Cloning of a 2.5 kb Murine Bone Sialoprotein Promoter Fragment and Functional Analysis of Putative Osf2 Binding Sites

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Abstract
Bone sialoprotein (BSP) is an extracellular matrix protein that is intimately associated with the process of biomineralization. Osf2, a member of the Cbf/runt family of transcription factors, is required for the development of osteoblasts in vivo and has been reported to stimulate the transcription of BSP when overexpressed in mesenchymal cell lines. To investigate the role of Osf2 in BSP expression, we cloned a 2.5 kb fragment of a 5' untranscribed sequence from the murine BSP gene and evaluated it for putative Osf2 binding sites. This promoter, which was able to direct 5- to 10-fold higher levels of luciferase reporter expression in osteoblastic cells than in nonbone cell lines, contains two consensus core binding sites for members of the Cbf/runt family. One, at -61 relative to the start of transcription, is within a region having 75% overall sequence identity with the rat and human BSP promoters. The other is located at -1335, outside this highly conserved region. Neither site is completely conserved in the rat or human sequences. Only the -1335 site was able to bind a protein in nuclear extracts of osteoblastic cells, and this protein was identified as Osf2. Despite this in vitro binding ability, we detected no significant enhancer activity in the -1335 element when placed in front of a minimal osteocalcin promoter driving a luciferase reporter gene in osteoblastic cells nor any loss in transcriptional activity of a 5' promoter deletion which eliminated this element as compared with the full-length 2.5 kb promoter. These results suggest that Osf2 binding to the BSP promoter is not essential for its osteoblast-selective expression.