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(Received for publication, March 14, 1996, and in revised form, June 17, 1996)
From the Osteopontin is an arginine-glycine-aspartic
acid-containing cell adhesion protein, which is frequently expressed in
transformed cells and is thought to play a role in tumorigenesis. v-Src
is a transforming viral oncogene product encoded by Rous sarcoma virus
(RSV). We report that v-Src expression in HT1080 fibrosarcoma cells
significantly stimulates mouse osteopontin promoter activity. We also
determined the v-Src response element in the osteopontin promoter as an
inverted CCAAT box located at
Volume 271, Number 37,
Issue of September 13, 1996
pp. 22713-22717
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
and
Department of Bone Biology and Osteoporosis
Research, Merck Research Laboratories, West Point, Pennsylvania 19486 and § Department of Biological Sciences, Rutgers University,
Piscataway, New Jersey 08855-1059
53 to 49 from the transcription start
site. Mutations of the CCAAT box disrupts protein-DNA interaction and
diminishes both v-Src stimulation and basal promoter activity. A CCAAT
box-containing fragment corresponding to 155 to 122 of RSV long
terminal repeat competed with the 72 to 38 fragment of mouse
osteopontin promoter for specific protein binding in the gel shift
assay. A polyclonal antibody against CBF, a CCAAT box-binding factor,
supershifted in gel shift assays the protein-DNA complex formed by
nuclear extract of HT1080 with either the RSV CCAAT box fragment or
with the osteopontin 72 to 38 fragment. Moreover, both osteopontin
mRNA levels and enhancer activity of CCAAT box-containing 72 to
38 fragment were significantly elevated in
v-src-transformed NIH 3T3 cells relative to parental cells.
These findings suggest that the elevated osteopontin expression in
transformed cells could be due, at least in part, to v-Src stimulation
of the osteopontin promoter and that this effect is mediated by a
CBF-like factor.
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