Impaired osteoblastic differentiation, reduced bone formation, and severe osteoporosis in noggin-overexpressing mice

Abstract

We describe the effects of the overexpression of noggin, a bone morphogenetic protein (BMP) inhibitor, on osteoblast differentiation and bone formation. Cells of the osteoblast and chondrocyte lineages, as well as bone marrow macrophages, showed intense beta-gal histo- or cytostaining in adult noggin+/- mice that had a LacZ transgene inserted at the site of noggin deletion. Despite identical BMP levels, however, osteoblasts of 20-month-old C57BL/6J and 4-month-old senescence-accelerated mice (SAM-P6 mice) had noggin expression levels that were approximately fourfold higher than those of 4-month-old C57BL/6J and SAM-R1 (control) mice, respectively. U-33 preosteoblastic cells overexpressing the noggin gene showed defective maturation and, in parallel, a decreased expression of Runx-2, bone sialoprotein, osteocalcin, and RANK-L. Noggin did not inhibit the ligandless signaling and pro-differentiation action of the constitutively activated BMP receptor type 1A, ca-ALK-3. Transgenic mice overexpressing noggin in mature osteocalcin-positive osteoblasts showed dramatic decreases in bone mineral density and bone formation rates with histological evidence of decreased trabecular bone and CFU-osteoblast colonies at 4 and 8 months. Together, the results provide compelling evidence that noggin, expressed in mature osteoblasts, inhibits osteoblast differentiation and bone formation. Thus, the overproduction of noggin during biological aging may result in impaired osteoblast formation and function and hence, net bone loss.

Figure 1

Noggin expression assessed by β-gal staining in adult bone tissues. Spine (a, b, c, and d) and calvaria (e and f) from adult 4-month-old noggin^+/– mice (a, c–f) or noggin^+/+ mice (as a negative control) (b) were stained for β-gal to investigate localization of cells expressing noggin. d and f represent polarized images corresponding to c and e, respectively. In a, yellow and blue arrows indicate noggin-expressing hypertrophic chondrocytes and mesenchymal osteoblastic cells in the capsule around the vertebral disc, respectively. In c, blue and yellow arrows indicate noggin-expressing osteoblasts/osteocytes in cortical bone and osteoblasts in trabecular bone, respectively. In e, blue and yellow arrows indicate noggin-expressing osteoblasts of outer and inner calvaria, respectively.

Figure 2

Noggin expression assessed by β-gal staining in bone marrow cell cultures. Bone marrow cells from adult noggin^+/– mice were cultured for either 1 week (a–c) or 2 weeks (d–f) and stained for β-gal alone (blue, a and d) or β-gal with either ALP (red, b and e) or NSE (black in c and f) to identify cell types in the culture. Yellow arrowheads indicate cells that costain with β-gal and ALP or β-gal and NSE.

Figure 3

Noggin expression in bone marrow–derived macrophages. Bone marrow–derived macrophages (Mφ) isolated from bone marrow cell cultures in the presence of M-CSF examined for noggin and BMP-4 expression by real-time PCR (a) and Western blot (b and c). Expression levels in macrophages in a were described as a ratio to those in U-33 preosteoblasts. In b, noggin expression in macrophages and U-33 cells were detected by Western blotting. In c, macrophages were cultured in the presence or absence of 300 ng/ml BMP-2 for 3 days, and noggin expression was analyzed by Western blotting. *P < 0.05, significantly different from U-33 cells by ANOVA. Cont, control.

Figure 4

Time course of noggin expression and cell differentiation in calvarial osteoblasts. Calvarial osteoblasts from neonatal noggin^+/– mice were cultured for up to 6 days in the presence of various osteotropic factors. (a) Isolated osteoblasts were cultured for 3 and 6 days in the presence or absence of BMP-2. (b) Time course of β-gal–positive cell numbers in the absence or presence of BMP-2 and TGF-β1. (c) β-gal and ALP activities were measured after 3 days of culture. For these experiments, 300 ng/ml BMP-2, -4, and -6, 5 ng/ml TGF-β1, 200 ng/ml IGF-1, 10^–8 M 1-34 human parathyroid hormone (PTH), 300 ng/ml noggin, and 10^–8 M 1,25(OH)₂D₃ (1,25D₃) were applied. *P < 0.05, significantly different from 0 timepoint (b) or control group (c).

Figure 5

Effects of overexpression of noggin in U-33 cells. Overexpression of noggin in U-33 cells generated by a virus infection system (see Methods) was detected by Western blotting (a) and immunocytostaining (b) using an anti-noggin monoclonal antibody. Cells infected with empty viral vector (Vector) were used as control. Arrow indicates 30 kDa protein under reducing conditions (a). Expression of Runx-2, bone sialoprotein (BSP), and osteocalcin (c), osteoclast-supporting activity (d), and expression of RANK-L in the presence of 10^–8 M 1,25(OH)₂D₃ (e) were compared in the control and noggin-infected U-33 cells. The expression levels of Runx-2, BSP, osteocalcin, and RANK-L were measured by real-time PCR and expressed as a ratio to untreated control (Vector) cells. TRAP-positive osteoclast number and RANK-L expression are expressed as mean ± SEM. *P < 0.05, significantly different from untreated control cells.

Figure 6

Effects of BMP and ca-ALK-3 overexpression on osteoblast differentiation. (a) Concentration-dependent stimulation of ALP activity in mature OB-6 osteoblasts by BMP-2 and BMP-4, expressed as absolute values. (b) Concentration-dependent inhibition of ALP activity by noggin or PEG-noggin in the presence of a fixed dose of BMP-2 or BMP-4 (100 ng/ml), expressed as a percentage of the control value. (c) Anti-HA–immunostained U-33 cells documenting constitutively active BMP type 1A receptor (ca-ALK-3) expression; the left panel (Vector) is a negative control. (d) Effect of increasing concentrations (0–100 ng/ml for 3 days) of PEG-noggin in vector or ca-ALK-3–overexpressing U-33 cells as assessed by ALP activity (given as mean ± SEM), expressed as absolute values. *P < 0.05, significantly different from untreated control (Vector) cells; ^#P < 0.05, significantly different from ca-ALK-3 at 0 concentration, by ANOVA.

Figure 7

Phenotype of a 4-month-old noggin-overexpressing mouse. Expression of noggin mRNA detected by RT-PCR in liver (L), kidney (K), and bone (B), and by Western blotting in bone (a) in WT and noggin transgenic (TG) littermates; GAPDH was used as a control. (b) Radiology of the femur; (c) bone mineral density of the femur, tibia, spine (L4–L6), and total body; and (d) H&E staining of femur were performed to show differences between WT and noggin transgenic mice. Comparison of bone marrow cell cultures isolated from WT and noggin transgenic mice to evaluate ALP-positive CFU-F, CFU-OB (e), and TRAP-positive osteoclast formation (f). Results are expressed as mean ± SEM. *P < 0.05, significantly different from WT mice by ANOVA.

Figure 8

Phenotype of an 8-month-old noggin-overexpressing mouse. (a) Radiology of the femur; (b) bone mineral density of the femur, tibia, spine, and total body; and (c) H&E staining of femur were performed to show differences in WT and noggin transgenic mice. (d) Comparison of bone marrow cell cultures isolated from WT and noggin TG mice to evaluate ALP-positive CFU-F, CFU-OB, and (e) TRAP-positive osteoclast formation. (f) The percentage of doubly-labeled bone surface to total bone surface and (g) the rate of bone formation as determined by the distance between calcein labeling lines in long bone (femur and tibia), spine, and calvaria from 8-month-old WT or noggin-overexpressing TG mice. Calvarial sections stained with H&E were analyzed to determine marrow space as a percentage of total bone area (h). Results are expressed as mean ± SEM. Statistics by ANOVA between WT and noggin TG mice. *P < 0.05.

Figure 9

Noggin expression in SAM-P6 mice and aged mice. Cell lysates (100 μg protein) prepared from long bone (a) and calvaria (b and c) of SAM-R1 (R1) and SAM-P6 (P6) mice (a and b) or from young (4-month-old) and old (20-month-old) mice (c) were subjected to Western blot to detect noggin protein expression. Band intensities of noggin expression in the top panels, which represent samples from individual animals, were quantified for β-actin expression and expressed in the graphs as a ratio of noggin to β-actin. (d and e) Noggin and BMP-4 expression in bone marrow cell cultures of young and old mice (C57BL/6J) or SAM-R1 (R1) and SAM-P6 (P6) mice were analyzed by real-time RT-PCR after culturing with 1 mM ascorbic acid–2-phosphate for 4 weeks. Gene expression is indicated as a ratio to the young or R1 control mice. *P < 0.05, significantly different from SAM-R1 or young control mice.