Establishment and characterization of a stable cell line to evaluate cellular Runx2 activity

Kim, Hyun-Jung; Park, Hee-Dae; Kim, Junghwan; Cho, Je‐Yoel; Choi, Je‐Yong; Kim, Jung-Keun; Shin, Hong‐In; Ryoo, Hyun‐Mo

doi:10.1002/jcb.20038

Cited by 24 publications

(25 citation statements)

References 37 publications

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“…We previously reported that phosphorylation (Kim et al, 2003; Kim et al, 2004) and subsequent protein stabilization of Runx2 (Park et al, 2010) is required for its transactivation activity for the stimulation of osteoblast differentiation. As a matter of fact, it was reported that the genetic insufficiency of Runx2 could be overcome by osteoblast-specific overexpression of constitutively active MEK1 (MEK1-Ca) and could be exaggerated by the expression of dominant-negative MEK1 (Ge et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Pin1‐mediated Runx2 modification is critical for skeletal development

Yoon

Islam

Cho

et al. 2013

Journal Cellular Physiology

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Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/− mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.

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Section: Discussionmentioning

confidence: 99%

Pin1‐mediated Runx2 modification is critical for skeletal development

Yoon

Islam

Cho

et al. 2013

Journal Cellular Physiology

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“…[16] To see if the Runx2 promoter responded to SAHA, a transient reporter assay was conducted by using 6XOSE as previously described. [14] As shown in Figure 1, luciferase activity was generated in a dose-dependent manner, with maximal activity seen with 10 µM SAHA. The EC50 was calculated as 1.0 µM.…”

Section: Resultsmentioning

confidence: 99%

“…[14] The cells were treated with SAHA at the indicated concentrations with FGF-2 as a control at 24 hr after transfection. Luciferase activity was measured by using the dual-luciferase assay system (Promega, Madison, WI, USA) in a Dynex luminometer.…”

Section: Methodsmentioning

confidence: 99%

A Novel Osteogenic Activity of Suberoylanilide Hydroxamic Acid is Synergized by BMP-2

Lee

Kim²,

Ryoo

2015

J Bone Metab

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BackgroundMany histone deacetylase (HDAC) inhibitors are well recognized as potential anti-cancer drugs. Inhibition of HDACs induces temporal transcription or epigenetic control, thus regulating many different biological responses. Here, we investigated the osteogenic effect of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat).MethodsThe effects of SAHA on osteoblast differentiation were examined in the 6XOSE-Luc reporter assay for determination of runt-related transcription factor 2 (Runx2) activity and alkaline phosphatase (ALP) activity and in an immunoprecipitation assay to determine the Runx2 acetylation state. The osteogenic activity of SAHA in vivo was studied in and receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoporotic mouse model.ResultsSAHA increased the transcriptional activity of Runx2 in a dose-dependent manner in the 6XOSE-Luc reporter assay. SAHA by itself was unable to induce ALP activity; however, SAHA enhanced ALP activity induced by bone morphogenetic protein-2 (BMP-2). The degree of acetylation of Runx2 was increased with SAHA treatment, which suggests that the increase in Runx2 transcriptional activity might be dependent on stabilization by acetylation. Also, SAHA successfully reversed soluble RANKL-induced osteoporotic bone loss.ConclusionsOur study shows an intriguing osteogenic potential of SAHA in a BMP-2-dependent manner and suggests that SAHA could be used at lower doses along with BMP-2 to treat osteoporosis.

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“…As described previously (Kim et al ., ; Kim and Kim, ), the p6 × osteoblast‐specific cis‐acting element (OSE) 2‐luc reporter vector was used for measuring runt‐related transcription factor 2 (Runx2) activity. Briefly, it was transiently transfected into C2C12 cells (4 × 10 3 cells/well) cultured in a 96‐well plate for 24 h. Then, the culture medium was changed with DMEM‐containing 5% FBS, and cells were treated with SCE.…”

Section: Methodsmentioning

confidence: 99%

In vitro and in vivo Bone‐Forming Activity of Saururus chinensis Extract

Moon

Choi

Park

et al. 2015

Phytotherapy Research

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Bone is maintained by osteoclast-mediated resorption and osteoblast-mediated formation. Recently, anti-osteoporotic activity of Saururus chinensis extract (SCE) and anti-osteoclastogenic activity of its components have been reported, but the effect of SCE on bone formation has not been studied well. Therefore, in this study, we investigated whether Saururus chinensis SCE exhibits in vitro osteogenic and in vivo bone-forming activity. extract strongly enhanced the bone morphogenetic protein (BMP)-2-stimulated induction of alkaline phosphatase, an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. In vitro osteogenic activity of SCE was accompanied by enhanced expression of BMP-2, BMP-4, BMP-7 and BMP-9 mRNA. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of SCE. Moreover, the BMP dependency and the involvement of p38 activation in the osteogenic action of SCE were confirmed by the treatment of noggin, an antagonist of BMP. Saururus chinensis extract also exhibited to induce runt-related transcription factor 2 activation at the high concentration. Furthermore, the in vivo osteogenic activity of SCE was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its use for bone formation. In conclusion, we suggested that in vivo anti-osteoporotic activity of SCE could be because of its dual action in bone, anti-osteoclastogenic and anabolic activity.

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Establishment and characterization of a stable cell line to evaluate cellular Runx2 activity

Cited by 24 publications

References 37 publications

Pin1‐mediated Runx2 modification is critical for skeletal development

Pin1‐mediated Runx2 modification is critical for skeletal development

A Novel Osteogenic Activity of Suberoylanilide Hydroxamic Acid is Synergized by BMP-2

In vitro and in vivo Bone‐Forming Activity of Saururus chinensis Extract

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