EFFECT OF BISPHOSPHONATE ON OSTEOBLAST DIFFERENTIATION

Bisphosphonate가 조골세포 분화에 미치는 영향

Bisphosphonates inhibit bone resorption in vivo and in vitro. Currently proposed mechanism of action of bisphosphonates involves both direct effect on osteoclasts and indirect effect through the mediation of osteoblasts. Recent understanding of molecular mechanism of osteoclastogenesis indicates that osteoclast differentiation is quite tightly regulated by signaling molecules from differentiating osteoblasts. Therefore this investigation was designed to elucidate the effect of bisphosphonate on osteoblast differentation. For this purpose, in vitro effects of etidronate and alendronate on the expression of Cbfa1 a master control gene of osteoblast differentiation, several bone marker genes, and formation of calcified nodules were evaluated. To evaluate the effect of bisphosphonate on calcified nodule formation, osteoblasts isolated from rat calvaria were cultured in a-MEM containing $10^{-4},\;10^{-5},\;10^{-6}M$ of etidronate or $10^{-6},\;10^{-7},\;10^{-8}M$ of alendronate for 15 days, and then stained by alizarin red to determine mineralization. To evaluate the effect of bisphosphonate on osteoblast differentiation, osteoblast cells were cultured in a-MEM containing $10^{-4},\;10^{-5},\;10^{-6}M$ of etidronate or $10^{-6}$ M of alendronate for 8 days. And then total RNA was extracted and northern blot analysis was done to examine the expression of Cbfa1, type I collagen, alkaline phosphatase, osteopontin and osteocalcin. The results were as follows: 1. Etidronate suppressed the calcification of bone nodule in dose dependent manner, while alendronate didn't. 2. The expression of Cbfa1 was decreased dose dependently by etidronate, but increased by alendronate. 3. Etidronate suppressed the expression of type I collagen, osteopontin and osteocalcin in dose dependent manner however alendronate promote the expression of osteoblast marker gene. 4. The expression of alkaline phosphatase was not affected either etidronate nor alendronate. These results suggest that etidronate suppressed the expression of Cbfa1 in dose dependent manner, and consequently the expression of osteoblast marker genes, such as type I collagen, osteopontin and osteocalcin were also suppressed in similar manner. And finally this decreased expression of osteoblastic marker gene prevent calcined bone nodule formation.

본 실험은 bisphosphonate가 조골세포 분화 및 파골세포 분화에 미치는 영향을 알아보고자, etidronate와 alendronate를 조골세포에 투여하여 조골세포 전사인자인 Cbfa1, 조골세포 표시 인자의 발현, 석회화된 골결절 형성을 평가하였다. Bisphosphonate가 조골세포의 석회화된 골결절 형성에 미치는 영향을 평가하기 위하여 배양액에 $10^{-6},\;10^{-5},\;10^{-4}M$의 etidronate 및 $10^{-8},\;10^{-7},\;10^{-6}M$의 alendronate를 첨가하였으며, 배양 15일 후에 alizarin red로 염색하여 관찰하였다. 또 조골세포의 분화에 미치는 bisphosphonate의 영향을 평가하고자 백서 두개관에서 얻은 조골세포에 etidronate $10^{-6},\;10^{-5},\;10^{-4}M$ 및 alendronate $10^{-6}$ M을 투여하고 배양 8일 후 총RNA를 수집하였고, 전기영동 및 Northern blot hybridization하여 Cbfa1, alkaline phosphatase, type I collagen, osteopontin, osteocalcin의 발현을 조사하였다. 이상의 실험결과 다음과 같은 결론을 얻었다. 1. Etidronate는 농도 의존적으로 골결절 석회화를 억제하였으나, alendronate는 골석회화를 억제하지 않았다. 2. Etidronate는 Cbfa1의 발현을 농도 의존적으로 억제하였으나, alendronate는 오히려 촉진하였다. 3. Etidronate는 type I collagen, osteocalcin 및 osteopontin의 발현을 농도 의존적으로 억제하였으나, alendronate는 오히려 증가시켰다. 4. Alkaline phosphatase의 발현은 사용된 etidronate와 alendronate에 의해 영향 받지 않았다. 이상의 결과에서 etidironate는 조골세포의 전사인자인 Cbfa1의 발현을 억제하며, 이에 의하여 조골세포의 분화표지인자인 type I collagen, osteopontin 및 osteocalcin의 합성이 억제되고, 결과적으로 석회화된 골결절의 형성을 억제하는 것으로 사료된다.