Bisphosphonates가 Osteoclast 분화에 미치는 기전 연구
Study on the Mechanism of Action of Bisphosphonate in Osteoblast Differentiation
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목적: Alendronate는 대표적인 제3세대 아미노-비스포스토네이트로서 일차적인 작용이 파골세포의 골 흡수 억제라 할지라도, 최근 비스포스포네이트가 조골세포에도 영향을 준다는 연구들이 제시되고 있다. 그러나 alendronate가 조골세포와 파골세포의 형성 및 분화에 미치는 정확한 기전에 대한 분자생물학적 수준에서의 연구는 매우 제한적이다. 그러므로 본 연구는 Bisphosphonates가 osteoclast 분화에 미치는 기전 연구 작용기전에 대해 조사하고자 한다. 방법: C2C12와 MC3T3-E1세포를 FBS가 포함된 DMEM, 배양액에 배양하면서 alendronate를 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: 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 발현량이 감소하였다. Id 유전자 프로모터 내 전사인자 가운데 CREB와 C/EBPβ는 Id 프로모터의 활성도를 각각 2배, 4배로 증가시켰으며, C/EBPβ와 alendronate를 병합 처리 시 Id-1 프로모터 활성도는 각각을 단독으로 처리했을 때보다 유의하게 증가하였다. 결론: alendronate에 의한 분화 촉진 효과는 C/EBPβ 전사인자와 상호 작용하여 Id 유전자의 발현 증가를 야기시키고 있음을 시사하고 있다.
Abstract
Objectives: Alendronate is a representative third generation bisphosphonates that is widely used in clinical treatment of various metabolic bone diseases and the primary action is the inhibition of osteoclastic bone resorption. Some studies suggest a possible role of bisphosphonates in osteoblastic activity. However, little is known about the mechanism of action of alendronate in osteoblast formation and differentiation has not yet been clearly elucidated. Therefore, this study was aimed to investigate the effect of alendronate on osteoblast proliferation and differentiation. Methods: 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: 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. Among transcription factors related to the promoter of Id genes, CREB and C/EBPβ increased promoter activity of Id genes to 2-fold and 4-fold, respectively. Co-treatment of C/EBPβ and alendronate resulted in a significant increased promoter activity of Id genes compared to each single treatment. Conclusion: This study shows that alendronate may be associated with the increased expression of Id genes via a synergistic interaction with C/EBPβ.
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