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

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.

Dihydroartemisinin attenuates osteoclast formation and bone resorption via inhibiting the NF‑κB, MAPK and NFATc1 signaling pathways and alleviates osteoarthritis

“…With the formation of mature osteoclasts, bone minerals represented by hydroxyapatite are dissolved by acids secreted from osteoclasts, and the organic matrix is degraded by certain specific proteolytic enzymes, including CTSK, TRAP, CTR and MMP-9 (47). In addition, the numbers of certain receptors located on the surface of osteoclast precursor cells that, are associated with cell migration and differentiation, such as CXCR4 and RANK, are increased during osteoclast formation (48,49). Consistent with previous reports, the present study revealed that, during osteoclastogenesis, the expression levels of certain osteoclast-associated genes, such as CTSK, CTR, TRAP, MMP-9, RANK and CXCR4, were upregulated.…”

Rab34 plays a critical role as a bidirectional regulator of osteoclastogenesis

“…36 Notably, we have recently revealed an important role of lysosomes in promoting the proteolytic degradation of c-fms and RANK surface receptors through the axis of early endosomes-late endosomes-lysosomes, and this lysosomal function was significantly facilitated by Rab11 overexpression in OCs. [37][38][39] In this study, we identify that Rab34, which is localized in early endosomes, late endosomes, and lysosomes in OCs, plays a bidirectional role in regulating osteoclastogenesis. While Rab34 serves as a negative regulator of OC differentiation by abolishing the surface levels of c-fms and RANK receptors via a specific mechanism underlying Rab34-mediated lysosomal proteolysis of c-fms and RANK receptors in OCs, it is also associated with the transport of lysosome-related proteases consisting of MMP9 and CTSK across the OC plasma membrane, which regulate the bone-resorbing activity of OCs.…”

“…Western blot (WB) analysis was performed as described previously. 37 Briefly, cell lysates were prepared using radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8.0], 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 150 mM NaCl) supplemented with proteinase inhibitor cocktail (Sigma-Aldrich), put on ice for 30 min, and subsequently centrifuged for 20 min at 15,000 rpm. The protein concentrations were subsequently determined by Bio-Rad assay (Thermo Pierce), according to the instructions of the manufacturer.…”

“…The protocol of small interfering RNA (siRNA) transfection into RAW-D cells was described previously. 37 Briefly, 10 pmol of covering the targeted sequence: 5ʹ-CCUGCUCAGUAUUCCCUAA-3ʹ (siRab34; Invitrogen Custom Primers; Invitrogen) or with 10 pmol of nontargeting siControl (siCtrl; Stealth RNAiTMsiRNA Negative Control; Invitrogen) diluted in Opti-MEM I (Life Technologies) with Lipofectamine RNAiMAX™ transfection reagent (Invitrogen) at 37°C overnight, according to the manufacturer's instructions. On first day of posttransfection, the cells were replaced and cultured with new culture media containing RANKL (100 ng/ml) for 3 days.…”

“…The protocol of subcellular fractionations using NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific) was described previously. 37 Briefly, cells were lysed with CER I with protease inhibitors, then added with ice-cold CER II, vortexed for 10 s, incubated on ice for 1 min, and centrifuged at 500g for 10 min at 4°C. The pellets were put on ice, and the supernatants were further centrifuged at 16,000g for 20 min at 4°C 16,000g supernatants were collected and referred to as the cytoplasmic fraction.…”