Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability

Abstract

Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-β signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4Δ(Os) mice, in which Smad4 was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4Δ(Os) mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5'-bromo-2'deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4Δ(Os) mice. Apoptosis in isolated calvaria cells from Smad4Δ(Os) mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4Δ(Os) mice. Conversely, osteoblast cells overexpressing Smad4 showed increased apoptosis. In an apoptosis induction model of Smad4Δ(Os) mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.

Figure 1

Delayed growth in targeted Smad4-disrupted mice. (a) Disruption of Smad4 protein in Smad4Δ^Os mice was verified by western blotting using total protein lysates from 3-week-old control and Smad4Δ^Os mouse femur, heart, liver, spleen, kidney and muscle. (b) Total RNA was isolated from the bones of each littermate mouse 3 weeks postnatally, and the mRNA level of Smad4 was determined using real-time RT-PCR analysis. (c) Characterization of Cre activity in 3-week-old Col1a1-Cre:R26R double transgenic mice. The left panel indicates primary spongiosa in the distal femur. The right panel indicates cortical bone in the femur shaft. (d) The 3-week-old mice were imaged with simple radiography. (e) A 3D μCT reconstruction of the head in 3-week-old littermates. (f) 3D μCT reconstruction of femora at 3 weeks, 6 weeks and 5 months of age. The reconstruction site was the center of the femur in the sagittal plane. Values are presented as the means±s.e.m. (n=5). ^**P<0.01 versus control. Scale bar (black), 50 μm (c, left), 25 μm (c, right). Scale bar (white), 2 mm.

Figure 2

Increased bone mass in Smad4Δ^Os mice. (a, b) 3D μCT reconstructions of distal femora of 3-week-old littermates and hematoxylin and eosin stain. (c) Total RNA was isolated from the bones of each littermate mouse at 3 weeks postnatal, and the mRNA levels of markers were determined by real-time RT-PCR analysis. Changes in the bone formation markers Runx2, Alp, Col1a1, OPN, OC, BSP and Dmp1. (d) Changes in the bone resorption markers Rankl, Opg, Trap and Ctsk. (e, f) TRAP staining in primary spongiosa of distal femora of 3-week-old mice, and counted osteoclasts per unit area. Values are presented as the means±s.e.m. (n=5). *P<0.05 and ^**P<0.01 versus control. Scale bars, 400 μm (b), 25 μm (e).

Figure 3

Regulation of osteoblast/osteocyte viability by Smad4. (a, b) Histological examination of primary spongiosa and cortical bone of distal femur stained with hematoxylin and eosin in control and Smad4Δ^Os mice at 3 weeks of age and counted osteoblasts and osteocytes per unit area. (c) Proliferation was evaluated using a BrdU labeling and detection kit at P10. (d) BrdU-labeled cells in trabecular osteoblasts and cortical osteocytes were counted per unit area. (e) Apoptotic osteocytes detected by TUNEL staining at 3 weeks of age. TUNEL-positive cells were counted and expressed as a percentage of all osteocytes per unit area. (f) Protein extracts of prepared bone and the levels of apoptosis-related proteins were examined by western blotting. C, cortical bone, M, bone marrow. Values are presented as the means±s.e.m. (n=5). ^**P<0.01 versus control. Scale bar, 25 μm (a, c).

Figure 4

The effect of Smad4 on apoptosis of osteoblasts in vitro. (a, b) Primary osteoblasts were isolated from calvaria of control mice and Smad4Δ^Os mice at postnatal day 3. (a) Isolated cells were stained with alizarin red solution on day 14 after initial mineralization. (b) The levels of apoptosis-related proteins were examined by western blotting on day 21 after initial mineralization. (c–f) 7F2 cells were transfected with either empty vector or Smad4 for 24 h and then treated with 10 mM β-glycerophosphate and 50 μg ml⁻¹ ascorbic acid. (c) Smad4 overexpression was analyzed by western blotting after transfection. (d) The levels of apoptosis-related proteins were examined by western blotting at the indicated time points after mineralization. (e) Apoptosis in 7F2 cells was assessed using an APOPercentage apoptosis assay kit. Apoptotic cells appear bright pink. (f) The absorbance of the accumulated APOPercentage dye was measured with a spectrophotometer at 550 nm. Values are presented as the means±s.e.m. (n=3). *P<0.05 versus empty vector. Scale bar, 100 μm.

Figure 5

Protection against unloading-induced reduction of bone mass and apoptosis in Smad4Δ^Os mice. (a) Five-month-old control and Smad4Δ^Os mice were subjected to skeletal unloading by tail suspension or kept on the ground for 2 weeks. Three-dimensional μCT reconstructions of diaphyseal femora. (b) Histological examination of cortical femora stained with hematoxylin and eosin in control and Smad4Δ^Os mice at 2 months of age following 2 weeks of unloading by tail suspension or staying on the ground. Arrowheads indicate endosteum-lining osteoblasts. (c) Osteocytes were counted per unit area. Oblique line bars indicate grounded mice, and dotted bars indicate tail-suspended mice during 2 weeks of treatment. (d) Apoptotic osteocytes detected by TUNEL staining. (e) TUNEL-positive cells were counted and expressed as a percentage of all osteocytes per unit area. Hatched bars indicate grounded mice, and dotted bars indicate tail-suspended mice. Values are presented as the means±s.e.m. (n=4). *P<0.05 versus control grounded; ^#P<0.01 versus control grounded; ^$P<0.01 versus control tail-suspended. Scale bar, 25 μm (b, d).