 and the relative level is shown. n = 3. Similar results were obtained in three independent experiments and representative data are shown. Frequencies of TUNEL-positive cells during culture. Primary osteoblasts from calvariae of 6 wild-type and 10 Bcl22/2 mice were stained for TUNEL before confluence, at confluence, and at 3 and 9 days after confluence. n = 425. Similar results were obtained in two independent experiments and representative data are shown. Differentiation of primary osteoblasts from calvariae of Bcl22/2 mice. Primary osteoblasts were seeded at a concentration of 26105/cm2, 50 mg/ml ascorbic acid and 10mM b-glycerophosphate were added at day 1, ALP activity was examined at day 2, mineralization was examined at day 9, and the osteoblast marker gene expression was examined at day 2 and 9 by real-time RT-PCR. n = 7 in G; n = 5 in H; n = 10212 in I. Similar results were obtained in three independent experiments and representative data are shown. vs. wild-type primary osteoblasts. P,0.05; , ##P,0.01; p,0.001. doi:10.1371/journal.pone.0086629.g003 TM) was introduced into primary osteoblasts. FoxO3a-TM enhanced ALP activity, mineralization, and the expression of Runx2, Osterix, ALP, and osteocalcin. Further, retroviral introduction of shRNA of either FoxO1 or FoxO3a into MC3T3-E1 cells reduced mineralization. These findings suggest that FoxOs may be involved in the enhanced osteoblast differentiation in Bcl22/2 mice. Discussion The proliferation was reduced and apoptosis was increased, but the differentiation was accelerated in osteoblasts and mature osteoblasts were increased in Bcl22/2 mice. Therefore, our findings indicate that bone mass was increased in Bcl22/2 mice not only due to the decrease in osteoclast number but also due to the acceleration of osteoblast differentiati.
