Figure 1.
Schematic representation of the pGL3-TRAP-1B/C-OA expression plasmid.
The pGL3-TRAP-1B/C-OA expression plasmid was generated by cloning the full-length mouse OA cDNA into the Hind3/XbaI restriction site of the pGL3-basic vector. The TRAP-1B/C promoter was then cloned into the Kpn1/Hind3 restriction sites.
Figure 2.
Expression levels of glycosylated OA protein species (A &B) and relative OA mRNA levels (C) in primary marrow-derived osteoclasts and calvaria-derived osteoblasts derived from 4-week-old female OA-Tg mice or WT littermates.
A: Top panel shows a representative Western blot of the OA proteins level in osteoclasts from two OA transgenic (OA-Tg) mice or from two age- and sex-matched WT littermates. Bottom panel summarizes the quantitative analysis of the relative density of the two predominant glycosylated OA protein species, normalized against the density of the corresponding actin protein band (shown as mean ± SEM). B: Top panel shows the Western blot of the OA proteins level in primary osteoblasts of two OA-Tg mice and two WT littermates. Bottom panel summarizes the quantitative analysis of the relative density of the ∼80 kD glycosylated OA protein species (normalized against the density of the corresponding actin band). Results are shown as mean ± SEM. C: Relative OA mRNA levels in cultured osteoclasts (left bar) and osteoblasts (right bar) of 4 weeks old female OA-Tg mice compared to age-matched female WT mice. Results are shown as mean ± SEM (n = 4 for each).
Table 1.
Comparison of bone and pQCT parameters of 8 weeks old OA transgenic (Tg) mice with targeted OA overexpression in osteoclastic cells to those of 8 weeks old sex-matched WT littermates (mean±SEM).
Figure 3.
Age effect on the relative overexpression levels of OA mRNA in femurs of female OA-Tg mice.
Total RNA was isolated from femurs of female OA-Tg mice of age of 4-, 8-, and 15.3-week-old and corresponding WT littermates (3–5 mice per group) and cDNA was prepared as described in Methods. The relative level of OA mRNA, determined by real-time RT-PCR and normalized against respective β-actin mRNA level), is reported as fold of that in corresponding age-matched WT osteoclasts (indicated by the dashed line) and shown as mean ± SEM (n = 3–5).
Figure 4.
Age effects on relative differences in pQCT bone parameters between OA-Tg mice and corresponding WT littermates.
The femur length (A), total BMD (B), cortical bone area (C), cortical thickness (D), cortical BMD (E), and subcortical BMD (F) at midshaft of femur of female OA-Tg mice and corresponding WT littermates of 4, 8, and 15.3 weeks of age were each plotted against corresponding age of the animals. Statistical significance of age, genotype, and age×genotype interaction was determined by two-factor ANOVA.
Table 2.
Comparison of bone pQCT parameters of weanling young (4-week-old) female OA-Tg mice and mature adult (15.3-week-old) female OA-Tg mice with those of corresponding age-matched female WT littermates (mean ± SEM).
Figure 5.
Comparison of μ-CT bone parameters at the secondary spongiosa of 4-week-old female OA-Tg mice with those of 4-week-old female WT littermates.
Top panels show the three-dimensional reconstruction of bone structure by μ-CT at the secondary spongiosa of two representative OA-Tg mice (right) and two WT littermates (panel). Bottom summarizes and compares the various μ-CT bone parameters of a group of four OA-Tg mice with a group of four WT littermates.
Table 3.
Comparison of static histomorphometric trabecular bone parameters at the secondary spongiosa of 4-week-old female OA-Tg mice with age- and sex-matched WT littermates (mean ± SEM).*
Figure 6.
Targeted overexpression of OA in cells of osteoclastic lineage increased circulating levels of c-telopeptide of type I collagen.
Plasma c-telopeptide levels of 8-week-old male young adult OA-Tg mice (n = 17) and WT littermates (n = 12) were measured with a commercial ELISA assay, and results are shown as mean ± SEM.
Figure 7.
Effects of targeted overexpression of OA in osteoclastic cells on the size and number of nuclei of osteoclasts in vivo.
Top panel of Fig. 7 shows photomicrographs of the TRAP-expressing osteoclasts (counter-stained with hematoxylin) on the trabecular bone surface at secondary spongiosa of a WT mouse and an OA-Tg mouse. Bottom panels show the quantitative differences in osteoclast size per osteoclast (left), osteoclast surface per osteoclast (OC.PM/OC, middle), and number of nuclei per osteoclast (right) between six OA-Tg mice and four WT littermates. Results are shown as mean ± SEM.
Figure 8.
Effects of overexpression of OA on relative size of marrow-derived osteoclasts (A and B), and TRAP expression levels (C), size of resorption pits formed in vitro (D), expression levels of bone resorption genes (E), cellular Src tyrosine-527 phosphorylation level (F), and number of TRAP-expressing osteoclast-like cells formed in response to the RANKL and m-CSF treatment (G).
In A–F, marrow-derived osteoclasts were generated from treatment of unattached marrow cells of 12-weeks-old male adult OA-Tg mice and corresponding WT littermates with RANKL and m-CSF for 7 days. In A, to identify the size of osteoclasts, actin rings were stained with FITC-Palloidin and visualized under a fluorescent microscope. Results are shown in mean ± SEM (n = 3 each for each parameter). In G, marrow-derived osteoclasts were generated by treating unattached marrow cells of five 4-weeks-old female OA-Tg mice or four 4-weeks-old female WT littermates. Results are shown as mean ± SEM.
Figure 9.
Effects of siRNA-mediated OA suppression on average cell size (A), in vitro bone resorption activity (B), and expression of osteoclastic genes (C) in RAW264.7 cell-derived osteoclast-like cells.
The dosage of OA siRNAs (29 pM) used in this experiment suppressed OA expression in RAW264.7 cells by greater than 70% (data not shown). RAW264.7 cells were treated with OA siRNAs or control siRNA in the presence of RANKL for 5 days. A shows the relative size of the derived TRAP positive, multinucleated osteoclast-like cells. Top is a representative photomicrograph of the derived osteoclast-like cells, and bottom summarizes the relative size (in relative percentage of the control siRNA-treated cells). B shows the bone resorption activity of the derived osteoclast-like cells determined by an in vitro resorption pit formation assay; and C summarizes the effects of OA siRNA on the relative expression levels of MMP9, CALCR, and NFATc1 mRNA (determined by real-time RT-PCR and normalized by the respective expression level of β-actin). Results are shown as percentage of respective control siRNA-treated RAW264.7 cell-derived osteoclast-like cells and in mean ± SEM (n = 3 or 4 for each parameter). The dashed line represents the 100% of the control siRNA-treated controls.
Table 4.
Effects of targeted overexpression of OA overexpression in osteoclastic cells on histomorphometric bone formation parameters in 8-week-old male OA-Tg and also in 15.3-week-old female OA-Tg mice (mean±SEM).
Table 5.
Comparison of osteoblast parameters at secondary spongiosa* of 4-week-old female OA-Tg mice with 4-week-old female WT littermates (mean ± SEM).
Figure 10.
Effects of targeted overexpression of OA in osteoclastic cells on plasma levels of biomarkers of bone formation in vivo.
In A, plasma levels of osteocalcin of female OA-Tg mice and WT littermates of 8 or 15.3 weeks of age were measured with a commercial ELISA kit. Results are shown as mean ± SEM with the indicated the number of mice per group. In B, plasma levels of pro-collagen type I N-terminal peptide (PINP) of both male and female 15.3-week-old OA-Tg mice and corresponding age- and sex-matched WT littermates were measured with a commercial ELISA kit. Results are shown as mean ± SEM with the indicated number of mice per group.
Table 6.
Sequence of primers used in real-time RT-PCR.