Molecular mechanism of receptor activator of NF-kB ligand-induced Ca2+ oscillations in osteoclastogenesis

Abstract

[한글]파골세포는 전구 세포로부터 분화하여 거대 다핵 세포를 형성함으로써 골을 흡수한다. 파골세포는 interleukin-1, tumor necROSis factor (TNF)-α와 같은 여러 외부 신호인자들에 의해 분화가 이루어지는데, 그 중 receptor activator of NF-κB ligand (RANKL)가 파골세포로의 분화에 중요한 역할을 담당하는 것으로 알려져 있다. RANKL은 조골세포에서 발현되는 단백질로서, 분비 후 파골세포막에 있는 RANK 수용체와 결합한 후 TNFR-associated factor 6 (TRAF6) 로부터 NF-κB, ERK, JNK, p38, PI3K 등의 신호를 활성화시켜 다핵 파골세포로의 분화를 촉진시킨다. 이외 RANKL 자극 후 분화과정 24시간 이후부터 유발된 세포 내 칼슘 신호가 calcineurin를 활성화시키며, NF-κB, ERK, JNK와 더불어 칼슘신호가 nuclear factor of activated T cell 1 (NFATc1)을 활성화시켜 최종 분화를 결정짓는 것으로 알려져 있다. 이중 칼슘신호와 NFATc1의 활성화는 분화 중기 이후의 신호 전달로서, 특히 NFATc1과 같은 전사인자에 의해 기타 다른 단백질 발현이 또 다른 분화 관련 신호 전달을 이루고 있음을 시사하고 있다. 그러나 아직까지 분화 중기 이후 나타나는 칼슘 신호의 발생 기작과 특성에 대해서 밝혀진 바가 없다. 이에 본 연구에서는 RANKL에 의해 발생하는 칼슘 oscillations의 특성과 RANKL 유도성 칼슘신호의 발생 기작에서의 활성산소종과 칼슘 신호전달과의 관계에 대해서 각 각 나누어 증명하고자 한다. 파골세포의 전구세포인 RAW264.7 세포 주, 또는 생쥐로부터 일차 분리 배양시킨 파골전구세포에 Fura-2 AM과 DCFH-DA와 같은 형광 염료를 사용하여 세포내 칼슘 농도와 활성산소종의 강도를 측정하였으며, tartrate resistant acidic phosphatase (TRAP) 염색법과 pit assay를 실시하여 분화도와 기능적 활성도를 측정하였다.파골전구세포를 RANKL 처리 시 24시간 이후부터 72시간 사이에 세포내 칼슘 농도의 주기적 변동 (oscillations)이 유도되었는데, RANKL 처리는 각 시간대에서 칼슘 신호전달관련 단백들인 inositol 1,4,5-triphosphate (IP3) 수용체, plasma membrane Ca2+ ATPase (PMCA), sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) 등의 발현에는 영향을 미치지 않았다. 세포막 인지질 가수분해 효소인 phospholipase C (PLC)의 활성을 억제하거나 세포내 칼슘저장고 이온통로인 IP3 수용체를 봉쇄시킨 결과 RANKL 유도성 칼슘신호가 발생되지 않았다. 또한 세포 외부로부터의 칼슘 유입의 억제도 칼슘신호를 유도하지 않았으며, 세포 내외부의 칼슘이온 통로의 특이 억제제를 통한 봉쇄는 RANKL 유도성 파골세포 분화와 골 흡수 기능을 억제하였다. 이는 세포내부와 외부로부터의 칼슘 농도의 증가가 RANKL 유도성 칼슘신호에 필수적임을 의미한다.RANKL 유도성 칼슘신호는 전구 세포에 항산화제를 처리하면 소실되었으며, RANKL 유도성 활성산소종의 강도 증가는 칼슘신호 유발과 동일한 시간대인 RANKL 처리 후 24시간에서 72시간까지 나타났다. 활성산소종 발생단백의 조절자인 Rac1 단백의 억제와 세포내 활성산소종 제거 효소인 peroxiredoxin II (PrxII)의 과발현은 RANKL 유도성 칼슘신호와 파골세포 분화를 억제하였다. 반대로 Rac1 단백의 지속적 활성화, 또는 PrxII의 기능 억제는 RANKL과 무관하게 칼슘신호를 유발하였으며, 이와 같은 칼슘신호는 세포 외부로부터의 칼슘 유입에 의존적이었다. 따라서, 활성산소종에의해 활성이 조절된다고 알려져 있는 칼슘 채널을 검색하고 그 후보물질로서 transient receptor potential M7 (TRPM7)을 선택하여 확인한 결과, 세포막 칼슘 이온통로인 TRPM7의 발현 억제는 RANKL 유도성 칼슘신호와 파골세포 분화를 억제하였다.이상의 실험 결과는 파골 전구세포에 RANKL 결합 후 분화 중기에 발생한 활성산소종이 PLC의 활성과 이에 따른 IP3 수용체로 부터의 칼슘 유리, 그리고 세포 외부로부터의 칼슘 유입에 필수적인 신호전달체계임을 의미한다.

[영문]Osteoclasts resorb Ca2+ ions from bone and provide it to plasma. Resorbed Ca2+ ions are delivered to each tissue and are mainly used to support the life. Osteoclasts are differentiated from bone marrow-derived monocytes (BMMs) by cell-to-cell contacts with osteoblasts. Osteoblasts express receptor activator of nuclear factor-κB ligand (RANKL) on plasma membrane which is reported as an essential factor to differentiate BMMs into osteoclasts. In recent years, it has been known that RANKL stimulation on osteoclast precursor cells transmits a differentiation-related signals to TNFR-associated factor 6 (TRAF6), then sequentially activates NF-κB, ERK, JNK pathways. In recently, it was reported that RANKL induces Ca2+ oscillations that activate calcineurin, a Ca2+/calmoduline dependent phosphatase, and subsequently regulate the translocation of nuclear factor of activated T cells (NFAT) into nucleus. Among them, Ca2+ signaling and NFATc1 activation are particular events in the middle period of differentiation, it suggests the existence of another signal pathway which is induced by RANKL-induced protein expression. However, it is not well known about the characteristics and generation mechanism of RANKL-induced Ca2+ oscillations. Here, we demonstrate the characteristics of RANKL-induced Ca2+ oscillations and the relationship between the reactive oxygen species (ROSs) and RANKL-induced Ca2+ oscillations, respectively. In addition, we suggest the involvement of transient receptor potentials M7 (TRPM7) in RANKL-induced osteoclastogenesis. To investigate this, RAW264.7 cells, mouse monocyte cell line, and bone marrow-derived monocytes (BMMs) were used as a sample. And fura-2 and DCFH-DA fluorescence dye were used to measure intracellular Ca2+ and ROSs. Furthermore, tartrate resistant acidic phosphatase (TRAP) stain assay and pit assay were performed to measure the differentiation rate and functional activities of osteoclasts.RANKL-induced Ca2+ oscillations were observed from 24 h to 72 h after RANKL stimulation in RAW264.7 cells and BMMs. block of Ca2+ release from inositol 1,4,5-triphosphate (IP3) receptor-sensitive Ca2+ stores by xestospongin C, a specific blocker of IP3Rs, and inactivation of phospholipase C (PLC) by U73122, an inhibitor of PLC, resulted the elimination of RANKL-induced Ca2+ oscillations. In addition, the involvement of PLCγ-1 in osteoclastogenesis is reconfirmed through knockdown of PLC-γ1. Moreover, elimination of RANKL-induced Ca2+ oscillations was resulted from removal of extracellular Ca2+ and block of Ca2+ entry with gadolinium (Gd) and 2-aminoethoxydiphenyl borate (2APB), general inhibitors of plasma membrane Ca2+ channel. Thus inhibited Ca2+ oscillations subsequently affected on the NFATc1 activities and differentiation rate into multi-nucleated cells (MNCs). These results indicate that internal Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ entry are simultaneously necessary for the generation of RANKL-induced Ca2+ oscillations and it sequentially activates NFATc1 which determines the late stage of differentiation.Next, we investigated the relationship between the ROSs and Ca2+ oscillations in osteoclastogenesis. Recently, it was reported that ROSs were produced by RANKL as a second messenger in macrophagic/monocytic cells and were also involved in differentiation into osteoclasts. Thus, we hypothesized that ROSs are produced by RANKL in particular time and are involved in generating RANKL-induced Ca2+ oscillations that trigger the late differentiation.ROSs production was observed not only within 25 m but also from 24 h to 72 h after RANKL stimulation. Furthermore, it was confirmed that scavenging the ROSs, generated from 24 h to 72 h not within 25 m, decisively affected on differentiation rate into MNCs at that time. Moreover, we demonstrated that Rac1 is involved in generation of ROSs and RANKL-induced Ca2+ oscillations. We additionally found that H2O2 among ROSs plays a crucial role in the generation of RANKL-induced Ca2+ oscillations through the expression of peroxiredoxin II (PrxII), a specific scavenger against H2O2. It was confirmed that thus inhibited Ca2+ oscillations and ROSs production by several factors give rise to decrease activity of fully-differentiated osteoclasts. On the other hand, we could observe the Ca2+ oscillations without RANKL when the H2O2 is produced endogenously by expression of DN-PrxII and RacV12, a dominant negative form of PrxII and constitutively active form of RacV12. Furthermore, it was considered that thus generated Ca2+ oscillations are dependent on extracellular Ca2+ influx. Then, as a candidate which permits the extracellular Ca2+ influx, we demonstrated that TRPM7 is involved in extracellular Ca2+ influx. These results indicate that ROSs production which is induced from 24 h, regulates the RANKL-induced Ca2+ oscillations which triggers and regulates the late stage of osteoclastogenesis.This study shows that ROSs which is produced by Rac1 activation regulates RANKL-induced Ca2+oscillations which are composed with extracellular Ca2+ influx through TRPM7 and Ca2+ release from IP3R-sensitive Ca2+ store simultaneously. These finding may support the another modality to regulate differentiation into osteoclasts which is concerned to various disorder of bone tissue.