Loss of Runx2 in committed osteoblasts impairs postnatal skeletogenesis

Abstract

The Runx2 transcription factor is critical for commitment to the osteoblast lineage. However, its role in committed osteoblasts and its functions during postnatal skeletogenesis remain unclear. We established a Runx2-floxed line with insertion of loxP sites around exon 8 of the Runx2 gene. The Runx2 protein lacking the region encoded by exon 8 is imported into the nucleus and binds target DNA but exhibits diminished transcriptional activity. We specifically deleted the Runx2 gene in committed osteoblasts using 2.3-kb col1a-Cre transgenic mice. Surprisingly, the homozygous Runx2 mutant mice were born alive. The Runx2 heterozygous and homozygous null were grossly indistinguishable from wild-type littermates at birth. Runx2 deficiency did not alter proliferative capacity of osteoblasts during embryonic development (E18). Chondrocyte differentiation and cartilage growth in mutants was similar to wild-type mice from birth to 3 months of age. Analysis of the embryonic skeleton revealed poor calcification in homozygous mutants, which was more evident in bones formed by intramembranous ossification. Runx2 mutants showed progressive retardation in postnatal growth and exhibited significantly low bone mass by 1 month of age. Decreased bone formation was associated with decreased gene expression of osteoblast markers and impaired collagen assembly in the extracellular matrix. Consequently, Runx2 mutant bones exhibited decreased stiffness and structural integrity. By 3 months of age, bone acquisition in mutant mice was roughly half that of wild-type littermates. In addition to impaired osteoblast function, mutant mice showed markedly decreased osteoclast number and postnatal bone resorption. Taken together, functional deficiency of Runx2 in osteoblasts does not result in failed embryonic skeletogenesis but disrupts postnatal bone formation.

Keywords: ADULT BONE SYNTHESIS; BONE REMODELING; OSTEOBLAST DIFFERENTIATION; OSTEOBLASTS; POSTNATAL SKELETOGENESIS; RUNX2.

Figure 1. Recombination of Runx2-floxed allele in osteoblasts generates a mutant protein that lacks transcriptional activity

(A) Schematic illustration of Runx2-floxed allele and Runx2 recombined allele (Runx2 ΔE8). Eight exons derived from P1 promoter are indicated; black region represents exons that encode Runt Homology Domain. Light grey boxes represent untranslated regions. Black arrows represent loxP sites. (B) Nuclear extracts were isolated from hindlimbs of 1-month old Runx2^+/+ and Runx2^ΔE8/ΔE8 mice. Blots were probed with Runx2 antibody, stripped, and re-probed for Lamin B antigen, used as an internal control. Full length Runx2 (WT) and mutant Δ369 proteins are indicated. (C) Rat osteoblastic (ROS17/2.8) cells were transfected with WT and Δ369 Runx2 expression plasmids. Cells were processed for in situ immunofluorescence and imaged at 60× magnification. Representative images of nuclei stained with Runx2 antibody and DAPI are shown. Inset shows respective phase images. (D) Schematic illustration of −0.2kb OC and −2.0kb SOST promoters. Gray lines indicate position of Runx binding motifs in each promoter. HEK293T cells were co-transfected with OC or SOST promoter luciferase plasmid and indicated Runx2 expression vectors. Luciferase activity was determined 24 hours later and pooled data from 3 independent experiments with 4 replicates each is shown in graph.

Figure 2. Runx2 function in committed osteoblasts is not essential for embryonic skeletogenesis

(A) Skeletons from Runx2^+/+, Runx2^+/ΔE8 and Runx2^ΔE8/ΔE8 newborn mice were double-stained with Alizarin red and Alcian blue. A representative picture of skeletons stained and imaged together is shown. (B) Hindlimbs were disjointed and skeletal elements were imaged with a Nikon dissecting microscope. The length of Alizarin red stained region between proximal and distal epiphyseal cartilages in each skeletal element was measured using NIS-Elements software. Graph represents pooled value from 4 independent litters. (C) Femur, lumbar spine and feet were dissected from WT and homozygous littermates at birth and imaged simultaneously. The degree of calcification was quantified by Alizarin red signal using NIS-Elements software. (D) Hindlimbs from newborn WT and homozygous littermates were sectioned laterally and processed together for Masson’s trichrome staining. Representative images of femurs captured at 10× and 40× (boxed region) magnification are shown. Collagen stained in blue showed a decreased signal in Runx2^ΔE8/ΔE8 mutants. (E) Pregnant mothers at E18 were injected with BrdU, sacrificed 3 hours later and processed for immunohistochemistry. BrdU and DAPI stained femurs from hypertrophic zone to mid-diaphyseal region are shown. BrdU positive osteoblasts were counted from 6 randomly selected 100µm² regions containing both cortical and trabecular bone. Pooled data from 3 independent litters with SD is shown. HC; hypertrophic chondrocytes, TB; trabecular bone, BM; bone marrow. Scale bars are 100 µm.

Figure 3. Intramembranous ossification is impaired in Runx2 deficient mice

(A) Dorsal view of Alizarin red/Alcian blue stained skulls from newborn littermates is shown. Dotted lines mark the borders of calcification in occipital bones and open fontanels at the sagittal plane. (B) Coronal sections from rostral part of newborn calvaria were stained with H&E. Frontal bones are shown at 10× magnification. Double arrows indicate thickness of the frontal bone in the boxed region. (C) Calvarial thickness was determined at 6 randomly selected areas along the frontal bone. Pooled data from 3 litters is shown in graph. Statistical significance was calculated by Student’s t test. Asterisks represent p < 0.05. Scale bars are 100 µm.

Figure 4. Runx2 deficiency in committed osteoblasts disrupts postnatal growth and structural integrity of bones

(A) Body weights of wild-type and mutant littermates of both genders were monitored weekly from birth to 3 months of age. Graph represents average weight of 4 pairs of male mice. (B) Femurs from 1-month old wild-type and homozygous littermates were scanned for 3D µ-CT imaging. Longitudinal section through center of femur is shown. Scale bar; 1.0mm. (C) Femur of 1-month old littermates were embedded in plastic, sectioned along the frontal plane and stained with H&E. Images were captured immediately beneath the growth plate with bright and polarized light to assess orientation of collagen in bone. Magnification 20×. Scale bars are 200 µm. (D) 3-point bend test was performed on femurs of 1-month old wild-type and homozygous littermates. The line graph shows data from 2 wild-type and 2 homozygous mice tested in the same group. Bar graphs show the average of 3 wild-type and 3 homozygous mice. Runx2 mutant mice exhibited a decrease in stiffness as well as reduced ability to withstand mechanical loading. Asterisks represent p < 0.05.

Figure 5. Runx2 deletion results in impaired osteoblast function and low postnatal bone mass

(A) Representative µ-CT images of 1-month old femurs taken immediately beneath the growth plate are shown in cross-section (top). The 3D reconstructions of cortical bone at the mid-diaphyis (bottom) and (B) trabecular bone beneath the growth plate are shown. Pooled data from 4 independent litters were utilized for analyses of indicated bone parameters for cortical bone and trabecular bone. BV; bone volume, TV; total volume, Tb.N; trabecular number, Tb.Th; trabecular thickness and Tb.Sp; trabecular space. Statistical significance was calculated by Student’s t test. Asterisks represent p < 0.05. Scale bar; 100µm. (C) Total RNA was isolated from the diaphyseal region of hindlimbs of 1 month old mice. The CT values from 6 replicates were normalized to β-actin and relative mRNA levels are shown for indicated genes. Nuclear extracts were obtained from 1-month old whole limbs for western blot analysis. Blots were probed with rabbit polyclonal Sp7 antibody. Lamin B is shown as loading control.

Figure 6. Runx2 function in osteoblasts is required for postnatal bone acquisition

(A) Undecalcified hindlimbs from 3 month old littermates were embedded in plastic, sectioned laterally and stained with Goldner’s trichrome. Representative images of the endosteal surfaces of cortical bone are shown at 40× magnification. (B) Histomorphometric analysis was performed on 3 independent stained sections and pooled data for bone parameters is shown in graphs. N.Ob/BS; number of osteoblasts per bone surface, Ob.S./BS; osteoblast surface per bone surface. (C) Male littermates at 3 months of age received 2 intraperitoneal injections of calcein 7 days apart. Undecalcified hindlimbs were embedded in plastic and sectioned laterally. Images show areas of double-labeled bone surfaces in WT and Runx2 mutant femurs. Representative phase and FITC fluorescent images taken at the same exposure time are shown. Boxed region of endosteal surfaces is shown at 40× magnification. (D) Dynamic histomorphometry was performed on double-calcein labeled bones from 3 independent litters. Pooled data from 3 independent WT and homozygous femurs show mineralizing parameters in graphs. MS/BS; mineralizing surface to bone surface, BFR; bone formation rate, MAR; mineral apposition rate. Scale bars are 200 µm.

Figure 7. Mice with osteoblast-specific deficiency of Runx2 exhibit low osteoclast activity

(A) Frontal sections of 1-month old WT and homozygous femurs were stained simultaneously for TRAP activity. Images of metaphyseal region captured with the same exposure time are shown at 4× and 10× (boxed region) magnifications. Scale bars are 200 µm. (B) Total RNA was isolated from the diaphyseal region of hindlimbs of 1-month old mice. The CT values from two independent WT and homozygous mice with 4 replicates each were normalized to β-actin. Relative mRNA levels are shown for RANKL and OPG. (C) Undecalcified hindlimbs from 3 month old littermates were embedded in plastic, sectioned laterally and stained with Goldner’s trichrome. Histomorphometric analyses of osteoclast and bone resorption parameters were measured from 3 independent stained sections and pooled data for bone parameters is shown in graphs. N.Oc/BS; number of osteoclasts per bone surface, Oc.S/BS; osteoclast surface per bone surface, ES/BS; erosion surface per bone surface, QS/BS; quiescent surface to bone surface. Asterisks represent p < 0.05.