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Department of Internal Medicine, Seoul National University College of Medicine, 28 Yungun-Dong, Chongno-Gu, Seoul 110-744, Republic of Korea

(Requests for offprints should be addressed to C S Shin; Email: csshin{at}snu.ac.kr)

(D H Kim is now at Department of Internal Medicine, Dankook University College of Medicine, Cheonan 330-714, Korea)

CCAAT/enhancer-binding proteins (C/EBPs) are involved in the regulation of cell proliferation, differentiation, and control of metabolic function. Although the roles of C/EBPs in osteoblasts are largely unknown, both C/EBPß and - have been shown to enhance rat osteocalcin promoter activity through the synergistic activation of Runx2 at the C/EBP element. Here we show that in the mouse, C/EBP increases the expression of osteocalcin whereas C/EBPß does not. This increased expression was found to occur at the transcriptional level, as demonstrated by the increased transcriptional activity from mouse osteocalcin II (OG2) promoter by C/EBP. Although we found three putative C/EBP sites in the –637/±34 region of the OG2 promoter, none of these sites showed binding activity with in vitro translated C/EBP proteins. Notably, we show that C/EBP physically interacts with Runx2 and that C/EBP overexpression increases binding between the Runx2–C/EBP complex and the OSE2 element, a critical osteoblast-specific cis-acting element in the OG2 promoter. Consistent with these DNA binding data, a mutation in OSE2 abrogated the stimulatory effect of C/EBP on this promoter activity. Finally, chromatin immunoprecipitation analysis in MC3T3-E1 cells showed in vivo occupancy of the OG2 promoter by Runx2 and C/EBP. In conclusion, C/EBP was found to regulate mouse osteocalcin OG2 promoter activity indirectly by interacting with Runx2 in the context of the OSE2 element and this subsequently resulted in the cooperative activation of the OG2 promoter.

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