Smad2 overexpression enhances Smad4 gene expression and suppresses CBFA1 gene expression in osteoblastic osteosarcoma ROS17/2.8 cells and primary rat calvaria cells.
Mothers against decapentaplegic-related proteins (Smads) are essential intracellular components for the signal transduction of transforming growth factor-beta (TGF-beta) family members. Smad1 mediates bone morphogenetic protein (BMP) signals, whereas Smad2 functions downstream of TGF-beta. TGF-beta 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-beta treatment enhanced Smad4 expression in PRC cells, and this TGF-beta effect was blocked by the cotreatment with BMP, indicating that TGF-beta 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.
Li
J
J
Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10 Kanda-Surugadai 2-Chome, Chiyoda-ku, Tokyo 101, Japan.
Tsuji
K
K
Komori
T
T
Miyazono
K
K
Wrana
J L
JL
Ito
Y
Y
Nifuji
A
A
Noda
M
M
D000818:Animals; D002454:Cell Differentiation; D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005246:Feedback; D005786:Gene Expression Regulation; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000166:cytology*; D015675:Osteocalcin / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D012886:Skull / Q000166:cytology*; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism
D050659:Core Binding Factor Alpha 1 Subunit; D050660:Core Binding Factors; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D015675:Osteocalcin