Md. Benson et al., Cloning of a 2.5 kb murine bone sialoprotein promoter fragment and functional analysis of putative Osf2 binding sites, J BONE MIN, 14(3), 1999, pp. 396-405
Bone sialoprotein (BSP) is an extracellular matrix protein that is intimate
ly associated with the process of biomineralization. Osf2, a member of the
Cbf/runt family of transcription factors, is required for the development o
f osteoblasts in vivo and has been reported to stimulate the transcription
of BSP when overexpressed in mesenchymal cell lines. To investigate the rol
e of Osf2 in BSP expression, we cloned a 2.5 kb fragment of a 5' untranscri
bed sequence from the murine BSP gene and evaluated it for putative Osf2 bi
nding sites. This promoter, which was able to direct 5- to 10-fold higher l
evels of luciferase reporter expression in osteoblastic cells than in nonbo
ne 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 wi
thin a region having 75% overall sequence identity with the rat and human B
SP 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 oste
oblastic cells, and this protein was identified as Osf2. Despite this in vi
tro binding ability, we detected no significant enhancer activity in the -1
335 element when placed in front of a minimal osteocalcin promoter driving
a luciferase reporter gene in osteoblastic cells nor any loss in transcript
ional activity of a 5' promoter deletion which eliminated this element as c
ompared with the full-length 2.5 kb promoter. These results suggest that Os
f2 binding to the BSP promoter is not essential for its osteoblast-selectiv
e expression.