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. 2001 Oct 1;155(1):157-66.
doi: 10.1083/jcb.200105052.

Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures

W Liu  1 , S Toyosawa, T Furuichi, N Kanatani, C Yoshida, Y Liu, M Himeno, S Narai, A Yamaguchi, T Komori
Affiliations

Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures

W Liu et al. J Cell Biol. .

Abstract

Targeted disruption of core binding factor alpha1 (Cbfa1) showed that Cbfa1 is an essential transcription factor in osteoblast differentiation and bone formation. Furthermore, both in vitro and in vivo studies showed that Cbfa1 plays important roles in matrix production and mineralization. However, it remains to be clarified how Cbfa1 controls osteoblast differentiation, bone formation, and bone remodelling. To understand fully the physiological functions of Cbfa1, we generated transgenic mice that overexpressed Cbfa1 in osteoblasts using type I collagen promoter. Unexpectedly, Cbfa1 transgenic mice showed osteopenia with multiple fractures. Cortical bone, which was thin, porous, and enriched with osteopontin, was invaded by osteoclasts, despite the absence of acceleration of osteoclastogenesis. Although the number of neonatal osteoblasts was increased, their function was impaired in matrix production and mineralization. Furthermore, terminally differentiated osteoblasts, which strongly express osteocalcin, and osteocytes were diminished greatly, whereas less mature osteoblasts expressing osteopontin accumulated in adult bone. These data indicate that immature organization of cortical bone, which was caused by the maturational blockage of osteoblasts, led to osteopenia and fragility in transgenic mice, demonstrating that Cbfa1 inhibits osteoblast differentiation at a late stage.

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Figures

Figure 1.
Figure 1.
Generation of transgenic mice. (A) Diagrams of the DNA constructs used to generate pro-α1 (I) collagen promoter Cbfa1 transgenic mice. DNA fragments covering the entire coding region of the mouse type II Cbfa1 isoform and 2.3 kb pro-α1 (I) collagen gene promoter region were used. *SV40 splice donor/acceptor signals; **SV40 polyadenylation signal. (B and C) α-Galactosidase staining in pro-α1 (I) collagen promoter α-galactosidase transgenic mice at E15.5. (C) Longitudinal section of forelimb. Staining is observed specifically in osteoblasts around the diaphysis and in immature osteoblastic cells around the metaphysis. Sections were counterstained with eosin. (D) Northern blot hybridized with Cbfa1 probe. RNA was extracted from tissues of 4-wk-old Cbfa1 transgenic mice, and 20 μg of total RNA was loaded per lane. St, stomach; Te, testis; Lu, lung; Sp, spleen; Li, liver; Th, thymus; Ki, kidney; He, heart; Br, brain; Mu, muscle; Bo, bone; Ca, cartilage; Sk, skin. (E) Northern blot analysis comparing the transgene and endogenous Cbfa1 levels of expression in long bones of newborn Cbfa1 transgenic mice. 20 μg of total RNA was loaded. Bar, 100 μm.
Figure 2.
Figure 2.
Radiological analysis. (A–C) X-ray analysis of 6-wk-old Cbfa1 transgenic mice from the established line. Whole skeletons of transgenic mice are proportionally shorter and more radiolucent (A). The transgenic mouse suffered from fractures in the tibia, fibula, and calcanei, and fracture healing is observed in these regions (A and B). The radiolucency is caused mainly by thinner cortices because metaphyseal trabeculation is similarly observed in femurs (C). (D) Radiograph of 9-wk-old F0 transgenic mice. Whole skeletons are also more radiolucent, and fracture healing is observed in tibiae, fibulae, and calcanei in both lower limbs. WT, wild-type mouse; Tg, transgenic mouse.
Figure 3.
Figure 3.
Histological appearance of transgenic bone. Longitudinal sections through the proximal tibia of wild-type (A and C) and transgenic (B and D) mice at 3 (A and B) and 6 wk of age (C and D). Cortical bone in transgenic mice is thin and porous at both 3 and 6 wk of age. Transgenic mice show reduced trabeculation at 3 wk of age but not at 6 wk of age. Undecalcified sections were stained with toluidine blue. Bar, 1 mm.
Figure 4.
Figure 4.
pQCT analysis. Diaphyses of femurs from wild-type and transgenic female mice at 3 mo of age were analyzed by pQCT. (A and B) pQCT images from wild-type (A) and transgenic (B) mice. Note the increased width of the marrow cavity and the concomitant reduction in cortical thickness in transgenic mice. Mineral densities are shown as different colors according to the standard mineral density gradients. (C–F) Cortical thickness (C), endosteal circumference (D), periosteal circumference (E), and bone mineral density (F) were measured for wild-type (white bars) and transgenic (black bars) mice. Error bars show means ± SEM (n = 4). *P < 0.05 and **P < 0.01 between wild-type and transgenic mice as determined by one-way ANOVA. Bars, 1 mm.
Figure 4.
Figure 4.
pQCT analysis. Diaphyses of femurs from wild-type and transgenic female mice at 3 mo of age were analyzed by pQCT. (A and B) pQCT images from wild-type (A) and transgenic (B) mice. Note the increased width of the marrow cavity and the concomitant reduction in cortical thickness in transgenic mice. Mineral densities are shown as different colors according to the standard mineral density gradients. (C–F) Cortical thickness (C), endosteal circumference (D), periosteal circumference (E), and bone mineral density (F) were measured for wild-type (white bars) and transgenic (black bars) mice. Error bars show means ± SEM (n = 4). *P < 0.05 and **P < 0.01 between wild-type and transgenic mice as determined by one-way ANOVA. Bars, 1 mm.
Figure 5.
Figure 5.
Decreased osteocytes, osteoclast invasion, collagen structure, and osteopontin deposition in cortical bone of transgenic mice. (A and B) TRAP staining of cortical bone in tibiae of wild-type (A) and transgenic (B) mice at 6 wk of age. Many TRAP-positive osteoclasts are observed in the cavities of cortical bone in transgenic mice (B). Note that osteocytes are diminished greatly in transgenic mice. (C and D) Polarized microscopy of cortical bone in tibias of wild-type (C) and transgenic (D) mice at 3 mo of age. Cortical bone in transgenic mice shows the woven pattern instead of the lamellar collagen deposition seen in wild-type mice. (E and F) Immunohistochemical analysis in cortical bone of wild-type (E) and transgenic (F) mice at 3 mo of age using antiosteopontin antibody. Osteopontin is deposited heavily in cortical bone of transgenic mice. Note that osteocytes are spaced regularly in wild-type bone, but a few osteocytic cells are spaced irregularly in transgenic bone. Bars, 100 μm.
Figure 6.
Figure 6.
Bone volume, matrix deposition, and cell parameters in Cbfa1 transgenic mice. Trabecular bone volume (BV/TV; bone volume over tissue volume) (A), osteoid thickness (O.Th) (B), osteoblast number (N.Ob) (C), osteoblast surface (Ob. S) (D), osteocyte number (N. Ot) (E), osteoclast number (N. Oc) (F), osteoclast surface (Oc. S) (G), and eroded surface (ES) (H) are compared between wild-type (white bars) and transgenic mice (black bars) at 3 and 6 wk of age. The analyses were done using proximal parts of tibiae at secondary spongiosa except osteocyte number, which was counted at cortical bone of diaphysis. Bars show means ± SEM (n = 4). *P < 0.05 and **P < 0.01 between wild-type and transgenic mice as determined by one-way ANOVA.
Figure 7.
Figure 7.
Bone formation in trabecular and cortical bone. To assess osteoblast function in mineralization, wild-type and transgenic mice were double labeled with calcein at 3 and 6 wk of age. (A and B) Fluorescent micrographs of the two labeled mineralization fronts in femurs of wild-type (A) and transgenic (B) mice at 3 wk of age. The distances between the double labeling in transgenic mice are much less than those of wild-type mice. Note that wild-type bone shows a clear double-labeling pattern, but transgenic bone shows waved single and double labeling with interruption. Cross sections of mid-diaphyses of femurs are shown. (C–E) Measurement of mineral apposition rate (MAR) (C), mineralizing surface (MS) (D), and bone forming rate (BFR) (E) in trabecular bone of wild-type (white bars) and transgenic (black bars) mice at 3 and 6 wk of age. Longitudinal sections from proximal parts of tibiae were used for the analysis. (F–H) Measurement of MAR (F), MS (G), and BFR (H) in periosteum and endosteum of wild-type (white bars) and transgenic (black bars) mice at 3 wk of age. Cross sections from mid-diaphyses of femurs were used for the analysis. Error bars show means ± SEM (n = 4). *P < 0.05 and **P < 0.01 between wild-type and transgenic mice as determined by one-way ANOVA. Scale bar, 100 μm.
Figure 7.
Figure 7.
Bone formation in trabecular and cortical bone. To assess osteoblast function in mineralization, wild-type and transgenic mice were double labeled with calcein at 3 and 6 wk of age. (A and B) Fluorescent micrographs of the two labeled mineralization fronts in femurs of wild-type (A) and transgenic (B) mice at 3 wk of age. The distances between the double labeling in transgenic mice are much less than those of wild-type mice. Note that wild-type bone shows a clear double-labeling pattern, but transgenic bone shows waved single and double labeling with interruption. Cross sections of mid-diaphyses of femurs are shown. (C–E) Measurement of mineral apposition rate (MAR) (C), mineralizing surface (MS) (D), and bone forming rate (BFR) (E) in trabecular bone of wild-type (white bars) and transgenic (black bars) mice at 3 and 6 wk of age. Longitudinal sections from proximal parts of tibiae were used for the analysis. (F–H) Measurement of MAR (F), MS (G), and BFR (H) in periosteum and endosteum of wild-type (white bars) and transgenic (black bars) mice at 3 wk of age. Cross sections from mid-diaphyses of femurs were used for the analysis. Error bars show means ± SEM (n = 4). *P < 0.05 and **P < 0.01 between wild-type and transgenic mice as determined by one-way ANOVA. Scale bar, 100 μm.
Figure 8.
Figure 8.
Maturational stage of osteoblasts in Cbfa1 transgenic mice. To determine the maturational stage of osteoblasts, sections from tibiae of wild-type (A, C, E, G, I, K, M, and O) and transgenic (B, D, F, H, J, L, N, and P) mice at birth (A–H) and 3 m of age (I–P) were examined by in situ hybridization using type I collagen (A, B, I, and J), osteopontin (C, D, K, and L), osteocalcin (E, F, M, and N), and Cbfa1 (G, H, O, and P) probes. The Cbfa1 probe detects the expression of endogenous Cbfa1 but not the transgene. Note that the pattern of Cbfa1 expression is similar to that of osteopontin expression but not osteocalcin expression in wild-type mice (C, E, G, K, M, and O). Osteopontin-positive cells are increased markedly, but osteocalcin highly positive cells are decreased markedly in transgenic mice (K, L, M, and N). Ct, cortical bone; Tb, trabecular bone. Bars: (A–H) 200 μm; (I–P) 100 μm.
Figure 9.
Figure 9.
Expression of genes related to bone matrix, mineralization, and osteoclastogenesis. RNA was extracted from long bones without fractures at 4 wk of age for Northern blot analysis and at 4 and 11 wk of age for RT-PCR. 20 μg of total RNA was loaded and hybridized with probes of pro-α1 (I) collagen (Col1(I)), osteocalcin (OC), MMP13, ALP, bone sialoprotein (BSP), and osteopontin (OP). Glyceraldehyde-3-phosphate-dehydrogenase was used as an internal control. Representative data from four independent samples are shown. The expression of pro-α 2(I) collagen (Col2(I)), RANKL, and OPG was examined by RT-PCR. HPRT was used as an internal control. Duplicate PCRs were performed in four independent samples. Representative data are shown. WT, wild-type mouse; Tg, transgenic mouse.
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