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. 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 2.3kb 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.

Loss of Osteoblast Runx3 Produces Severe Congenital Osteopenia

“…Furthermore, the specific ablation of Runx3 in chondrocytes (Runx3CHN mice), the main drivers of longitudinal bone growth (34), did not produce a notable defect in bone development or structure. This finding could be explained by the dominance of Runx2 in chondrocyte maturation (33), a notion that corresponds with perinatal lethality in chondrocytespecific Runx2-deficient mice (14,15).…”

“…Thus, Runx2-null mice are completely devoid of OBLs and bones (9)(10)(11), and transgenic expression of Runx2 (12) or its two isoforms, Runx2-I and Runx2-II (13), in OBLs results in severe osteopenia and bone fragility due to the inhibition of OBL maturation. Interestingly, recent findings suggest that Runx2 is not required for precommitted OBL lineage cell generation, even though it is mandatory for progenitor cell commitment to the osteogenic lineage (14,15). This indicates that as-yet-unidentified participants are involved in osteoblastogenesis proper.…”

Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation

“…21 AIC was also completely blocked likely due to the inhibition of oxidative stress-dependent osteogenic differentiation and osteoblast-osteoclast crosstalk. 21 These findings are likely explained by the truncated Runx2 protein acting via a dominant negative effect 42,45 on the Runt-related family members in addition to Runx2, especially Runx1 and Runx3 that share the same highly conserved Runt DNA binding domain and are involved in regulating monocyte/macrophage biology. [46][47][48] Along these lines, recent studies have shown transdifferentiation of SMCs into a lipid-loaded, foam cell-like state during atherosclerosis.…”

Dwarfism in homozygous Agc1CreERT mice is associated with decreased expression of aggrecan

“…Mice were deskinned, eviscerated, and muscles partially removed for alizarin red/alcian blue double staining. Cartilage and bone tissue staining was performed essentially as described previously (Adhami et al, 2015). Stained skeletons were cleared of soft tissues by gentle shaking in 1% KOH for 2-weeks for 1-month old and for 4-weeks for 3-month old animals.…”