Metformin induces osteoblast differentiation via orphan nuclear receptor SHP-mediated transactivation of Runx2☆
Research Highlights
► Metformin increases AMPK activation in osteoblast cells. ► SHP expression is induced by AMPK-activated USF-1 expression in osteoblast cells. ► AMPK increases Runx2 expression. ► SHP enhances Runx2 transactivity in osteoblast cells.
Introduction
Bone formation is a tightly regulated process of lineage-specific differentiation events, and bone homeostasis is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts [1]. Osteoblasts derived from pluripotent mesenchymal stem cells with the capacity to also differentiate into myocytes, adipocytes, and chondrocytes possess the necessary components to form bone matrix and allow subsequent mineralization [2]. Osteoblast differentiation is controlled by a range of hormones and cytokines, such as bone morphogenetic proteins (BMPs), and multiple transcription factors, such as Runx2, Osx, Dlx5, Msx2, AP1(c-Fos/c-Jun), and ATF4 [3], [4]. Recent studies demonstrated a link between the glucose metabolism and bone homeostasis [5], [6]. For example, osteocalcin (OC) is the most abundant noncollagenous protein of the extracellular matrix of bone and is synthesized only by osteoblasts. OC knockout mice show increased accumulation of visceral fat associated with insulin resistance and glucose intolerance in early life [7].
Metformin is an anti-hyperglycemic drug used extensively to treat type 2 diabetes [8], [9], and can reduce the level of hepatic glucose production and improve the peripheral insulin sensitivity and glucose uptake via the activation of AMP-activated protein kinase (AMPK) in the liver and peripheral tissue [9], [10], [11]. AMPK is a serine/threonine kinase that consists of a catalytic α subunit and two regulatory β and γ subunits [12]. AMPK senses the AMP/ATP ratio within the cell, and once activated switches on the catabolic pathways and switches off the anabolic pathway. Tumor suppressor kinase (LKB1), calmodulin kinase kinase (CaMKK), and transforming growth factor-beta-activated kinase (TAK1) are known as AMPK kinases [12], [13], [14]. AMPK functions as an intracellular energy sensor and has been implicated in the modulation of the glucose and fatty acid metabolism [15], [16]. Recently, AMPK was reported to be able to regulate osteoblast differentiation [17], [18], [19]. For example, metformin and 5-aminoimdazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) induced the osteoblastic differentiation and matrix mineralization in MC3T3E1 cells via activation of the AMPK signaling pathway, and also adiponectin stimulated the proliferation, differentiation and mineralization of osteoblasts through the adiponectin receptor type I (AdipoRI) and AMPK signaling pathways in autocrine and/or paracrine manner [18], [20]. However, these results are in contrast to recent findings that osteoblast differentiation is functionally associated with decreased AMPK activity [21].
The small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor lacking a conventional DNA-binding domain with a putative ligand binding domain [22]. SHP regulates the transcriptional activity of a range of transcription factors involved in regulating several metabolic pathways, including bile acid homeostasis and glucose metabolism [23], [24]. Recently, it was demonstrated that metformin or hepatocyte growth factor (HGF) inhibits hepatic gluconeogenesis through the AMPK-dependent regulation of SHP [25], [26]. Upstream stimulatory factor-1 (USF-1) specifically mediated the HGF effect on SHP gene expression. USF-1 functions as a transcription factor regulating several genes involved in glucose and lipid metabolism [27], [28], [29]. In addition, BMP2-induced SHP gene expression stimulates osteoblast differentiation via an interaction with Runx2 [30]. However, it is still unclear if metformin or AMPK regulates SHP expression in osteoblast cells.
Runx2, also known as Cbfa1, is a multifunctional factor that controls skeletal development by regulating ALP, OC and BSP gene expression [31], [32]. In addition, Runx2 increases alkaline phosphatase (ALP) activity and mineralization in mesenchymal cells and osteoblast cells in vitro [33], [34], [35]. Runx2 interacts with a range of transcription factors [36], and recruits both the co-activator (p300, HES-1 and YAP) and co-repressor (TLE, mSin3a, and HDACs) to form a complex on its target gene promoter during osteoblast differentiation [37], [38], [39], [40]. Recently, reduced Runx2 expression and the down-regulation of Runx2 target genes were observed in insulin-deficient hyperglycemic diabetic mice [41], [42]. Interestingly, delayed wound healing was observed in Runx2 heterozygous knockout mice [43], [44]. More recent reports demonstrated that Runx2 is suppressed under hyperglycemic conditions through the aldose reductase (AR) polyol pathway [45]. These findings indicate that Runx2 also may be a link between the bone metabolism and glucose metabolism.
This study examined the effect of metformin on osteoblast differentiation using MC3T3E1 and primary mouse calvarial cells. Metformin was found to regulate SHP and Runx2 expression via AMPK activation, and stimulated the transcriptional activity of the OC gene through an interaction between SHP and Runx2.
Section snippets
Reagents and antibodies
Metformin (1,1-dimethylbiguanide hydrochloride), 5-aminoimdazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), ascorbic acid 2-phosphate (AA), β-glycerophosphate (β-GP), and compound C were purchased from Sigma Aldrich Co. (St. Louis, MO). Recombinant human BMP2 was obtained from R&D (Minneapolis, MN). The antibodies specifically recognizing AMPK and phospho-AMPKα (Thr 172) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). The antibodies against SHP, Runx2, and β-actin were
Effects of metformin on osteoblast differentiation
Although metformin has been reported to regulate the osteogenic differentiation of MC3T3E1 cells [17], [18], [21], the role of metformin in osteoblast differentiation and mechanistic signaling is largely unknown. To confirm the effect of metformin on osteogenic differentiation, alkaline phosphatase (ALP) staining was performed and the level of osteocalcin (OC) production was measured in MC3T3E1 cells. Interestingly, metformin increased the level of ALP staining and OC production at a dose of 100
Discussion
This study demonstrated that metformin stimulated osteoblast differentiation through activating AMPK, inducing SHP and Runx2 expression, and enhancing the transcriptional activity of OC gene. Our studies provide a mechanism wherein metformin-induced SHP stimulates the DNA-binding activity of Runx2 via a protein–protein interaction leading to increased Runx2-dependent gene expression.
Metformin is known to regulate the target gene expression and cell function via the activation of AMPK. For
Acknowledgments
This study was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) to J.T.K. (KRF-2007-313-E00469), and H.S.C. was supported by grant from the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A100588).
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Conflict of interest: All authors have no conflict of interest.
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W.G. Jang and E. J. Kim contributed equally to this paper.