title: Metformin induces osteoblast differentiation via orphan nuclear receptor SHP-mediated transactivation of Runx2.
abstract: Metformin is an oral anti-diabetic drug of the biguanide class that is commonly used to treat type 2 diabetes mellitus. This study examined the molecular mechanism for the action of metformin on osteoblast differentiation. Metformin-induced mRNA expression of the osteogenic genes and small heterodimer partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR. Metformin increased significantly the expression of the key osteogenic genes, such as alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP) as well as SHP. Transient transfection assays were performed in MC3T3E1 cells to confirm the effects of metformin on SHP, OC and Runx2 promoter activities. Metformin increased the transcription of the SHP and OC genes, and the metformin effect was inhibited by dominant negative form of AMPK (DN-AMPK) or compound C (an inhibitor of AMPK). The adenoviral overexpression of SHP increased significantly the level of ALP staining and OC production. However, metformin did not have any significant effect on osteogenic gene expression, ALP staining and activity, and OC production in SHP null (SHP-/-) primary calvarial cells. Moreover, upstream stimulatory factor-1 (USF-1) specifically mediated metformin-induced SHP gene expression. In addition, metformin-induced AMPK activation increased the level of Runx2 mRNA and protein. However, USF-1 and SHP were not involved in metformin-induced Runx2 expression. Transient transfection and chromatin immunoprecipitation assays confirmed that metformin-induced SHP interacts physically and forms a complex with Runx2 on the osteocalcin gene promoter in MC3T3E1 cells. These results suggest that metformin may stimulate osteoblast differentiation through the transactivation of Runx2 via AMPK/USF-1/SHP regulatory cascade in mouse calvaria-derived cells.
authors.affiliation: Dental Science Research Institute and BK21, School of Dentistry, Chonnam National University, Gwangju 500-757, Republic of Korea. jangwg@chonnam.ac.kr
authors.forename: Won Gu
authors.initials: WG
authors.lastname: Jang
authors.forename: Eun Jung
authors.initials: EJ
authors.lastname: Kim
authors.forename: In-Ho
authors.initials: IH
authors.lastname: Bae
authors.forename: Kkot-Nim
authors.initials: KN
authors.lastname: Lee
authors.forename: Yong Deuk
authors.initials: YD
authors.lastname: Kim
authors.forename: Don-Kyu
authors.initials: DK
authors.lastname: Kim
authors.forename: Sun-Hun
authors.initials: SH
authors.lastname: Kim
authors.forename: Chul-Ho
authors.initials: CH
authors.lastname: Lee
authors.forename: Renny T
authors.initials: RT
authors.lastname: Franceschi
authors.forename: Hueng-Sik
authors.initials: HS
authors.lastname: Choi
authors.forename: Jeong-Tae
authors.initials: JT
authors.lastname: Koh
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D047369:Chromatin Immunoprecipitation; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D007004:Hypoglycemic Agents; C493716:Runx2 protein, mouse; D008687:Metformin