TGFbeta inducible early gene-1 directly binds to, and represses, the OPG promoter in osteoblasts

Abstract

TGFbeta inducible early gene-1 (TIEG) is a member of the Krüppel-like family of transcription factors (KLF10) that plays an important role in TGFbeta mediated Smad signaling. In order to better understand the role of TIEG in bone, we generated TIEG knockout (KO) mice. Calvarial osteoblasts (OBs) isolated from these mice exhibit a reduced ability to support osteoclastogenesis in vitro. Gene expression studies revealed decreased receptor activator of NF-kappaB ligand (RANKL) and increased osteoprotegerin (OPG) expression in TIEG KO OBs, suggesting a potential role for TIEG in regulating the expression of these genes. Since OPG and RANKL are two important regulators of osteoclast (OC) differentiation, we sought to determine if TIEG directly regulates their expression. Luciferase constructs, containing fragments of either the mouse OPG promoter (-1486 to +133 bp) or the RANKL promoter (-2000 to +1 bp) were each cloned into the pGL3 basic reporter vector and transiently transfected into TIEG KO calvarial OBs with and without a TIEG expression vector. No significant changes in the activity of the RANKL promoter were detected in the presence of TIEG. However, OPG promoter activity was inhibited in the presence of TIEG protein suggesting that TIEG directly represses the expression of OPG in OBs. In order to determine the region of this promoter through which TIEG acts, sequential 5'-deletion constructs were generated. Transient transfection of these constructs revealed that the TIEG regulatory element(s) reside within a 200 bp region of the OPG promoter. Transient ChIP analyses, using a TIEG-specific antibody, revealed that TIEG binds to this region of the OPG promoter. Since we have previously shown that TIEG regulates target gene expression through Sp-1 sites, we examined this region of the OPG promoter for potential TIEG binding elements and identified four potential Sp-1 binding sites. Site-directed mutagenesis was used to determine if TIEG utilizes these Sp-1 elements to regulate the activity of the OPG promoter. The data demonstrate that two Sp-1 sites are likely to be involved in TIEG's repression of the OPG promoter. Taken together, these results confirm that TIEG directly binds to and inhibits OPG promoter activity in OBs, partially explaining the inability of TIEG KO OBs to fully support osteoclast differentiation.