The Osteoblast-specific Transcription Factor Cbfa1 Contributes to the Expression of Osteoprotegerin, a Potent Inhibitor of Osteoclast Differentiation and Function

Abstract

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblastsecreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE 2 )), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the ␤-galactosidase reporter gene (pOPG5.9␤gal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9␤gal reporter construct led to 39-, 7-, and 16-fold increases in ␤-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE 2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE 2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequencespecific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.Bone growth, development, and maintenance in mammals is a highly regulated process. The level of bone mass is dependent on the balance between bone formation and resorption. At the cellular level, this balance involves the coordinate regulation and interaction of the component cell types: bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are derived from mesenchymal stem cells, and their formation and function are under the control of an osteoblast-specific transcription factor known as core binding factor a1 (Cbfa1) or osteoblast specific factor 2 (Osf2) 1 (1-3). Cbfa1 has been shown to regulate the expression of genes that characterize the osteoblast phenotype, including osteocalcin, osteopontin, type I collagen, bone sialoprotein, and collagenase-3, by binding to DNA sequence elements called OSE 2 that are present in the control regions of these genes (1, 4 -6). Cbfa1 knock out animals completely lack bones because of a maturational arrest of osteoblasts and die soon after delivery (2, 3). Further, haploinsufficiency of the Cbfa1 gene product in mice and humans heterozygous for the Cbfa...

Periodontal Ligament Stem Cells Modulate Root Resorption of Human Primary Teeth via Runx2 Regulating RANKL/OPG System

“…Therefore, we believe that during root resorption, Runx2 was able to bind with the same site to increase the expression of RANKL in PDLSCs. Additionally, previous study demonstrated that 12 putative Cbfa1/Runx2-binding elements (osteoblast-specific element 2-OSE 2 ) were presented in human OPG promoter sequence and Runx2 could bind to the proximal OSE 2 to increase OPG expression level [36]. Further, when doxycycline was used to induce increased expression of Runx2 in ST2 cells (a bone marrow-derived mesenchymal pluripotent cell line), the differentiation of cocultured splenocytes into osteoclasts was enhanced along with the inhibition of OPG [37], which result was consistent with our data.…”

Overexpression of osteoprotegerin promotes preosteoblast differentiation to mature osteoblasts

“…22 Another study shows that Runx2 contributes to OPG activities. 21 The reason is probably that Runx2 regulates its target genes together with other factors in different osteoblast maturation stages and different cell lineages. Akt1 is an important transcription gene for controlling posttranscriptional levels of osteoblast differentiation 7 ; it interacts with Figure 3.…”

“…5 The effect of Runx2 function on osteoblast differentiation and OPG expression is controversial. [21][22][23] Some studies show that Runx2 induces RANKL expression and suppresses OPG expression, 24 and osteoblast differentiation was arrested in Runx2 overexpression cells. 22 Another study shows that Runx2 contributes to OPG activities.…”

Expression of OPG, RANKL, and RUNX2 in rabbit periodontium under orthodontic force

“…Orthodontic treatment caused an inflammatory reaction in periodontal tissues under the action of the external force, and orthodontic force was transferred to the periodontal membrane, which induced periodontal tissue remodeling (Thirunavukkarasu et al, 2000;Hendesi et al, 2015). This process required osteoclasts and cementoblasts.…”