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Abstract
Objective
Alendronate, a widely used bisphosphonates, acts to inhibit bone resorption by interfering with the activity of osteoclasts. Recently, it has been reported that alendronate also may increase bone proliferation and osteoblastic differentiation. However, little is known about mechanism of the action of alendronate on osteoblast differentiation, especially in transcription level. Inhibitors of DNA binding/differentiation (Ids) are helix-loop-helix (HLH) transcription factors and play an important role in BMP-induced osteoblast lineage-specific differentiation. Therefore, this study was aimed to investigate the effect of alendronate on osteoblast differentiation and expression of Id-1 and Id-2.
Methods
MC3T3-E1, pre-osteoblast cell line, were treated with alendronate of various concentrations (10-9 M-10-4 M) and time periods (24, 48 and 72 hours). And then, the effect of alendronate on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and RT-PCR for osteoblast differentiation markers such as ALP, type 1 collagen (Col 1), and osteocalcin (OCN). The expressions of Id-1 and Id-2 were measured by RT-PCR.
Figures and Tables
Figure 1
Effects of alendronate (Aln) on osteoblastic cell viability and differentiation. Alendronate were treated with various concentrations (10-9 M-10-4 M) for various duration (24, 48, and 72 hours) in MC3T3-E1 cells. (A) Cell viability was determined by MTT assay. (B) For evaluation of osteoblast differentiation, ALP activity was measured at 405 nm. The experiment was repeated three times and each performed with triple samples. *P <0.05, †P <0.01 (vs control).
Figure 2
Effect of alendronate (Aln) of different concentrations and times on osteogenic gene marker expression. (A) MC3T3-E1 cells were treated with increasing concentrations (10-6, 10-7 and 10-8 M) for 48 hours. (B) MC3T3-E1 cells were treated with different time periods (24, 48, and 72 hours) at 10-8 M. ALP, Col 1, OCN, and CTSK mRNA levels were detected by RT-PCR with β-actin as loading control. Data shown are representative of three independent experiments. The relative density of each gene was quantitated and statistically analyzed by ImageJ version 1.35d (NIH Image). *P <0.01, †P <0.005 (vs control).
Figure 3
Expression of Id-1 and Id-2 genes during alendronate (Aln)-induced osteoblast differentiation. MC3T3-E1 cells were treated with increasing concentrations (10-6, 10-7 and 10-8 M) for 48 hours (A) and different time periods (24, 48, and 72 hours) at 10-8 M (B). Total RNA was prepared and analyzed for the expression of the Id-1 and Id-2 mRNA by RT-PCR as described. Data shown are representative of four independent experiments. The relative density of each gene was quantitated and statistically analyzed by ImageJ version 1.35d (NIH Image). *P <0.05, †P <0.01, ‡P <0.005 (vs control).
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