The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation

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 MLK3 mRNA expression is highest in BMs, BMMs and OCs. Moreover, we found that the mRNA expression of MLK2 and 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. [Korean Journal of Bone Metabolism, 19(1): 21-28, 2012]