Figure 1.
Bone morphometric analysis, BrdU and TUNEL staining, and real-time RT-PCR analysis of apoptosis-related genes in Bcl2−/− mice.
(A) Bone histomorphometric analysis. The trabecular bone volume (bone volume/tissue volume, BV/TV), number of osteoblasts (N.Ob/B.Pm), and number of osteoclasts (N.Oc/B.Pm) were compared in femurs between 6 wild-type and 4 Bcl2−/− mice at 2 weeks of age. B.Pm, bone perimeter. (B–H) BrdU labeling (B, C) and TUNEL staining (D, E) of sections of femurs from wild-type mice (B, D) and Bcl2−/− mice (C, E). Bars = 50 µm. BrdU-positive osteoblastic cells (F), TUNEL-positive osteoblastic cells (G), and TUNEL-positive osteocytes (H) were counted and shown as a percentage of the number of osteoblastic cells or osteocytes. wild-type mice, n = 7; Bcl2−/− mice, n = 5 in F. wild-type mice, n = 8; Bcl2−/− mice, n = 5 in G and H. (I) Real-time RT-PCR analysis of apoptosis-related genes. RNA was directly extracted from newborn calvariae of wild-type and Bcl2−/− mice. wild-type mice, n = 6; Bcl2−/− mice, n = 15. *vs. wild-type mice. *P<0.05, **P<0.01.
Figure 2.
Expression of bone matrix protein genes in Bcl2−/− mice.
(A) Real-time RT-PCR analysis of Bcl2, Runx2, Osterix, Col1a1, osteopontin, and osteocalcin. RNA was directly extracted from newborn calvariae of wild-type (wt) and Bcl2−/− mice. The values of wild-type mice were defined as 1, and relative levels are shown. wild-type mice, n = 6; Bcl2−/− mice, n = 15. *vs. wild-type mice. *P<0.05, **P<0.01, ***P<0.001. (B–U) In situ hybridization analysis of Col1a1, osteopontin, and osteocalcin. The sections of femurs from Bcl2+/− mice (B, D, F, H, J), Bcl2−/− mice (C, E, G, I, K, M, O, Q, S, U), and wild-type mice (L, N, P, R, T) at birth (B–K) and at 2 weeks of age (L–U) were stained with H–E (B, C, L, M) or hybridized with Col1a1 (D, E, N, O), osteopontin (F, G, P, Q), and osteocalcin (H–K, R–U) 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 (data not shown). Bars: 100 µm (B–I, L–S); 50 µm (J, K, T, U).
Figure 3.
Analyses of proliferation, differentiation, and apoptosis of Bcl2−/− primary osteoblasts.
(A) MTT assay. Primary osteoblasts from calvariae of 4 wild-type and 3 Bcl2−/− mice were cultured for 3 days, the cells were replated, and MTT assay was performed 24 hrs later. n = 16. Similar results were obtained in two independent experiments and representative data are shown. (B–D) Differentiation of primary osteoblasts from Bcl2−/− mice. Primary osteoblasts were prepared from newborn calvariae of wild-type and Bcl2−/− mice, and ALP and von Kossa staining (B), quantification of mineralization (C), and real-time RT-PCR analysis (D) were performed. Primary osteoblasts were seeded at a concentration of 2.5×104/cm2 (day 0). 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate were added at day 3, ALP activity and the osteoblast marker gene 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. (E) Frequencies of TUNEL-positive cells during culture. Primary osteoblasts from calvariae of 6 wild-type and 10 Bcl2−/− mice were stained for TUNEL before confluence, at confluence, and at 3 and 9 days after confluence. n = 4−5. Similar results were obtained in two independent experiments and representative data are shown. (F–I) Differentiation of primary osteoblasts from calvariae of Bcl2−/− mice. Primary osteoblasts were seeded at a concentration of 2×105/cm2 (day 0), 50 µg/ml ascorbic acid and 10mM β-glycerophosphate were added at day 1, ALP activity was examined at day 2 (F), mineralization was examined at day 9 (G), and the osteoblast marker gene expression was examined at day 2 (H) and 9 (I) by real-time RT-PCR. n = 7 in G; n = 5 in H; n = 10−12 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.
Figure 4.
Expression and activation of FoxOs in Bcl2−/− calvariae.
(A) Real-time RT-PCR analysis of the expression of FoxOs. RNA was directly extracted from newborn calvariae of wild-type and Bcl2−/− mice. wild-type mice, n = 6; Bcl2−/− mice, n = 15. *vs. wild-type mice, **p<0.01, ***p<0.001. (B) Reporter assay of Gadd45a promoter using wild-type and Bcl2−/− primary osteoblasts. Similar results were obtained in two independent experiments and representative data are shown. (C, D) Western blot analysis. Protein was extracted from newborn calvariae of wild-type and Bcl2−/− mice. The intensities of the bands were normalized against each β-actin, the normalized values in wild-type mice were set as 1, and relative levels are shown. Similar results were obtained in three independent experiments and representative data are shown. (E) Real-time RT-PCR analysis. RNA was directly extracted from calvariae of wild-type and Bcl2−/− newborn mice. wild-type mice, n = 6; Bcl2−/− mice, n = 15. *vs. wild-type mice. *P<0.05, **P<0.01. (F) p53, Pten, and Igfbp3 expression in primary osteoblasts. The cDNA in Fig. 3I was used for real-time PCR analysis. n = 10−12. *vs. wild-type primary osteoblasts. **P<0.01. (G) Induction of Pten by p53. p53−/− osteoblasts were infected with p53-expressing retrovirus or empty retrovirus. Next day, the cells were plated at the concentration of 1.5×105/well in 48 well plates (day 0). 50 µg/ml ascorbic acid and 10mM β-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 = 12−13. *vs. empty retrovirus. **P<0.01, ***p<0.001. (H) 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 (G). Dotted arrows indicate that the activation did not occur in Bcl2−/− mice (C).
Figure 5.
Induction of osteoblast differentiation by FoxOs.
(A–C) Induction of osteoblast differentiation by FoxO3aTM. Primary osteoblasts from calvariae of wild-type mice were infected with adenovirus expressing GFP or FoxO3aTM, and ALP staining at 2 days and von Kossa staining at 6 days after infection (A), quantification of mineralization (B), and osteoblast marker gene expression (C) are shown. The value in GFP-introduced cells was set as 1 and the relative level is shown in B. Similar results were obtained in three independent experiments and representative data are shown. (D–F) Inhibition of the mineralization of MC3T3-E1 cells by shFoxO1 and shFoxO3a. MC3T3-E1 cells were infected with retrovirus expressing GFP, shFoxO1, or shFoxO3a, and cultured in the presence of BMP2 (100ng/ml). The expression of FoxO1 and FoxO3a was examined by real-time RT-PCR (D) and mineralization was examined by von Kossa staining (E) and its quantification (F) after culture for 2 weeks. The value in shGFP-introduced cells was set as 1 and the relative levels are shown in F. Similar results were obtained in three independent experiments and representative data are shown.