J Bone Metab.  2015 May;22(2):51-56. 10.11005/jbm.2015.22.2.51.

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

• Lee ZH
• Kim HJ
• Ryoo HM

• KMID: 2170088
• DOI: http://doi.org/10.11005/jbm.2015.22.2.51

Abstract

BACKGROUND
Many 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).
METHODS
The 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.
RESULTS
SAHA 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.
CONCLUSIONS
Our 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.

Keyword

Bone morphogenetic protein 2; Histone deacetylase inhibitors; Osteogenesis

MeSH Terms

Acetylation
Alkaline Phosphatase
Animals
Bone Morphogenetic Protein 2
Epigenomics
Histone Deacetylase Inhibitors
Histone Deacetylases
Hydroxamic Acids*
Immunoprecipitation
Mice
Osteoblasts
Osteogenesis
Osteoporosis
RANK Ligand
Transcription Factors
Alkaline Phosphatase
Bone Morphogenetic Protein 2
Histone Deacetylase Inhibitors
Histone Deacetylases
Hydroxamic Acids
RANK Ligand
Transcription Factors

Figure

• 

  Fig. 1 Induction of transcription of runt-related transcription factor 2 (Runx2) by suberoylanilide hydroxamic acid (SAHA). A Runx2-promoter-driven luciferase vector previously named 6XOSE was transiently transfected into C2C12 cells. After 24 hr these cells were stimulated with various concentrations of SAHA or with fibroblast growth factor-2 (FGF-2) as a control. Cell lysates were subjected to luciferase assay. A plot of luminescence vs. SAHA concentration was constructed.

• 

  Fig. 2 Induction of alkaline phosphatase (ALP) activity by suberoylanilide hydroxamic acid (SAHA). C2C12 cells were cultured in 96-well plates and stimulated with various concentrations of SAHA as indicated in the presence or absence of a fixed bone morphogenetic protein-2 (BMP-2) concentration (30 ng/mL). After 48 hr these cells were washed and fixed. ALP staining was conducted. As controls, several BMP-2 concentrations were used for comparison.

• 

  Fig. 3 Effect of suberoylanilide hydroxamic acid (SAHA) on the stability of acetylated runt-related transcription factor 2 (Runx2). A 3xMyc-tagged Runx2 expression vector was transfected into 293T cells, which were then treated with various concentrations of SAHA for 24 hr as indicated. Cell lysates were immunoprecipitated with anti-acetylated-Lys followed by immunoblotting with anti-Myc antibody. The same blot was reprobed with anti-β actin.

• 

  Fig. 4 Osteogenic potential associated with suberoylanilide hydroxamic acid (SAHA) in soluble receptor activator of nuclear factor-kappa B ligand (sRANKL)-induced osteoporosis model. ICR mice were treated with various concentrations of SAHA. sRANKL was administered on day 1 and day 2, after which the mice were left untreated until day 4. Starting on day 4, parathyroid hormone (PTH) as the control or various concentrations of SAHA were administered daily until day 13. Mice were sacrificed on day 14. (A) The osteogenic activity of SAHA was assessed with 3-dimensional (3D) micro-computed tomography (CT) image analysis of femurs. (B) Trabecular bones were isolated and bone measurement parameters were enumerated, including bone volume, bone thickness, and bone numbers. Femoral bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and number of trabecular bones (Tb.N) were measured 0.6 to 1.0 mm proximal to the distal growth plate by histomorphometric analysis. N=7.RL, sRANKL. *P<0.01 vs. RL. **P<0.05 vs. RL. ***P<0.005 vs. RL.

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