조골세포 분화 세포유도주를 이용한 비스포스포네이트의 골에 미치는 작용 기전
The Bone Action Mechanism of Bisphosphonate using Osteoblast Differentiation Cell Line
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목적: 조골세포 분화 유도 세포주인 MC3T3-E1세포를 이용하여 조골세포의 증식과 분화과정 동안의 alendronate의 작용기전에 대해 조사하고자 한다. 방법: MC3T3-E1세포를 FBS가 포함된 DMEM, 배양액에 배양하면서 alendronate를 다양한 농도(10-9 M, 10-8 M, 10-7 M, 10-6 M, 10-5 M, 10-4 M)로 첨가하고 1, 2, 3일 동안 배양한 후 MTT 분석, alkaline phosphatase(ALP) 활성도 및 RT-PCR 방법을 이용한 ALP, osteocalcin(OCN), collagen I α1(Col 1), inhibitor of differentiation(Id) 및 cathepsin K(CTSK) 등의 조골세포와 파골세포 분화 관련 유전자의 발현을 조사하였다. Id 유전자의 발현 증가에 관여하는 전사인자를 TRANSFEC program을 이용하여 동정하고 이들 전사인자가 Id 유전자의 발현에 미치는 효과를 promoter 분석을 통하여 조사하였다. 결과: Alendronate의 농도에 따른 세포 독성은 없었다. ALP 활성은 MC3T3-E1에서는 ALP 활성이 10-8 M에서 48시간까지 급격하게 증가되나 10-8 M 이상의 농도에서는 시간경과에 상관없이 급격히 감소되었다. ALP, Col 1 및 OCN의 유전자 발현은 alendronate 처리 24시간까지는 두 세포주 모두에서 alendronate의 농도가 감소함에 따라 증가하는 경향을 보였다. Id-1의 유전자의 발현은 대조군에 비해 10-6 M에서 증가하여 10-8 M까지 유지되었으나, Id-2는 alendronate 농도에 따라 점차적으로 증가하였으며, 72시간에서는 Id-1과 Id-2 유전자 모두 발현양이 감소하였다. Id 유전자 프로모터 내 전사인자 가운데 CREB와 C/EBP β는 Id 프로모터의 활성도를 각각 2배, 4배로 증가시켰으며, C/EBPβ와 alendronate를 병합 처리 시 Id-1 프로모터 활성도는 각각을 단독으로 처리했을 때보다 유의하게 증가하였다. 결론: 본 연구는 alendronate가 농도에 의존적으로 조골세포의 분화를 촉진시키고 있음을 보여주고 있으며, 이러한 alendronate에 의한 분화 촉진 효과는 C/EBPβ 전사인자와 상호 작용하여 Id 유전자의 발현 증가를 야기하고 있음을 시사하고 있다.
Abstract
Objectives: To investigate the effect of alendronate on osteoblast proliferation and differentiation using mosue preosteoblast cell line, MC3T3-E1 and myoblastic cell line. Material & Methods: MC3T3-E1 cell lines were cultured in DMEM media with various concentrations(10-9 M, 10-8 M, 10-7 M, 10-6 M, 10-5 M, 10-4 M) of alendronate for one, two, or three days. After the indicated culture times, MTT assay and ALP activity were examined. The expression of genes for ALP, osteocalcin (OCN), collagen I α1(Col 1), inhibitor of differentiation(Id) and cathepsin K(CTSK) were examined by RT-PCR method. Transcription factors related to the increased expression of Id genes were identified with TRANSFEC program. The effect of these transcription factors on the expression of Id genes were tested by promoter assay. Results:Alendronate had no cytotoxicity regardless of the concentrations used. In MC3T3-E1, it was increased greatly at 10-8 M before 48 hours of treatment and it was decreased rapidly at more than 10-8 M concentration regardless of times of treatment. Expressions of ALP, Col 1 and OCN genes had a tendency to increase with decreasing of alendronate concentrations before 24 hours of treatment in both cell lines. The expression of Id-1 gene began to increase from 10-6 M of alendronate compared to the control group and kept to 10-8 M. However, Id-2 gene expression was increased gradually with increasing of alendronate concentrations. Conclusion: This study shows that alendronate has a dose-dependent effect in osteoblast differentiation. The effect may be associated with the increased expression of Id genes.
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