2 DISTINCT OSTEOBLAST-SPECIFIC CIS-ACTING ELEMENTS CONTROL EXPRESSIONOF A MOUSE OSTEOCALCIN GENE

Osteoblasts are cells of mesodermal origin that play a pivotal role du ring bone growth and mineralization. The mechanisms governing osteobla st-specific gene expression are still unknown. To understand these mec hanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA tra nsfection experiments in osteoblastic and nonosteoblastic cell lines a nd by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luc iferase chimeric gene showed that a region located between -147 and -3 4 contained most if not all of the regulatory elements required for os teoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bo und a factor present only in nuclear extracts of osteoblastic cell lin es and nonmineralizing primary osteoblasts. The B element, located bet ween bp -110 and -83, bound a ubiquitously expressed factor. The C ele ment, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and m ineralizing primary osteoblasts. When cloned upstream of a minimum ost eocalcin promoter or a heterologous promoter, multimers of the A eleme nt strongly increased the activities of these promoters in osteoblasti c cell lines at two different stages of differentiation but in no othe r cell line; we named this element osteocalcin-specific element 1 (OSE 1). Multimers of the C element increased the activities of these promo ters predominantly in a differentiated osteoblastic cell line; we name d this element OSE2. This study demonstrates that two distinct cis-act ing elements are responsible for osteoblast expression of mOG2 and pro vides for the first time a functional characterization of osteoblast-s pecific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.