The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors.

Manh Tien Tran,Eiko Sakai,Yuka Okusha,Masatoshi Morimatsu,Keiji Naruse,Yunxia Feng,Chiharu Sogawa,Tomoko Kadowaki,Kuniaki Okamoto,Takayuki Tsukuba,Hirohiko Okamura,Takanori Eguchi,Penggong Wei

doi:10.3390/ijms21249352

Abstract

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

Highlights

Osteoclasts, multinucleated cells differentiated from macrophage-monocyte cells, play a critical role in bone tissue destruction [1,2]
Albeit the mRNA level of Rab11b was insignificantly altered (Figure 1A), the protein level thereof was strongly increased from day two to five (Figure 1C and Figure S1A) in RAW-D cells upon five days of receptor activator of nuclear factor kappa-B ligand (RANKL) stimulation
Our tartrate-resistant acid phosphatase (TRAP) staining results showed that numbers of TRAP-positive multinucleated osteoclasts (MNCs) formed reached their maximum at days three and four in both cases of RAW-D cells (Figure 1E and Figure S1C,D) and Bone marrow-derived macrophages (BMMs) (Figure 1F and Figure S1E), but somewhat reduced at day five in RAW-D cell-derived osteoclasts, suggesting that mature osteoclasts, unless otherwise specified, were formed in vitro after three days of RANKL stimulation

Summary

Introduction

Osteoclasts, multinucleated cells differentiated from macrophage-monocyte cells, play a critical role in bone tissue destruction [1,2]. Osteoclast differentiation, called osteoclastogenesis, is directly induced via binding of the receptor activator of nuclear factor kappa-B ligand (RANKL) to the cell surface RANK receptor. This binding stimulates six various signaling cascades essential for osteoclast differentiation including (i) the nuclear factor of activated T cells cytoplasmic-1 (NFATc-1); (ii) nuclear factor kappa B (NF-κB); (iii) phosphatidylinositol 3-kinase (PI3K/Akt); (iv) Jun N-terminal kinase (JNK); (v) extracellular signal-regulated kinase (Erk); and (vi) p38 mitogen-activated protein kinase (MAPK) [3], thereby boosting the secretion of bone-resorbing enzymes such as tartrate-resistant acid phosphatase (TRAP), Cathepsin K (CTSK), and matrix metalloproteinase 9 (MMP9), into the extracellular environment [4,5]. Subcellular localization of Rab11b was identified to localize in perinuclear recycling compartments wherein functionally it was engaged in ruffled border membrane turnover and in osteoclast motility [14]

Methods

Results

Conclusion