Identification of a DNA Sequence Involved in 0steoblast-specific Gene Expression via Interaction with Helix-Loop-Helix (HLH)-type Transcription Factors

To elucidate regulatory mechanism(s) underlying differentiation of osteoblasts, we examined involvement of helix-loop-helix (HLH)-type transcription factors in osteoblast-specific expression of a phenotypic marker gene which encodes osteocalcin, a major noncollagenous bone matrix protein, exclusively expressed in osteoblasts. Overexpression of a dominant negative HLH protein, Id-1, decreased the activity of the 1.1-kb osteocalcin gene promoter cotransfected into rat osteoblastic osteosarcoma ROS17/2.8 cells. Analysis of deletion mutants revealed that a 264-bp fragment of osteocalcin promoter (-198 to +66) was sufficient for the Id-l-dependent suppression. Furthermore, the activity of the same promoter fragment (-198 to +66) was enhanced when antisense Id-1 expression vector was cotransfected. This osteocalcin gene promoter region contains two sites of an E-box motif, a consensus binding site for HLH proteins, which we refer to as OCE1 (CACATG, at -102) and OCE2 (CAGCTG, at -149), respectively. Mutagenesis in OCE1 but not OCE2 led to greater than 50% reduction in transcriptional activity of the osteocalcin gene promoter. Electrophoresis mobility shift assay indicated that factors in nuclear extracts prepared from ROS17/2.8 cells bound to the 30-bp oligonucleotide probe containing the E-box motif of OCE1. This binding was competed out by OCE1 oligonucleotide but neither by OCmE1 oligonucleotide in which E-box motif was mutated nor by OCE2. The OCEl-binding activity in the nuclear extracts of ROS17/2.8 cells was reduced by 70% when bacterially expressed Id-1 protein was added to the reaction mixture, suggesting the involvement of HLH proteins in the DNA/protein complex formation. In contrast to the osteoblast-like cells, OCEl-binding activity in the nuclear extracts of C3H10T1/2 fibroblasts was very low. However, when these fibroblasts were treated with recombinant human bone morphogenetie protein-2 which induced expression of osteocalcin as well as other phenotypic markers of osteoblasts, OCEl-binding activity was increased ,x,40-fold, indicating that OCE1 would be involved in the tissuespecific expression of the osteocalcin gene. These findings indicated for the first time that osteoblastspecific gene transcription is regulated via the interaction between certain E-box binding transcription factor(s) in osteoblasts and the OCE1 sequence in the promoter region of the osteocalcin gene. D VELOPMENT and differentiation of cells are under the control of various classes of transcription factors which have been identified through genetic and biochemical means. Some of these factors have been shown to play key roles during cell differentiation process. Extracellular signals including hormones, growth factors, cytokines, or extraceUular matrix components as well as their intracellular mediators regulate cell differentiation or expression of phenotypes by modulating the activities of transcription factors involved in expression of respective genes (for review Address all correspondence to Masaki Noda, M.D., Ph.D., Dept. of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10 Kanda-Surugadal 2-chome, Chiyoda-ku, Tokyo 101, Japan. see Stein and Lian, 1993). Osteoblasts are thought to be derived from so-called undifferentiated mesenchymal cells. Upon differentiation, osteoblasts express phenotype-related genes encoding proteins such as osteocalcin, osteopontin, alkaline phosphatase, type I collagen, parathyroid hormone receptor, and so on (for review see Rodan and Rodan, 1984). Among those, osteocalcin is considered to be the only protein expressed specifically in osteoblasts (for review see Gundberg et al., 1984). Osteocalcin (bone ~,-carboxyglutamic acid-containing protein) is the most abundant noncollagenous matrix protein in bone and is produced exclusively by relatively mature osteoblasts (for reviews see Gundberg et al., 1984; Hauschka, 1986). Serum osteocalcin level correlates well with histo© The Rockefeller University Press, 0021-9525/94/08/773/10 $2.00 The Journal of Cell Biology, Volume 126, Number 3, August 1994 773-782 773 on A ril 2, 2017 D ow nladed fom Published August 1, 1994