Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and Induces Osteocyte Apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis

SP7 Inhibits Osteoblast Differentiation at a Late Stage in Mice

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2−/− calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter

BrdU uptake and real-time RT-PCR analysis.

<p>(A) The percentage of osteoblasts positive for BrdU at 2 weeks of age. n = 4. (B, C) Real-time RT-PCR analysis. B, Expression of <i>Col1a1</i>, <i>Spp1</i>, <i>Ibsp</i>, and <i>Bglap2</i> was determined using RNA from whole bones of femurs and tibiae in wild-type (wt), tg1, and tg2 mice at 4 weeks of age. n = 5–8. *P<0.05 and **P<0.01 vs. wild-type mice. C, Expression of <i>Dmp1</i>, <i>Mepe</i>, <i>Phex</i>, <i>Fgf23</i>, <i>Sost</i>, <i>Atf4</i>, <i>Tnfsf11</i>, <i>Tnfrsf11b</i> was determined using RNA from the osteocyte fraction of femurs and tibiae in wild-type and tg2 mice at 12 weeks of age. n = 5. **P<0.01 vs. wild-type mice. The values of the wild-type mice were defined as 1, and relative levels are shown in B and C.</p

Osteoblast differentiation <i>in vitro</i>.

<p>(A–D) Real-time RT-PCR analysis. A, Induction of <i>Sp7</i> expression by RUNX2 in <i>Runx2</i>^−/− calvarial cells. The cells isolated using collagen gel were infected with EGFP or <i>Runx2</i>-EGFP expressing adenovirus (6×10⁶ pfu/well). RNA samples were obtained at 6, 12, 24 and 48 hours after infection. Expression levels before infection (0) were defined as 1 and relatives levels are shown. <i>Runx2</i>^−/− calvarial cells express an aberrant transcript of <i>Runx2</i> at a low level. n = 3. B, <i>Sp7</i> expression after the infection of adenovirus carrying EGFP or <i>Sp7</i>. C, <i>Sp7</i> expression after the infection of adenovirus carrying EGFP or sh-<i>Sp7</i>. Adenoviruses were used at 3×10⁶ pfu/well or 6×10⁶ pfu/well as indicated. D, <i>Alpl</i>, <i>Col1a1</i>, and <i>Bglap2</i> expressions were examined at 4 days or 9 days of culture after the infection. Adenoviruses were used at 3×10⁶ pfu/well. The levels in cells, which were infected with EGFP-expressing adenovirus at 3×10⁶ pfu/well, at 4 days of culture were defined as 1, and relative levels are shown. *p<0.05 and **p<0.001 vs. EGFP. n = 4. (E) Von Kossa staining in primary osteoblasts infected with an adenovirus carrying EGFP, <i>Sp7</i>, sh-<i>Sp7</i>, or <i>Runx2</i>. Three independent experiments were performed and representative data are shown in A–E.</p

X-ray and histological analyses of <i>Sp7</i> transgenic mice.

<p>(A) X-ray analysis of the femurs of a wild-type (wt), tg1, and tg2 mouse at 6 weeks of age. (B–G) Histological appearance of bone in transgenic mice. Longitudinal sections through the distal part (B–D) and diaphysis (E–G) of femurs in a wild-type (B, E), tg1 (C, F), and tg2 (D, G) mouse at 6 weeks of age. The sections were stained with H–E. (H–J) Polarized microscopic examination of the diaphyses of femurs from wild-type (H), tg1 (I), and tg2 (J) mice at 6 months of age. (K–T) TEM images of osteoblasts (K, L) and osteocytes (M, N), canalicular staining of tibiae (O, P), and SEM images of preosteocytes (Q, S) and young osteocytes (R, T) of wild-type (K, M, O, Q, R) and tg2 (L, N, P, S, T) mice at 10 weeks of age. In Q–T, bone and osteoid in cortical bone were dissolved and preosteocytes and young osteocytes just beneath the osteoblast layer in the endosteum were observed by SEM. Bars: (B–D) 500 μm; (E–J) 50 μm; (K–N), 1 μm; (O–T), 10 μm.</p

pQCT and bone histomorphometric analyses and bone resorption markers.

<p>(A–C) pQCT analyses of femurs from wild-type (wt) and tg2 mice at 10 weeks of age. A, pQCT images of the diaphyses of the femurs. Bar: 1 mm. B, The trabecular bone density in 13 equal cross-divisions of the metaphysial parts of the femurs was measured in wild-type (open circles) and tg2 (closed circles) mice. C, Cortical bone density, cortical thickness, endosteal circumference, and periosteal circumference of the diaphyses of the femurs were measured in wild-type (open columns) and tg2 (closed columns) mice. n = 5. (D) Bone histomorphometric analysis. Trabecular bone volume (bone volume/tissue volume, BV/TV), osteoid thickness (O.Th), osteoblast surface (Ob.S/BS), osteoclast surface (Oc.S/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) were compared at 10 weeks of age. BS, bone surface. n = 5. (E) Urinary deoxypyridinoline levels at 6 and 10 weeks of age. n = 7–11. *P<0.05 and **P<0.01 vs. wild-type mice.</p

Positive regulation of <i>Sp7</i> promoter by SP7.

<p>(A, B) Real-time RT-PCR analysis. A, Endogenous <i>Sp7</i> expression in wild-type and tg2 mice at 18 weeks of age. RNA was prepared from tibiae and femurs, in which hematopoietic cells were flushed out. *P<0.05 vs. wild-type mice. n = 4–5. B, Osteoprogenitors isolated from wild-type calvaria using collagen gel were infected with adenovirus expressing EGFP or <i>Sp7</i> at 6×10⁶ pfu/well, and endogenous <i>Sp7</i> expression was examined. **p<0.001 vs. EGFP-expressing cells. The level in wild-type mice or EGFP-expressing cells was set at 1, and relative values are shown. (C, D) Reporter assays using the <i>Sp7</i> promoter. C, The reporter activity using the 1.8-kb <i>Sp7</i> promoter-luciferase (Luc) construct and the deletion constructs. Reporter assays were performed using 293T cells with (closed columns) or without (open columns) the <i>Sp7</i> expression vector. The lines in the schematic diagram of the <i>Sp7</i> promoter indicate putative SP1-binding sites. D, The reporter activity using the 1.8-kb <i>Sp7</i> promoter in ATDC5 cells infected with adenovirus expressing EGFP or sh-<i>Sp7</i> at 6×10⁶ pfu/well. *p<0.05 and **p<0.001 vs. control. (E) ChIP assay. DNA before immunoprecipitation (input) and after immunoprecipitation with anti-SP7 antibody or anti-IgG antibody was amplified by PCR using primers that amplify the region containing the proximal 170 bp of <i>Sp7</i> promoter.</p