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J Biol Chem, Vol. 273, Issue 47, 31009-31015, November 20, 1998
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From the Mothers against
decapentaplegic-related proteins (Smads) are essential
intracellular components for the signal transduction of transforming
growth factor- Department of Molecular Pharmacology,
Medical Research Institute, Tokyo Medical and Dental University,
3-10 Kanda-Surugadai 2-Chome, Chiyoda-ku, Tokyo 101, Japan, the
§ Department of Medicine III, Osaka University Medical
School, 2-2 Yamada-oka Suita, Osaka 565, Japan, the ¶ Department
of Biochemistry, Cancer Institute, 1-37-1 Kamiikebukuro, Toshima-ku,
Tokyo 170, Japan, the Program in Developmental Biology, Division
of Gastroenterology, Hospital for Sick Children, Toronto, Ontario M5G
1X8, Canada, and the ** Laboratory of Cell Regulation, Department of
Viral Oncology, Institute for Virus Research, Kyoto University,
Sakyo-ku, Kyoto 606, Japan
(TGF-) family members. Smad1 mediates bone
morphogenetic protein (BMP) signals, whereas Smad2 functions downstream
of TGF-. TGF- is expressed in osteoblastic cells and acts as an
autocrine and/or paracrine factor in regulation of osteoblastic
functions. In this study, we examined the levels and functions of Smad2
in osteoblastic cells. Smad2 mRNA expression was hardly detectable
by Northern blot analysis in an osteoblast-like cell line, ROS17/2.8,
as well as in primary rat calvaria (PRC) cells. Overexpression of Smad2
gene enhanced endogenous Smad4 gene expression in both ROS17/2.8 and
PRC cells, while Smad3 levels were not altered. Smad2 overexpression
suppressed osteocalcin mRNA expression in ROS17/2.8 cells.
Furthermore, Smad2 overexpression also suppressed transcriptional
activity of the 1-kilobase pair osteocalcin gene promoter, which was
linked to chloramphenicol acetyltransferase reporter gene in both ROS
and PRC cells. Since core binding factor A1 (CBFA1) is involved in
osteocalcin gene expression, we further examined CBFA1 expression in
the Smad2-overexpressing ROS17/2.8 and PRC cells. The levels of CBFA1
mRNA were suppressed by the overexpression of Smad2 by about 50%
in both ROS17/2.8 and PRC cells. TGF- treatment enhanced Smad4
expression in PRC cells, and this TGF- effect was blocked by the
cotreatment with BMP, indicating that TGF- signaling pathway is
interfered by BMP. These data indicate that Smad2 regulates Smad4
specifically and that CBFA1 gene is one of the downstream targets of Smad2.
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