Cbfa1 isoform overexpression upregulates osteocalcin gene expression in non-osteoblastic and pre-osteoblastic cells

The mouse Cbfa1 gene potentially encodes several proteins that differ in th eir N-terminal sequences, including an osteoblast-specific transcription fa ctor, Cbfa1/Osf2, a Cbfa1 isoform (CDfal/iso), and the originally described Cbfa1 gene product (Cbfa1/org). Uncertainty exists about the function of t hese potential isoforms of the Cbfa1 gene. To examine the transactivation p otential of different Cbfa1 gene products, we compared the ability of Cbfa1 /Osf2, Cbfa1/iso, and Cbfa1/org overexpression to activate an osteocalcin p romoter/reporter construct in NIH3T3 fibroblasts, C3H10T1/2 pluripotent cel ls and MC3T3-E1 pre-osteoblasts. These three cell lines were transiently co transfected with a 1.3-kb mouse osteocalcin promoter luciferase-fusion cons truct (p1.3OC-luc) and different amounts of expression vectors containing t he respective full-length Cbfa1 isoform cDNAs. Using transfection protocols with lower amounts of expression plasmid DNAs, we found that all three Cbf a1 isoforms stimulated osteocalcin promoter activity in each of the cell ty pes, consistent with the their ability to induce expression of an osteoblas t-specific gene both in non-osteoblast cells and in osteoblast cell lines. However, using transfection protocols with higher amounts of expression pla smids containing Cbfa1 cDNAs, we found that the Cbfa1/Osf2 and Cbfa1/org ha d less transactivating potential compared with Cbfa1/iso. Our studies sugge st that the 87-amino acid N-terminus of Cbfa1/Osf2 is not crucial for optim al transactivation, whereas the 19-amino acid N-terminal sequence of Cbfa1/ iso augments transcriptional activation only at high doses of the expressio n plasmid. The physiological significance of these in vitro findings remain to be determined. (C) 1999 Wiley-Liss, Inc.