Expression and regulation of Runx2/Cbfa1 and osteoblast phenotypic markersduring the growth and differentiation of human osteoblasts

The runt family transcription factor (AML-3/PEBP2 alpha A1/Cbfa1/RUNX2) pla ys a crucial role in formation of the mineralized skeleton during embryogen esis and regulates maturation of the osteoblast phenotype. Because steroid hormones and growth factors significantly influence growth and differentiat ion properties of osteoblasts, we addressed Cbfa1 as a target gene for regu lation by dexamethasone (Dex), 1,25(OH)D-3 (vitamin D3), 17 beta -estradiol , and transforming growth factor-beta1 (TGF-beta1). The representation of f unctional protein levels by Western blot analyses and gel mobility shift as says was examined during the growth and mineralization of several condition ally immortalized human osteoblast cell lines HOB 04-T8, 03-CE6, and 03-CE1 0, each representing different stages of maturation. In situ immunofluoresc ence demonstrates Cbfa1 is associated with nuclear matrix in punctate domai ns, some of which are transcriptionally active, colocalizing with phosphory lated RNA polymerase II. Although each of the cell lines exhibited differen t responses to the steroid hormones and to TGF-beta1, all cell lines showed a similar increase in Cbfa1 protein and DNA binding activity induced only by Dex. On the other hand, Cbfa1 mRNA levels were not altered by Dex treatm ent. This regulation of Cbfa1 by steroid hormones in human osteoblasts cont rasts to modifications in Cbfa1 expression in primary rat calvarial osteobl asts and the mouse MC3T3-E1 osteoblast cell line. Thus, these results revea l multiple levels of regulation of Cbfa1 expression and activity in osteobl asts. Moreover, the data suggest that in committed human osteoblasts, const itutive expression of Cbfa1 may be required to sustain the osteoblast pheno type. (C) 2001 Wiley-Liss, inc.