조골세포에서 Rosiglitazone에 의한 RANKL과 OPG의 발현 조절
Differential Expression of RANKL and OPG by the PPAR gamma Agonist Rosiglitazone in Osteoblasts
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목적: Receptor activator of nuclear factor-kB ligand (RANKL)과 osteoprotegerin (OPG)는 조골세포에서 생성되는 사이토카인인데, RANKL은 파골세포 형성을 촉진시키고 OPG는 RANKL의 decoy 수용체로 작용함으로써 파골세포 형성을 억제한다. Peroxisome proliferation-activated receptor gamma (PPARγ)는 지방세포 분화에 중요한 역할을 하는 것으로 알려져 있지만 이것은 골 재형성에도 관여한다. 따라서 본 연구에서는 PPARγ 작용제인 rosiglitazone이 조골세포에서 RANKL과 OPG 발현을 어떻게 조절하는지 조사했다. 방법: MC3T3-E1 조골세포를 여러 농도의 rosiglitazone으로 24시간 처리하여 세포생존능을 MTT 방법으로 측정했다. PPARγ 유전자의 발현은 통상적인 PCR로 관찰했으며, RANKL과 OPG mRNA와 단백질의 발현은 real-time RT-PCR과 Western blot으로 각각 정량 분석했다. 결과: MC3T3-E1 조골세포에 존재하는 PPARγ 유전자 발현은 rosiglitazone 0.5μM 처리 시 대조군과 유사했으나 5μM 처리 시 현저히 증가했다. 세포생존능의 경우 rosiglitazone 1～10μM 범위에서 대조군과 차이를 보이지 않았다. Rosiglitazone (5μM) 처리에 의해 OPG mRNA의 발현은 대조군에 비해 2.3배 증가하였고 RANKL mRNA의 경우 1/5로 감소했다. 그러나 이러한 변화는 PPARγ 길항제인 GW9662 처리에 의해 모두 대조군 수준으로 회복되었다. Rosiglitazone에 의한 OPG와 RANKL 단백질 발현 또한 이들의 mRNA 발현 변화와 유사한 경향을 보였다. 결론: 조골세포에서 rosiglitazone에 의해 PPARγ가 활성화됨으로써 RANKL 발현은 감소되고 동시에 OPG 발현은 증가되는데, 이는 파골세포 형성을 억제할 것으로 사료된다.
Abstract
Objectives: Osteoblasts secrete receptor activator of nuclear factor-kB ligand (RANKL) and osteoprotegerin (OPG). RANKL stimulates osteoclastogenesis but OPG inhibits it by binding with RANKL. Although peroxisome proliferation-activated receptor gamma (PPARγ) has a major role in adipocyte differentiation, it is also involved in bone remodeling. The aim of this study was to examine whether rosiglitazone, the PPARγ agonist, regulates the expression of RANKL and OPG in osteoblasts. Methods: MC3T3-E1 osteoblasts were cultured and treated with different concentrations of rosiglitazone. Cell viability was assayed with MTT. A conventional PCR was used for the detection of PPARγ gene. Expression of RANKL and OPG mRNA and their protein levels was analyzed by quantitative real-time RT- PCR and Western blot, respectively. Results: PPARγ mRNA was expressed in MC3T3-E1 osteoblasts and its expression was greatly increased by treatment of 5μM rosiglitazone. Treatment with 1 to 10μM rosiglitazone did not affect cell viability. Compared with vehicle, rosiglitazone (5μM) up-regulated OPG mRNA expression by 2.3-fold and down- regulated RANKL mRNA expression by 5-fold, which was reversed by the PPARγ antagonist GW9662. The level of RANKL and OPG protein varied as compared to their mRNA expression. Conclusion: These results demonstrate that activation of PPARγ by rosiglitazone induces both down- regulation of RANKL and up-regulation of OPG in osteoblasts, which is likely to cause an inhibition of osteoclastogenesis.
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