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The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget's disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget's disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget's, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival., Competing Interests: No authors have competing interests.

Disulfiram (DSF), a cysteine modifying compound, has long been clinically employed for the treatment of alcohol addiction. Mechanistically, DSF acts as a modulator of MAPK and NF-κB pathways signaling pathways. While these pathways are crucial for osteoclast (OC) differentiation, the potential influence of DSF on OC formation and function has not been directly assessed. Here, we explore the pharmacological effects of DSF on OC differentiation, activity and the modulation of osteoclastogenic signaling cascades. We first analyzed cytotoxicity of DSF on bone marrow monocytes isolated from C57BL/6J mice. Upon the establishment of optimal dosage, we conducted osteoclastogenesis and bone resorption assays in the presence or absence of DSF treatment. Luciferase assays in RAW264.7 cells were used to examine the effects of DSF on major transcription factors activation. Western blot, reverse transcription polymerase chain reaction, intracellular acidification and proton influx assays were employed to further dissect the underlying mechanism. DSF treatment dose-dependently inhibited both mouse and human osteoclastogenesis, especially at early stages of differentiation. This inhibition correlated with a decrease in the expression of key osteoclastic marker genes including CtsK, TRAP, DC-STAMP and Atp6v0d2 as well as a reduction in bone resorption in vitro. Suppression of OC differentiation was found to be due, at least in part, to the blockade of several key receptor activators of nuclear factor kappa-B ligand (RANKL)-signaling pathways including ERK, NF-κB and NFATc1. On the other hand, DSF failed to suppress intracellular acidification and proton influx in mouse and human osteoclasts using acridine orange quenching and microsome-based proton transport assays. Our findings indicate that DSF attenuates OC differentiation via the collective suppression of several key RANKL-mediated signaling cascades, thus making it an attractive agent for the treatment of OC-mediated disorders.

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.

Vacuolar-type H(+)-ATPases (V-ATPases) are macromolecular proton pumps that acidify intracellular cargos and deliver protons across the plasma membrane of a variety of specialized cells, including bone-resorbing osteoclasts. Extracellular acidification is crucial for osteoclastic bone resorption, a process that initiates the dissolution of mineralized bone matrix. While the importance of V-ATPases in osteoclastic resorptive function is well-defined, whether V-ATPases facilitate additional aspects of osteoclast function and/or formation remains largely obscure. Here we report that the V-ATPase accessory subunit Ac45 participates in both osteoclast formation and function. Using a siRNA-based approach, we show that targeted suppression of Ac45 impairs intracellular acidification and endocytosis, both are prerequisite for osteoclastic bone resorptive function in vitro. Interestingly, we find that knockdown of Ac45 also attenuates osteoclastogenesis owing to a reduced fusion capacity of osteoclastic precursor cells. Finally, in an effort to gain more detailed insights into the functional role of Ac45 in osteoclasts, we attempted to generate osteoclast-specific Ac45 conditional knockout mice using a Cathepsin K-Cre-LoxP system. Surprisingly, however, insertion of the neomycin cassette in the Ac45-Flox(Neo) mice resulted in marked disturbances in CNS development and ensuing embryonic lethality thus precluding functional assessment of Ac45 in osteoclasts and peripheral bone tissues. Based on these unexpected findings we propose that, in addition to its canonical function in V-ATPase-mediated acidification, Ac45 plays versatile roles during osteoclast formation and function.

Macrophage-CSF (M-CSF) is critical for osteoclast (OC) differentiation and is reported to enhance mature OC survival and motility. However, its role in the regulation of bone resorption, the main function of OCs, has not been well characterised. To address this we analysed short-term cultures of fully differentiated OCs derived from human colony forming unit-granulocyte macrophages (CFU-GM). When cultured on dentine, OC survival was enhanced by M-CSF but more effectively by receptor activator of NFκB ligand (RANKL). Resorption was entirely dependent on the presence of RANKL. Co-treatment with M-CSF augmented RANKL-induced resorption in a concentration-dependent manner with a (200-300%) stimulation at 25 ng/mL, an effect observed within 4-6 h. M-CSF co-treatment also increased number of resorption pits and F-actin sealing zones, but not the number of OCs or pit size, indicating stimulation of the proportion of OCs activated. M-CSF facilitated RANKL-induced activation of c-fos and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but not NFκB nor nuclear factor of activated T-cells, cytoplasmic-1 (NFATc1). The mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 partially blocked augmentation of resorption by M-CSF. Our results reveal a previously unidentified role of M-CSF as a potent stimulator of mature OC resorbing activity, possibly mediated via ERK upstream of c-fos.

Microtubule organization and lysosomal secretion are both critical for the activation and function of osteoclasts, highly specialized polykaryons that are responsible for bone resorption and skeletal homeostasis. Here, we have identified a novel interaction between microtubule regulator LIS1 and Plekhm1, a lysosome-associated protein implicated in osteoclast secretion. Decreasing LIS1 expression by shRNA dramatically attenuated osteoclast formation and function, as shown by a decreased number of mature osteoclasts differentiated from bone marrow macrophages, diminished resorption pits formation, and reduced level of CTx-I, a bone resorption marker. The ablated osteoclast formation in LIS1-depleted macrophages was associated with a significant decrease in macrophage proliferation, osteoclast survival and differentiation, which were caused by reduced activation of ERK and AKT by M-CSF, prolonged RANKL-induced JNK activation and declined expression of NFAT-c1, a master transcription factor of osteoclast differentiation. Consistent with its critical role in microtubule organization and dynein function in other cell types, we found that LIS1 binds to and colocalizes with dynein in osteoclasts. Loss of LIS1 led to disorganized microtubules and aberrant dynein function. More importantly, the depletion of LIS1 in osteoclasts inhibited the secretion of Cathepsin K, a crucial lysosomal hydrolase for bone degradation, and reduced the motility of osteoclast precursors. These results indicate that LIS1 is a previously unrecognized regulator of osteoclast formation, microtubule organization, and lysosomal secretion by virtue of its ability to modulate dynein function and Plekhm1.