Runt-related transcription factor 1 is required for murine osteoblast differentiation and bone formation

Jun Tang,Jing Xie,Wei Chen,Chenyi Tang,Jinjin Wu,Yiping Wang,Xue-Dong Zhou,Hou-De Zhou,Yi-Ping Li

doi:10.1074/jbc.ra119.007896

Abstract

Despite years of research investigating osteoblast differentiation, the mechanisms by which transcription factors regulate osteoblast maturation, bone formation, and bone homeostasis is still unclear. It has been reported that runt-related transcription factor 1 (Runx1) is expressed in osteoblast progenitors, pre-osteoblasts, and mature osteoblasts; yet, surprisingly, the exact function of RUNX1 in osteoblast maturation and bone formation remains unknown. Here, we generated and characterized a pre-osteoblast and differentiating chondrocyte-specific Runx1 conditional knockout mouse model to study RUNX1's function in bone formation. Runx1 ablation in osteoblast precursors and differentiating chondrocytes via osterix-Cre (Osx-Cre) resulted in an osteoporotic phenotype and decreased bone density in the long bones and skulls of Runx1f/fOsx-Cre mice compared with Runx1f/f and Osx-Cre mice. RUNX1 deficiency reduced the expression of SRY-box transcription factor 9 (SOX9), Indian hedgehog signaling molecule (IHH), Patched (PTC), and cyclin D1 in the growth plate, and also reduced the expression of osteocalcin (OCN), OSX, activating transcription factor 4 (ATF4), and RUNX2 in osteoblasts. ChIP assays and promoter activity mapping revealed that RUNX1 directly associates with the Runx2 gene promoter and up-regulates Runx2 expression. Furthermore, the ChIP data also showed that RUNX1 associates with the Ocn promoter. In conclusion, RUNX1 up-regulates the expression of Runx2 and multiple bone-specific genes, and plays an indispensable role in bone formation and homeostasis in both trabecular and cortical bone. We propose that stimulating Runx1 activity may be useful in therapeutic approaches for managing some bone diseases such as osteoporosis.

Highlights

Bone marrow mesenchymal stem cells are multipotent progenitors that give rise to osteoblasts, chondrocytes, and adipocytes upon specific stimulation for cell differentiation
We found that Runx1 deletion in osteoblast precursors and differentiating chondrocytes leads to a severe osteoporotic phenotype with a 50% reduction in both total bone volume and cortical bone, and a 40% reduction in trabecular bone
These results demonstrated that Runx1 ablation in osteoblasts and chondrocyte precursors results in an osteoporotic phenotype characterized by short stature and impaired bone mineralization

Summary

Introduction

Bone marrow mesenchymal stem cells are multipotent progenitors that give rise to osteoblasts, chondrocytes, and adipocytes upon specific stimulation for cell differentiation. Upon staining to detect proliferating cell nuclear antigen (PCNA), we found that the expression was significantly decreased in both the proliferative and hypertrophic zones of Runx1f/fOsx-Cre mice (Fig. 4, E and F).

Results

Conclusion