파골세포 형성에 있어서 MLKs-JNK 경로의 역할 규명
The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation
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연구목적: 파골전구세포는 M-CSF 존재하에서 조혈모세포로부터 형성되며, M-CSF와 RANKL 존재하에서 성숙 파골세포로 분화된다. 본 실험에서는 성숙 파골세포 형성에 있어서 MLKs-JNK 경로의 역할을 알아보고자 하였다. 연구방법: 파골세포 형성 측정법과 RT-PCR 분석법을 사용하였다. 결 과: 먼저 파골세포 형성에 있어서 JNK의 역할을 조사한 결과, JNK 저해제는 파골전구세포와 성숙 파골세포의 형성을 모두 억제하는 것으로 나타났다. JNK의 활성은 MLK에 의해 조절된다는 보고가 있으므로 MLKs mRNA의 발현을RT-PCR 방법으로 조사하였다. MLKs 중, MLK3의 발현이 골수, 파골전구, 성숙 파골세포에서 가장 높은 것으로 나타났다. 또한 MLK2와 MLK3의 발현은 골수에서 파골전구세포로 분화시 증가하였으며, 성숙 파골세포에서도 발현이 높게 유지되었다. siRNA를 사용한 MLK3 유전자의 knock-down은 파골세포 분화를 일부 억제하였다. 결 론: JNK는 파골전구세포와 성숙 파골세포로의 분화에 필수적이며, MLK3는 파골세포 분화에 일부 관여한다.
Abstract
Objectives: Osteoclasts (OCs) are bone-resorbing multinucleated cells derived from hematopoietic progenitors of the monocyte-macrophage lineage. OC precursors, such as bone marrow-derived macrophages (BMMs), are formed in the presence of macrophage colony-stimulating factor (M-CSF) and differentiate into OCs in response to M-CSF and receptor activator of nuclear factor κB ligand (RANKL). In this study, we investigated the role of mixed lineage kinases (MLKs)-c-Jun amino-terminal kinase (JNK) pathways in OC formation. Methods: We performed an OC formation assay and reverse transcription polymerase chain reaction (RT-PCR)analysis. Results: We first explored the role of JNK on osteoclst formation using mouse bone marrow (BM) culture system. We found that OC formation was impaired when the JNK inhibitor was added either in early or late stage, suggesting the requirement for JNK activation during OC formation. MLKs are serine/threonine kinases that regulate signaling by the JNK. Since the JNK activity is specifically required for osteoclastogenesis, we examined the messenger ribonucleic acid (mRNA) levels of MLKs in BMs, BMMs and OCs by RT-PCR. Among MLKs, the level of MLK3mRNA expression is highest in BMs, BMMs and OCs. Moreover, we found that the mRNA expression of MLK2and MLK3 is increased with the differentiation of BMs to BMMs, and is sustained in OCs. Finally we investigated the role of MLK3 in OC differentiation using gene knock-down techniques. The silencing of MLK3 in BMMs partly attenuated RANKL-induced OC differentiation. Conclusions: These data suggest that JNK and MLK3 may positively regulate OC formation.
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