One Mass and Osteoblast Apoptosis in Bcl22/2 Mice As Bcl22/2 mice died at approximately 23 weeks of age, bone histomorphometric analysis was performed on the trabecular bone of femurs at 2 weeks of age. The bone volume was increased in Bcl22/2 mice and the density of osteoblasts in Bcl22/ 2 mice was similar to that in wild-type mice. In 58-49-1 chemical information contrast, the density of osteoclasts was reduced in Bcl22/2 mice. The percentage of BrdU-positive osteoblastic cells in Bcl22/2 mice was less than that in wild-type mice, while the percentage of TUNEL-positive osteoblastic cells was increased in Bcl22/2 mice compared with wild-type mice. The percentage of TUNEL-positive osteocytes in Bcl22/2 mice was similar to that in wild-type mice. The expression of apoptosis-related genes, including Fas, FasL, p53, Noxa, Bax, Bid, Bim, Bad, Bnip3l, was increased in calvaria of Bcl22/2 mice compared with wild-type mice. ALP activity, mineralization, and the expression of ALP, Col1a1, osteopontin, and osteocalcin were similar to those from wild-type mice. However, apoptosis of the proliferating osteoblasts should affect the results of the MTT assay. Further, apoptosis during culture should affect osteoblast differentiation, because osteoblast differentiation in vitro is largely dependent on the cell density. Thus, we examined apoptosis during osteoblast proliferation and differentiation in vitro. Osteoblast apoptosis was significantly increased not only during proliferation but also during differentiation in Bcl22/2 osteoblasts. To minimize the reduction of cell density by apoptosis, primary osteoblasts isolated from wild-type, Bcl2+/2, and 16574785 Bcl22/2 mice were seeded at a higher concentration and ALP activity and the osteoblast marker gene expression were examined after 2 days. ALP activity and the expression of osteopontin, ALP, and Osterix were increased in Bcl22/2 primary osteoblasts compared with those in wild-type primary osteoblasts. After 8 days, the mineralization was similar between wild-type and Bcl22/2 primary osteoblasts, but osteocalcin mRNA was increased in Bcl22/2 primary osteoblasts. Although ALP activity was slightly increased in Bcl2+/2 primary osteoblasts compared with wild-type primary osteoblasts, the mineralization and the osteoblast marker gene expression were similar between Bcl2+/2and wild-type primary osteoblasts. Upregulation and Activation of FoxOs in Bcl22/2 Calvariae As Bcl2-deficiency enhanced osteoblast differentiation in vivo, we examined the mechanism of the accelerated osteoblast differentiation in vivo by directly analyzing the newborn calvariae. The expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated in Bcl22/2 calvariae. As FoxO1 enhances osteoblast differentiation,, FoxOs might be involved in enhanced osteoblast differentiation in Bcl22/2 mice. Thus, we first examined the expression and activity of FoxOs. The expressions of FoxO1, FoxO3a, and FoxO4 mRNAs were increased in Bcl22/2 calvariae compared with wild-type calvariae, and the promoter activity of Gadd45a was enhanced in Bcl22/2 primary osteoblasts compared with wild-type primary osteoblasts. FoxO proteins are inactivated through the Dimethylenastron phosphorylation by Akt. Akt itself is activated by phosphorylation ,,. Thus, we examined the activation state of Akt and FoxOs by examining their phosphorylation. The phosphorylation of Akt was markedly reduced in Bcl22/2 calvariae compared with wild-type calvariae, although similar levels of Akt protein were detec.One Mass and Osteoblast Apoptosis in Bcl22/2 Mice As Bcl22/2 mice died at approximately 23 weeks of age, bone histomorphometric analysis was performed on the trabecular bone of femurs at 2 weeks of age. The bone volume was increased in Bcl22/2 mice and the density of osteoblasts in Bcl22/ 2 mice was similar to that in wild-type mice. In contrast, the density of osteoclasts was reduced in Bcl22/2 mice. The percentage of BrdU-positive osteoblastic cells in Bcl22/2 mice was less than that in wild-type mice, while the percentage of TUNEL-positive osteoblastic cells was increased in Bcl22/2 mice compared with wild-type mice. The percentage of TUNEL-positive osteocytes in Bcl22/2 mice was similar to that in wild-type mice. The expression of apoptosis-related genes, including Fas, FasL, p53, Noxa, Bax, Bid, Bim, Bad, Bnip3l, was increased in calvaria of Bcl22/2 mice compared with wild-type mice. ALP activity, mineralization, and the expression of ALP, Col1a1, osteopontin, and osteocalcin were similar to those from wild-type mice. However, apoptosis of the proliferating osteoblasts should affect the results of the MTT assay. Further, apoptosis during culture should affect osteoblast differentiation, because osteoblast differentiation in vitro is largely dependent on the cell density. Thus, we examined apoptosis during osteoblast proliferation and differentiation in vitro. Osteoblast apoptosis was significantly increased not only during proliferation but also during differentiation in Bcl22/2 osteoblasts. To minimize the reduction of cell density by apoptosis, primary osteoblasts isolated from wild-type, Bcl2+/2, and 16574785 Bcl22/2 mice were seeded at a higher concentration and ALP activity and the osteoblast marker gene expression were examined after 2 days. ALP activity and the expression of osteopontin, ALP, and Osterix were increased in Bcl22/2 primary osteoblasts compared with those in wild-type primary osteoblasts. After 8 days, the mineralization was similar between wild-type and Bcl22/2 primary osteoblasts, but osteocalcin mRNA was increased in Bcl22/2 primary osteoblasts. Although ALP activity was slightly increased in Bcl2+/2 primary osteoblasts compared with wild-type primary osteoblasts, the mineralization and the osteoblast marker gene expression were similar between Bcl2+/2and wild-type primary osteoblasts. Upregulation and Activation of FoxOs in Bcl22/2 Calvariae As Bcl2-deficiency enhanced osteoblast differentiation in vivo, we examined the mechanism of the accelerated osteoblast differentiation in vivo by directly analyzing the newborn calvariae. The expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated in Bcl22/2 calvariae. As FoxO1 enhances osteoblast differentiation,, FoxOs might be involved in enhanced osteoblast differentiation in Bcl22/2 mice. Thus, we first examined the expression and activity of FoxOs. The expressions of FoxO1, FoxO3a, and FoxO4 mRNAs were increased in Bcl22/2 calvariae compared with wild-type calvariae, and the promoter activity of Gadd45a was enhanced in Bcl22/2 primary osteoblasts compared with wild-type primary osteoblasts. FoxO proteins are inactivated through the phosphorylation by Akt. Akt itself is activated by phosphorylation ,,. Thus, we examined the activation state of Akt and FoxOs by examining their phosphorylation. The phosphorylation of Akt was markedly reduced in Bcl22/2 calvariae compared with wild-type calvariae, although similar levels of Akt protein were detec.
