Int J Thyroidol.  2017 Nov;10(2):71-76. 10.11106/ijt.2017.10.2.71.

Enhancement of Osteogenic Differentiation by Combination Treatment with 5-azacytidine and Thyroid-Stimulating Hormone in Human Osteoblast Cells

• Sun HJ
• Song YS
• Cho SW
• Park YJ

• KMID: 2396809
• DOI: http://doi.org/10.11106/ijt.2017.10.2.71

Abstract

BACKGROUND AND OBJECTIVES
The role of thyroid-stimulating hormone (TSH) signaling on osteoblastic differentiation is still undetermined. The aim of this study was to investigate the effects of 5-aza-2"²-deoxycytidine (5-azacytidine) on TSH-mediated regulations of osteoblasts.
MATERIALS AND METHODS
MG63, a human osteoblastic cell-line, was treated with 5-azacytidine before inducing osteogenic differentiation using osteogenic medium (OM) containing L-ascorbic acid and β-glyceophosphate. Bovine TSH or monoclonal TSH receptor stimulating antibody (TSAb) was treated. Quantitative real-time PCR analyses or measurement of alkaline phosphatase activities were performed for evaluating osteoblastic differentiation.
RESULTS
Studies for osteogenic-related genes or alkaline phosphatase activity demonstrated that treatment of TSH or TSAb alone had no effects on osteoblastic differentiation in MG63 cells. However, treatment of 5-azacytidine, per se, significantly increased osteoblastic differentiation and combination treatment of 5-azacytidine and TSH or TSAb in the condition of OM showed further significant increase of osteoblastic differentiation.
CONCLUSION
Stimulating TSH signaling has little effects on osteoblastic differentiation in vitro. However, in the condition of epigenetic modification using inhibitor of DNA methylation, TSH signaling positively affects osteoblastic differentiation in human osteoblasts.

Keyword

Thyroid-stimulating hormone; 5-azacytidine; Osteogenic differentiation; Osteoblast

MeSH Terms

Alkaline Phosphatase
Ascorbic Acid
Azacitidine*
DNA Methylation
Epigenomics
Humans*
In Vitro Techniques
Osteoblasts*
Real-Time Polymerase Chain Reaction
Receptors, Thyrotropin
Social Control, Formal
Thyrotropin*
Alkaline Phosphatase
Ascorbic Acid
Azacitidine
Receptors, Thyrotropin
Thyrotropin

Figure

• 

  Fig. 1 The effects of TSH signaling on osteogenic differentiation in MG63 cells. MG63 cells were treated with TSH (20 mIU/L) or monoclonal stimulatory TSHR antibody, M22 (TSAb, 10 ng/mL) with or without osteogenic medium containing L-ascorbic acid and b-glycerophosphate for 7 days. (A–C) mRNA expressions of (A) ALP, (B) OC, and (C) Col-I were detected using quantitative reverse transcription-polymerase chain reaction. (D) ALP activities were measured with each condition. ALP: alkaline phosphatase, Col-I: collagen type I, OC: osteocalcin. *p<0.05 vs. 1st column; #p<0.05 vs. 4th column.

• 

  Fig. 2 The effects of TSH signaling on Wnt/b-catenin and BMP-2/Smad signaling. (A) MG63 cells were treated with TSH (20 mIU/L) or monoclonal stimulatory TSHR antibody, M22 (TSAb, 10 ng/mL) with or without Wnt-3a for 3 days. Western blot analyses were performed using anti-b-catenin. (B) MG63 cells were treated with TSH (20 mIU/L) or monoclonal stimulatory TSHR antibody, M22 (TSAb, 10 ng/mL) with or without BMP-2 for 3 days. Western blot analyses were performed using anti-b-catenin.

• 

  Fig. 3 The effects of combination treatment of 5-azacytidine and TSH on osteogenic differentiation. MG63 cells were treated with TSH (20 mIU/L) or monoclonal stimulatory TSHR antibody, M22 (TSAb, 10 ng/mL) with or without 5-azacytidine (1 or 5 µM) in osteogenic medium containing L-ascorbic acid and b-glycerophosphate for 4 days. (A) mRNA expressions of ALP were detected using quantitative reverse transcription-polymerase chain reaction. (B) ALP activities were quantified. (C) Staining of ALP activities was performed at 24-well plates. 5-azadC: 5-azacytidine, ALP: alkaline phosphatase, Col-I: collagen type I, OC: osteocalcin. *p<0.05 vs. 1st column; **p<0.01 vs. 1st column; #p<0.05 vs. 6th column.

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