The Role of AML! In Osteoclastogenesis and Osteoclast Gene Expression

The overall goal of this study is to understand the mechanisms underlying the transcription factors that regulate osteoclast lineage commitment, differentiation, and function.  RUNX1 also plays a key role in early embryonic development, angiogenesis, and skeletal development. This proposal is highly significant since elucidating these mechanisms has potential to define new therapeutic targets for bone disorders that involve osteoclast generation and function and to provide new insight to genetic diseases of human development. Despite the recent insights gained from the effects of targeted deletion of the c-fos, PU.1, NF-kB, and NFATc1 transcription factor genes, the mechanism underlying transcription factors that specify osteoclast (OC) lineage commitment, differentiation, and function remains unclear.  The expression of these transcription factors in various cell types suggests that they are unlikely to be the switch that dominates the osteoclast differentiation process. Further study is needed to determine the cascade of transcription factors that control OC differentiation. It is also necessary to clarify why M-CSF alone induces precursors to differentiate into macrophages, while both M-CSF and RANKL induce precursors to differentiate into osteoclasts. As a step towards understanding the factors which modulate osteoclast gene (e.g., cathepsin K) expression and osteoclast differentiation, we are characterizing the cathepsin K critical cis-regulatory elements (CCREs). We found a RUNX1 (alias AML1) binding site as a CCRE and confirmed RUNX1 as its trans-regulatory factor, or as the CCRE binding protein (CCREBP). It was demonstrated that RUNX1 is highly induced by RANKL and M-CSF.  Our Preliminary Studies showed that RUNX1 silencing in vitro blocked osteoclast differentiation. Furthermore, conditional knockout of RUNX1 inhibited osteoclast differentiation, but it did not inhibit macrophage differentiation. However, conventional knockout technology results in early embryonic lethality from constitutive Runx1 excision. Through in vitro studies of E12 Runx1-/- embryonic liver cells, we found that the Runx1 gene is indispensable for monocyte/macrophage precursor commitment and development, as well as for osteoclast lineage commitment and differentiation, during embryonic development (specifically during organogenesis). We found that RUNX1 may regulate the expressions of both osteoclast genes (e.g., cathepsin K) and osteoclast transcription factors, such as PU.1 and NFATc1. We proposed a RUNX1 working model in osteoclast cell lineage commitment, differentiation, and function for further RUNX1 function characterization. Based on our data, we hypothesize that RUNX1 regulates osteoclast lineage commitment, differentiation, and function in a dose dependent manner through interactions with other osteoclast transcription factors and regulation of osteoclast gene expression.