TY - JOUR
T1 - Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1 stimulate osteoblast-specific osteocalcin gene expression
AU - Xiao, Guozhi
AU - Jiang, Di
AU - Ge, Chunxi
AU - Zhao, Zhuoran
AU - Lai, Yumei
AU - Boules, Heidi
AU - Phimphilai, Mattabhorn
AU - Yang, Xiangli
AU - Karsenty, Gerard
AU - Franceschi, Renny T.
PY - 2005/9/2
Y1 - 2005/9/2
N2 - The role of ATF4 (activating transcription factor 4) in osteoblast differentiation and bone formation was recently described using ATF4-deficient mice (Yang, X., Matsuda, K., Bialek, P., Jacquot, S., Masuoka, H. C., Schinke, T., Li, L., Brancorsini, S., Sassone-Corsi, P., Townes, T. M., Hanauer, A., and Karsenty, G. (2004) Cell 117, 387-398). However, the mechanisms of ATF4 in bone cells are still not clear. In this study, we determined the molecular mechanisms through which ATF4 activates the mouse osteocalcin (Ocn) gene 2 (mOG2) expression and mOG2 promoter activity. ATF4 increased the levels of Ocn mRNA and mOG2 promoter activity in Runx2-containing osteoblasts but not in non-osteoblastic cells that lack detectable Runx2 protein. However, ATF4 increased Ocn mRNA and mOG2 promoter activity in non-osteoblastic cells when Runx2 was co-expressed. Mutational analysis of the OSE1 (ATF4-binding site) and the two OSE2s (Runx2-binding sites) in the 657-bp mOG2 promoter demonstrated that ATF4 and Runx2 activate Ocn via cooperative interactions with these sites. Pull-down assays using nuclear extracts from osteoblasts or COS-7 cells overexpressing ATF4 and Runx2 showed that both factors are present in either anti-ATF4 and anti-Runx2 immunoprecipitates. In contrast, pull-down assays using purified glutathione S-transferase fusion proteins were unable to demonstrate a direct physical interaction between ATF4 and Runx2. Thus, accessory factors are likely involved in stabilizing interactions between these two molecules. Regions within Runx2 required for ATF4 complex formation and activation were identified. Deletion analysis showed that the leucine zipper domain of ATF4 is critical for Runx2 activation. This study is the first demonstration that cooperative interactions between ATF4 and Runx2/Cbfa1 stimulate osteoblast-specific Ocn expression and suggests that this regulation may represent a novel intramolecular mechanism regulating Runx2 activity and, thereby, osteoblast differentiation and bone formation.
AB - The role of ATF4 (activating transcription factor 4) in osteoblast differentiation and bone formation was recently described using ATF4-deficient mice (Yang, X., Matsuda, K., Bialek, P., Jacquot, S., Masuoka, H. C., Schinke, T., Li, L., Brancorsini, S., Sassone-Corsi, P., Townes, T. M., Hanauer, A., and Karsenty, G. (2004) Cell 117, 387-398). However, the mechanisms of ATF4 in bone cells are still not clear. In this study, we determined the molecular mechanisms through which ATF4 activates the mouse osteocalcin (Ocn) gene 2 (mOG2) expression and mOG2 promoter activity. ATF4 increased the levels of Ocn mRNA and mOG2 promoter activity in Runx2-containing osteoblasts but not in non-osteoblastic cells that lack detectable Runx2 protein. However, ATF4 increased Ocn mRNA and mOG2 promoter activity in non-osteoblastic cells when Runx2 was co-expressed. Mutational analysis of the OSE1 (ATF4-binding site) and the two OSE2s (Runx2-binding sites) in the 657-bp mOG2 promoter demonstrated that ATF4 and Runx2 activate Ocn via cooperative interactions with these sites. Pull-down assays using nuclear extracts from osteoblasts or COS-7 cells overexpressing ATF4 and Runx2 showed that both factors are present in either anti-ATF4 and anti-Runx2 immunoprecipitates. In contrast, pull-down assays using purified glutathione S-transferase fusion proteins were unable to demonstrate a direct physical interaction between ATF4 and Runx2. Thus, accessory factors are likely involved in stabilizing interactions between these two molecules. Regions within Runx2 required for ATF4 complex formation and activation were identified. Deletion analysis showed that the leucine zipper domain of ATF4 is critical for Runx2 activation. This study is the first demonstration that cooperative interactions between ATF4 and Runx2/Cbfa1 stimulate osteoblast-specific Ocn expression and suggests that this regulation may represent a novel intramolecular mechanism regulating Runx2 activity and, thereby, osteoblast differentiation and bone formation.
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U2 - 10.1074/jbc.M500750200
DO - 10.1074/jbc.M500750200
M3 - Article
C2 - 16000305
AN - SCOPUS:24744458827
SN - 0021-9258
VL - 280
SP - 30689
EP - 30696
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 35
ER -