Loss of osteoblast Runx3 produces severe congenital osteopenia

Abstract

Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3(fl/fl)/Col1α1-cre), but not chondrocyte-specific (Runx3(fl/fl)/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. Human RUNX3 resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization.

FIG 1

Phenotypic analysis of Runx3 KO mouse bones. (A) Short stature and delayed ossification of the distal limb and mandibular bones of Runx3 KO E18.5 embryos. Alizarin red S (bone) (purple)-alcian blue (cartilage) (blue) staining of forelimbs (left) and hindlimbs (middle) shows calcification of tarsal, metatarsal, and digital limb bones in a WT embryo (red arrows) and a lack of calcification in a Runx3 KO embryo. Similar deficits are seen by comparative μCT imaging of the palmar bones (bottom left). Alizarin red S-alcian blue staining shows a complete lack of ossification of the angular process of the mandibular bone (right, white arrow) in an E18.5 Runx3 KO embryo versus its apparent ossification in a WT littermate embryo. (B) The ossification pattern of a 7-day-old (D7) Runx3 KO skeleton is indistinguishable from that of a WT skeleton. Alizarin red S-alcian blue staining shows a WT-like ossification pattern of the Runx3 KO skeleton. Note the apparently smaller Runx3 KO skeleton. (Top left) Front limbs; (bottom left) hind limbs; (right) skulls. (C) Gene dosage effect of Runx3 on bone ossification in Runx2^+/− mice. Shown is alizarin red S-alcian blue staining of E18.5 and D0.5 palm bones of Runx2^+/− mice with either the Runx3^+/+, Runx3^+/−, or Runx3^−/− genotype. Note the relationship between the Runx3 dosage and the degree of ossification of the palmar and plantar bones at E18.5 and D0.5. (D) Short stature and kyphosis in adult Runx3 KO mice. Pictures of 5-month-old Runx3 KO and WT littermate mice show significantly smaller size and kyphosis in the Runx3 KO mouse. (E) Runx3 KO mice have short bones (left), thin cortices (top right), and underdeveloped trabecular cross sections (bottom right). Shown are representative 3D-reconstructed μCT images of femora of 23-day-old Runx3 KO and WT mice. Dashed lines mark the cortical (red) and trabecular (blue) regions of interest (see Materials and Methods). (F) Runx3 KO mice are severely osteopenic. Comparative μCT analyses of femora of 23-day-old Runx3 KO and WT mice were performed. (a to f) Whole-bone and bone cortex parameters (n = 6 Runx3 KO mice; n = 11 WT littermate mice); (g to i) cancellous bone parameters (n = 4 Runx3 KO mice; n = 6 WT littermate mice). Values are means and SD. Asterisks indicate a P value of <0.01.

FIG 2

Temporal onset of Runx3 KO osteopenia and Runx3 expression in developing bone OBLs. (A) Runx3 KO osteopenia is congenital. Comparative μCT analyses of humeri from D1 Runx3 KO and WT littermate mice were performed. Images showing congenital osteopenia of both cortical and cancellous regions in humeri of Runx3 KO versus WT mice (left) (the color bar indicates mineral content) and quantitative μCT structural analysis of these bones (right) are depicted. (B) Altered bone ultrastructure in E18.5 Runx3 KO mice. Shown are μCT images of 50-μm-thick reconstructed midhumeral cross sections. Runx3 KO mice (top) are compared to WT (middle) and Runx3^+/− (bottom) littermate control mice. (C) Delayed ossification in E14.5 Runx3 KO embryos. von Kossa-eosin staining of an E14.5 WT femoral section shows calcification in the bone collar (bc) and primary spongiosa (ps) regions (arrow), versus the complete lack of calcification in bone from an age-matched Runx3 KO mouse (top row) (magnifications, ×40 for images and ×100 for inset). By E16.5, the ossification patterns in WT and Runx3 KO femora were qualitatively indistinguishable (bottom row) (magnifications, ×40 for images and ×200 for insets). Bars, 500 μm (images), 125 μm (top inset), and 100 μm (bottom insets). (D) Runx3 expression in developing bone OBLs. RNA in situ hybridization of an E16.5 humeral section demonstrates intense Runx3 expression in primary spongiosa and bone collar OBLs. At this embryonic stage, Runx3 expression is also detected in prehypertrophic (ph), but not in hypertrophic (h), chondrocytes (c) in the developing growth plate. Runx3 signal intensity in OBLs is as high as that in prehypertrophic chondrocytes. Bar, 200 μm.

FIG 3

The Runx3 KO osteopenic phenotype is recapitulated in Runx3OBL but not in Runx3CHN mice. (A) Runx3OBL mice recapitulate the Runx3 KO skeletal phenotype. The decreased body weight and short stature of Runx3OBL mice are indicated. Body weights of D3 mice (left) (n = 6 mice/genotype) and representative gross appearances at D23 (right) are shown. (B) Alizarin red S-alcian blue staining of D21 Runx3OBL and WT mice showing similar ossification patterns with distinct size differences. (Left) Front limb; (middle) hind limb; (right) skull. (C to E) Runx3OBL mice exhibit an osteopenic phenotype. μCT analysis of D23 Runx3OBL (n = 2) and control littermate (WT and Runx3^fl/+/Col1a1-cre) (n = 5) female mice was performed. (C) Bone thickness; (D) cortex area; (E) BMC. Values are means and SD. Asterisks indicate statistical significance (P < 0.01). (F) Runx3CHN mice are seemingly indistinguishable from WT littermate mice. Shown are 3-month-old mice. (G) Similar ossification patterns in Runx3CHN and WT mice. Shown are whole skeleton (left) and hind limbs (right) of alizarin red S-alcian blue-stained D0.5 mice. (H to J) Comparable bone parameters for Runx3CHN and WT littermate mice. μCT analysis of D23 Runx3CHN mice (n = 2) and WT littermate controls (n = 5) was performed. (H) Bone thickness; (I) cortex area; (J) BMC. Values are means and SD, and asterisks indicate statistical significance (P < 0.01).

FIG 4

Comparative histomorphometry and OBL proliferation/apoptosis analyses. (A) Reduced bone volume in Runx3 KO mice. Shown are images of von Kossa-stained vertebral sections of 3-month-old Runx3 KO and WT littermate mice (left) (magnification, ×40; bar, 500 μm) and trabecular bone volume quantification (right) (n = 3 mice/genotype). Values are means and SD, and asterisks indicate statistical significance (P < 0.01). (B to F) Bone formation rates in 3-month-old Runx3 KO and WT littermate mice indicating a significant reduction in the number of active OBLs and a diminished bone formation rate in Runx3 KO mice. (B) Osteoblast surface (Ob.S). BS, bone surface. (C) Number of osteoblasts (N.Ob). B.Pm, bone perimeter. (D) Mineral apposition rate. (E) Mineralizing surface/bone surface. (F) Bone formation rate (n = 3 mice/genotype for panels B and C; n = 4 mice/genotype for panels D to F). Values are means and SD, and asterisks indicate statistical significance (P < 0.01). (G) Impaired OBL proliferation rate in Runx3OBL mice. Comparative BrdU incorporation assays using calvaria sections from 9-day-old Runx3OBL (n = 1) and WT (n = 4) littermate mice show significant reductions in proliferating OBLs in Runx3OBL mice. BrdU incorporation was determined for ≥6 calvaria sections/mouse. Values are means and SD, and asterisks indicate statistical significance (P < 0.01). (H) Comparable OBL apoptosis rates in Runx3OBL and WT littermate mice. OBL apoptosis was determined by a TUNEL assay using calvaria sections from 14-day-old mice (n = 2 mice/genotype; ≥6 calvaria sections per mouse). Values are means and SD.

FIG 5

Growth rate and mineral deposition analysis of cultured Runx3-null OBLs. (A) Reduced number of active OBLs in Runx3OBL BM stromal cultures. Total BM cells were cultured to confluence for 21 days (21d) and stained with ALP. (Left) Whole-plate and magnified (×200) images. Bar, 200 μm. (Middle) Osteoprogenitor cell numbers were determined by counting ALP-positive colonies (>10 cells) in 10 microscope fields at a ×100 magnification. (Right) The number of OBL lineage cells generated from these osteoprogenitor colonies was deduced by determining the cumulative area of ALP-positive colonies per whole-plate area. Data shown represent data from three independent experiments with similar findings. Values are means and SD, and asterisks indicate statistical significance (P < 0.01). (B) Impaired mineralization capacity of cultured Runx3 KO BM OBLs. Following plating, the mineral deposition capacity of Runx3 KO and WT BM-derived OBL cultures was determined at 7-day intervals. Shown are whole-plate images (von Kossa staining) (left) and digital quantification of mineralized nodules (black) at 14 and 21 days of culture (right). Data shown represent data from two independent experiments with similar findings. (C) Cultured BM OBLs from Runx3OBL mice display a markedly reduced mineral deposition capacity. Shown are whole-plate images of 28-day cultures (von Kossa staining) (left) and digital enumeration (right). Data shown represent data from two independent experiments with similar findings. (D) Calvaria-derived OBL cultures exhibit a diminished mineralization capacity. Mineral staining (von Kossa) of 21-day-old confluent OBL cultures isolated from calvariae of WT and Runx3OBL mice was performed. Shown are images of whole plates (left), a microscopic view of an enlarged plate area (middle) (magnification, ×100; bar, 400 μm), and whole-plate digital quantification of mineral deposition (right). Data shown represent data from two independent experiments with similar findings. Values are means and SD. Note that both Runx3OBL and WT culture images were taken under the same settings. The difference in clarity is due to the fact that in the WT culture plate, the brown mineralized (∼35% of the plate) nodules cast shadows that enhanced the contrast of neighboring cells. This effect does not exist in the KO plate, where mineralization was significantly reduced (∼9% of the plate).

FIG 6

OBL markers and gene expression analysis. (A) Comparative expression analysis of OBL markers. Femoral sections from D3 Runx3 KO and WT mice were used for ³⁵S-RISH (magnification, ×40; bar, 100 μm). Decreased bone areas where marker genes are expressed in Runx3 KO bone sections reflect the lower numbers of OBLs in these mice. (B) Scatter plot of differentially expressed genes in calvaria-derived OBLs from Runx3OBL versus WT mice. Shown are gene expression levels (log₂ scale) in Runx3OBL versus WT OBLs. Significantly increased and decreased expression levels of genes are indicated in red and green, respectively. Enlarged circles indicate Runx3-responsive genes that are known to participate in osteoblastogenesis. (C) Validation of several differentially expressed OBL genes. RT-qPCR analyses of RNA isolated from WT and Runx3OBL calvaria cultures showed gene expression trends similar to those observed by gene expression chip analysis.