title: TIEG1/KLF10 modulates Runx2 expression and activity in osteoblasts.
abstract: Deletion of TIEG1/KLF10 in mice results in a gender specific osteopenic skeletal phenotype with significant defects in both cortical and trabecular bone, which are observed only in female animals. Calvarial osteoblasts isolated from TIEG1 knockout (KO) mice display reduced expression levels of multiple bone related genes, including Runx2, and exhibit significant delays in their mineralization rates relative to wildtype controls. These data suggest that TIEG1 plays an important role in regulating Runx2 expression in bone and that decreased Runx2 expression in TIEG1 KO mice is in part responsible for the observed osteopenic phenotype. In this manuscript, data is presented demonstrating that over-expression of TIEG1 results in increased expression of Runx2 while repression of TIEG1 results in suppression of Runx2. Transient transfection and chromatin immunoprecipitation assays reveal that TIEG1 directly binds to and activates the Runx2 promoter. The zinc finger containing domain of TIEG1 is necessary for this regulation supporting that activation occurs through direct DNA binding. A role for the ubiquitin/proteasome pathway in fine tuning the regulation of Runx2 expression by TIEG1 is also implicated in this study. Additionally, the regulation of Runx2 expression by cytokines such as TGFβ1 and BMP2 is shown to be inhibited in the absence of TIEG1. Co-immunoprecipitation and co-localization assays indicate that TIEG1 protein associates with Runx2 protein resulting in co-activation of Runx2 transcriptional activity. Lastly, Runx2 adenoviral infection of TIEG1 KO calvarial osteoblasts leads to increased expression of Runx2 and enhancement of their ability to differentiate and mineralize in culture. Taken together, these data implicate an important role for TIEG1 in regulating the expression and activity of Runx2 in osteoblasts and suggest that decreased expression of Runx2 in TIEG1 KO mice contributes to the observed osteopenic bone phenotype.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America. hawse.john@mayo.edu
authors.forename: John R
authors.initials: JR
authors.lastname: Hawse
authors.forename: Muzaffer
authors.initials: M
authors.lastname: Cicek
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Grygo
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Bruinsma
authors.forename: Nalini M
authors.initials: NM
authors.lastname: Rajamannan
authors.forename: Andre J
authors.initials: AJ
authors.lastname: van Wijnen
authors.forename: Jane B
authors.initials: JB
authors.lastname: Lian
authors.forename: Gary S
authors.initials: GS
authors.lastname: Stein
authors.forename: Merry Jo
authors.initials: MJ
authors.lastname: Oursler
authors.forename: Malayannan
authors.initials: M
authors.lastname: Subramaniam
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Spelsberg
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit / Q000502:physiology*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000502:physiology*; D051765:Early Growth Response Transcription Factors / Q000502:physiology*; D005260:Female; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D004247:DNA; D004268:DNA-Binding Proteins; D051765:Early Growth Response Transcription Factors; D051741:Kruppel-Like Transcription Factors; D014157:Transcription Factors; D053773:Transforming Growth Factor beta1; C098111:KLF10 protein, human; C493709:RUNX2 protein, human; C493716:Runx2 protein, mouse; C569958:KLF10 protein, mouse