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. 2002 Sep;22(17):6222-33.
doi: 10.1128/MCB.22.17.6222-6233.2002.

High bone resorption in adult aging transgenic mice overexpressing cbfa1/runx2 in cells of the osteoblastic lineage

Valérie Geoffroy  1 , Michaela Kneissel, Brigitte Fournier, Alan Boyde, Patrick Matthias
Affiliations

High bone resorption in adult aging transgenic mice overexpressing cbfa1/runx2 in cells of the osteoblastic lineage

Valérie Geoffroy et al. Mol Cell Biol. 2002 Sep.

Abstract

The runt family transcription factor core-binding factor alpha1 (Cbfa1) is essential for bone formation during development. Surprisingly, transgenic mice overexpressing Cbfa1 under the control of the 2.3-kb collagen type I promoter developed severe osteopenia that increased progressively with age and presented multiple fractures. Analysis of skeletally mature transgenic mice showed that osteoblast maturation was affected and that specifically in cortical bone, bone resorption as well as bone formation was increased, inducing high bone turnover rates and a decreased degree of mineralization. To understand the origin of the increased bone resorption, we developed bone marrow stromal cell cultures and reciprocal coculture of primary osteoblasts and spleen cells from wild-type or transgenic mice. We showed that transgenic cells of the osteoblastic lineage induced an increased number of tartrate-resistant acid phosphatase-positive multinucleated cells, suggesting that primary osteoblasts as well as bone marrow stromal cells from transgenic mice have stronger osteoclastogenic properties than cells derived from wild-type animals. We investigated the candidate genes whose altered expression could trigger this increase in bone resorption, and we found that the expression of receptor activator of NF-kappaB ligand (RANKL) and collagenase 3, two factors involved in bone formation-resorption coupling, was markedly increased in transgenic cells. Our data thus suggest that overexpression of Cbfa1 in cells of the osteoblastic lineage does not necessarily induce a substantial increase in bone formation in the adult skeleton but has a positive effect on osteoclast differentiation in vitro and can also dramatically enhance bone resorption in vivo, possibly through increased RANKL expression.

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Figures

FIG. 1.
FIG. 1.
Generation of transgenic mice overexpressing Cbfa1/Runx2. (A) Quantification of the expression of the transgene in the hindlimbs, forelimbs, and calvaria. RNAs were extracted from tissues isolated from 4-month-old mice. The level of expression of the transgene was determined by RT-PCR, using HPRT as internal control. (B) RT-PCR analysis of the temporal expression of the transgene in transgenic mice. RNAs were extracted from long bones of mice at the indicated ages. In all cases, HPRT was used as internal control. E, embryonic day. (C) Northern blot analysis (left pair of panels) showing the levels of expression of the endogenous Cbfa1/Runx2 gene and the transgene in long bones from 4-month-old wild-type (WT) and transgenic (TG) mice. The quantification of the Cbfa1/Runx2 and the transgene expression is shown in the right pair of panels. The results were normalized to the expression level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as internal control. Open bars represent the expression of endogenous Cbfa1/Runx2 in wild-type (WT) and transgenic (TG) mice; black bars represent the expression of the transgene.
FIG. 2.
FIG. 2.
Radiological analysis and mineralization parameters of Cbfa1-overexpressing mice. (A) Skeletal preparation of 1-week-old wild-type (WT) or Cbfa1-overexpressing (TG) mice, followed by Inouye's staining (left panels) and X-ray analysis of 16-month-old wild-type and Cbfa1-overexpressing mice (right panels). At 1 week of age, Cbfa1-overexpressing mice exhibited normal distribution of bone and cartilage tissues, but at 16 months of age, Cbfa1-overexpressing mice exhibited smaller stature and dramatically lower cortical thickness and mineral density (white arrows), which induced multiple fractures in the appendicular (a) and central (b) skeleton and resulted in widened and/or misshapen bone structures (c). Arrows indicate different sites of fracture or bone structure alteration. (B) Total bone mineral density measured in proximal tibia from Cbfa1-overexpressing mice (TG) and their wild-type littermates (WT) at 2, 4, and 14 months of age. The phenotype did not improve with increasing age. (C) BSE images of tail vertebrae from 4-month-old Cbfa1-overexpressing mice (TG) (top middle and top right panels) and from a wild-type littermate (WT) (top left panel). The bone mineralization characteristics, as measured by quantitative BSE imaging, show that the bone mineralization profile (center panel) is shifted towards lower densities in Cbfa1-overexpressing mice (red line) compared to that of wild-type mice (black line) and that the average level of bone mineralization (bottom panel) is reduced in 4-month-old Cbfa1-overexpressing mice (black bar) compared to that of their wild-type littermates (open bar). Magnification of a vertebral fracture site in transgenic mice is presented in the top three panels. The fracture-healing process was normal in transgenic mice, as calcified cartilage was produced normally. Bars represent means ± SEM; statistically significant differences between wild-type and transgenic mice are represented by stars (P < 0.05 by Student 's t test [n = 5 for TG; n = 10 for WT]).
FIG. 3.
FIG. 3.
Histological analysis of Cbfa1-overexpressing mice. (A) Giemsa staining (bar, 50 μm) of the cortexes of tail vertebrae from a 4-month-old female Cbfa1-overexpressing mouse (TG) and a wild-type littermate (WT). The cortex of the Cbfa1-overexpressing mouse is dramatically thinner than that of the wild-type mouse. Numerous osteoblasts (arrowheads) were present on the cortex of the transgenic bone, especially at the subperiosteal location (top of the cortex). (B) TRAP staining (bar, 50 μm) of the cortexes in tail vertebrae from a 4-month-old female Cbfa1-overexpressing mouse (TG) and a wild-type littermate (WT). The cortex of the transgenic bone is dramatically excavated due to a high level of endocortical bone resorption activity. TRAP-positive osteoclasts are indicated by arrowheads. (C) Movat staining (bar, 50 μm) of longitudinal tibia sections of wild-type (WT) and Cbfa1-overexpressing (TG) mice. The cortex thickness and the number of osteocytes per unit area were markedly reduced in Cbfa1-overexpressing mice compared to their wild-type littermates. (D) Evaluation of osteocyte apoptosis by TUNEL immunostaining (bar, 10 μm) on decalcified vertebrae. More apoptotic cells (stained in brown) were observed in the transgenic vertebrae than in the WT bone.
FIG. 4.
FIG. 4.
Histomorphometric bone formation and resorption parameters in Cbfa1-overexpressing mice. Parameters of bone formation and bone resorption as well as cell surface areas and numbers were measured in the spongiosa and at the endocortex and the periosteum of tail vertebrae in 4-month-old female Cbfa1-overexpressing mice (black bars, TG) and their wild-type littermates (open bars, WT). Ocl.Pm, osteoclast perimeter; Obl.Pm, osteoblast perimeter; B.Pm, bone perimeter; Md.Pm, mineralized perimeter. Bars represent means ± SEM; statistically significant differences between wild-type and transgenic mice are represented by stars (P < 0.05 by Student 's t test [n = 5 for TG; n = 10 for WT]).
FIG. 5.
FIG. 5.
Gene expression in Cbfa1-overexpressing mice. Northern blot (A) or RT-PCR Southern blot (B) analysis of gene expression in long bones of 1- and 4-month-old Cbfa1-overexpressing mice (TG) and their wild-type littermates (WT). Expression of genes involved in bone formation (OPN, BSP, collagen alpha I type I [Coll (I)], Cbfa1/Runx2, OC, and ALP) and in bone resorption (MMP-13, TRAP, M-CSF, OPG, RANK, and RANKL) were analyzed in Cbfa1-overexpressing mice and in their wild-type littermates. RNAs were extracted from hindlimb long bones. GAPDH and HPRT were used as internal controls for Northern blot and RT-PCR Southern blot analysis, respectively.
FIG. 6.
FIG. 6.
In vitro analysis of bone marrow stromal cells from Cbfa1-overexpressing mice. (A) Bone marrow cells from wild-type (WT) or Cbfa1-overexpressing (TG) mice were cultured in the presence of dihydroxyvitamin D3 (10−8 M) and ascorbic acid (50 μM) to induce osteoclastogenesis. After 21 days in culture, cells were stained for TRAP activity and counterstained with methyl green to visualize the nuclei. Arrows and arrowheads represent mononucleated and multinucleated TRAP-positive cells, respectively. Bar, 50 μm. (B) RT-PCR analysis of the expression of the transgene in bone marrow cells after 3 or 21 days in culture. The results were normalized to the expression level of HPRT used as internal control. (C) Expression of gene markers of osteoblasts (Cbfa1, OC, and ALP) and of genes involved in osteoclastogenesis (MMP-13, OPG, and RANKL) was analyzed by RT-PCR Southern blotting in bone marrow stromal cells from wild-type (WT) and transgenic (TG) mice after 3 (open bars) or 21 days (black bars) in culture (pairs of panels on right). Values were normalized against HPRT expression and are presented as severalfold induction in the transgenic cells relative to that in the wild-type cells. (Left panels; results represent means ± SEM of two independent experiments).
FIG. 7.
FIG. 7.
In vitro analysis of primary osteoblasts from Cbfa1-overexpressing mice (TG). (A) Primary osteoblasts prepared from calvaria from 2- to 4-day-old mice and spleen cells from wild-type (WT) or Cbfa1-overexpressing (TG) mice were cocultured in the presence of dihydroxyvitamin D3 (10−8M) and ascorbic acid (50 μM) to induce osteoclastogenesis. After 8 days, cells were stained for TRAP activity. Arrows and arrowheads represent mononucleated and multinucleated TRAP-positive cells, respectively. Bar, 50 μm. (B) RT-PCR analysis of the expression of the transgene in primary osteoblasts isolated from calvaria after 3 or 10 days of culturing in presence of ascorbic acid (50 μM). The results were normalized to the expression level of HPRT used as internal control. (C) Analysis of the expression of osteoblastic marker genes (Cbfa1, OC, and ALP) and of genes involved in osteoclastogenesis (MMP-13, OPG, and RANKL) by RT-PCR Southern blotting with primary osteoblasts from wild-type (WT) and transgenic (TG) 2- to 4 day-old mice after 3 days (open bars) or 10 days (black bars) in culture (pairs of panels on right). Values were normalized against HPRT expression and are presented as fold induction in the transgenic cells relative to that in the wild-type cells (left panels; results represent means ± SEM of two independent experiments).
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