To mechanistically evaluate the transition from osteoclast precursors to multinucleated osteoclasts, we incubated primary human monocytes with recombinant macrophage colony-stimulating factor (M-CSF) and then with M-CSF and recombinant RANKL (Whitlock et al., 2023; Asagiri and Takayanagi, 2007). While the time course and efficiency of multinucleated osteoclast formation vary from donor to donor (Whitlock et al., 2023), on average, we observed the appearance of small, multinucleated syncytia 2 days after RANKL application and fusion rapidly increased over the next 2 days, resulting in mature, resorption competent osteoclasts at 4–5 days post-RANKL addition (Whitlock et al., 2023). Previously, we demonstrated that the cell fusion stage of multinucleated osteoclast formation depends on La trafficking to the surface of the osteoclasts at days 2–4 post-RANKL application (Whitlock et al., 2023); however, the mechanisms that trigger La’s trip to the osteoclast surface and what molecular requirements must be met for its unconventional fusion role there remained open questions.

To address open questions concerning La’s surface trafficking and molecular function in osteoclast multinucleation, we evaluated the redox state of La in fusing osteoclasts using recently validated monoclonal α-La antibodies that recognize oxidized La (clone 7B6) or reduced La (clone 312B), or do not distinguish between these La species (Pan, clone 5B9) (Berndt et al., 2021a). At the time of fusion, immunofluorescence analysis of La’s localization in permeabilized osteoclasts with pan α-La antibody demonstrated that La is present in both the cytoplasm and nuclei (Figure 1a). In stark contrast, the cytoplasmic/plasma membrane-associated pool was recognized by the α-La antibody that recognizes oxidized La species, while the α-La antibody that recognizes reduced La species recognized the nuclear protein pool (Figure 1a; Figure 1—figure supplement 1). Under non-permeabilizing conditions – focusing on the exofacial surface of the plasma membrane – we readily observed La at the surface of fusing osteoclasts (see also Whitlock et al., 2023). We find that this surface pool of La is dramatically enriched in oxidized, rather than reduced, La species (Figure 1b). In further support of this conclusion, we found that application of the membrane-impermeable reducing reagent Tris (2-carboxyethyl) phosphine (TCEP) dramatically decreased the recognition of surface La by the α-La antibody that recognizes the oxidized species and increased the recognition of surface La by the α-La antibody that recognizes the reduced species (Figure 1b, c). In contrast, TCEP had no effect on the ability of the pan α-La antibody to recognize surface La (Figure 1b, c) or on the surface detection of La with another α-La antibody (Abcam #75927) that we have used previously to evaluate La at the surface of fusing osteoclasts (Whitlock et al., 2023) and now found to recognize both reduced and oxidized species of La (data not shown). These findings indicate that the surface pool of La that manages osteoclast size and resorptive function is primarily composed of an oxidized La molecular species. Interestingly, TCEP treatment did not dissociate cell surface La from plasma membrane, suggesting that the reduced species of La retains its ability to associate with the plasma membrane.

We further assessed the functional importance of surface La’s redox status in synchronized osteoclast fusion. As in earlier studies (Whitlock et al., 2023; Verma et al., 2014; Verma et al., 2018), we uncoupled the cell fusion stage of osteoclast formation from pre-fusion differentiation processes using lysophosphatidylcholine (LPC), a reversible inhibitor of an early stage of membrane rearrangement required for osteoclast fusion. LPC was applied for 16 hr following 2 days of RANKL elicited osteoclastogenesis. Ready-to-fuse cells, which could not fuse in the presence of LPC, rapidly fused after LPC wash out (Figure 2a, b). As we demonstrated previously with another pan α-La antibody (Whitlock et al., 2023), pan α-La antibody 5B9 applied at the time of LPC removal inhibited synchronized osteoclast fusion (Figure 2a, b). Similarly, α-La antibodies that recognize the oxidized species inhibited fusion, while antibodies that recognize the reduced La species had no effect on fusion (Figure 2a, b). These data strongly support the conclusion that the functional La species that promotes osteoclast fusion and function is an unconventional, cell surface-associated, oxidized species. In further support of the functional importance of surface La oxidation, and possibly other surface proteins, we find that reducing the surface of human osteoclasts via TCEP treatment – which reduces surface La but does not alter its membrane association (Figure 1c) – inhibits synchronized osteoclast fusion in a dose-dependent manner (Figure 2c).

To further validate the role of La’s redox status in osteoclast multinucleation and resorptive function, we chose to assess the functional impact of perturbing the oxidation status of recombinant La. Previously, we showed that the C-terminal half of La produced recombinantly and added to the medium bathing differentiating osteoclast precursors binds the surface of these cells and is sufficient to promote osteoclast fusion and resorptive function (Whitlock et al., 2023). We produced La 194–408 recombinantly and reduced a portion of the protein with TCEP followed by the application of iodoacetamide to block the free thiols in La 194–408, preventing their subsequent oxidation. When we compared the effects of La 194–408 vs reduced La 194–408 on primary human osteoclast multinucleation, we found that the reduction of recombinant La greatly diminished its ability to promote osteoclast multinucleation and resorption (Figure 3a–c).

Many of the effects of redox signaling are mediated by the oxidation of cysteine residues within proteins (van der Reest et al., 2018). Human La has three cysteine residues. However, we have previously demonstrated that the N-terminal half of La (La 1–187), containing one of La’s cysteines, is dispensable for La’s role in promoting osteoclast fusion (Whitlock et al., 2023). Therefore, we focused on the two cysteines in the C-terminal half of La: Cys 232 and Cys 245. We recombinantly produced a La 194–408 mutant where cysteines 232 and 245 were mutated to alanine residues (La Cys Mut) (Figure 4a, b; Figure 4—figure supplement 1a). Interestingly, we find that a fraction of La 194–408 migrates as a band ~twice the predicted size of La 194–408 despite separation via denaturing gel electrophoresis, suggesting the presence of La 194–408 dimers (Berndt et al., 2021a; Craig et al., 1997). In contrast, we observe no dimer band in La Cys Mut (Figure 4—figure supplement 1b, c). In addition, we find that the loss of Cys 232 and Cys 245 greatly diminishes the affinity of the α-La antibody that recognizes the oxidized La species, whereas both species are recognized by an α-6xHis monoclonal antibody via their N-terminal His tags (Figure 4b). Finally, we found that the loss of Cys 232 and Cys 245 strongly abrogates the ability of La 194–408 to promote osteoclast fusion (Figure 4c, d). From these data, we conclude that Cys 232 and Cys 245 – previously highlighted for their roles in redox-dependent structural changes in La (Berndt et al., 2021a) – are vital for La’s function in osteoclast multinucleation.

To summarize, La promotes osteoclast fusion as an unconventional, cell surface-associated, oxidized species. Moreover, our data suggest that the conformational transition from reduced to oxidized La species that is critical for the protein’s role in osteoclast formation and function depends on cysteine residues within its C-terminal half.

The La pool within the nuclei of eukaryotic cells is primarily a reduced species (Berndt et al., 2021a; Berndt et al., 2021b). Finding that the surface La pool, which promotes multinucleation in osteoclasts, is enriched in an oxidized species raised the question of where La becomes oxidized? Is La oxidation in osteoclasts a consequence of surface delivery or does La oxidation precede surface trafficking?

Finding that La in permeabilized differentiating osteoclasts is recognized by the α-La antibody that recognizes oxidized rather than reduced La species (Figure 1a and Figure 1—figure supplement 1) suggested that the oxidation of La takes place in the cytoplasm of forming osteoclasts prior to delivery to the oxidizing environment of their surface. To further address this question, we utilized a cell-permeable reducing reagent N-acetylcysteine (NAC) that has been widely used to inhibit intracellular ROS generation and suppress cytosolic redox signaling (Zafarullah et al., 2003; Sun, 2010). Since under some conditions complex indirect effects of NAC raise rather than lower ROS levels (Pedre et al., 2021; Mlejnek et al., 2021; Murphy et al., 2022), we verified the ROS-lowering effects of NAC in osteoclast precursors using a previously validated, cell-permeable ROS probe CellROX Deep Red (Lee et al., 2005). We found that NAC treatment suppresses ROS elevation stimulated by RANKL-initiated differentiation of human osteoclasts (Figure 5a, b). While early treatment with NAC has been reported to disrupt the pre-fusion stages of osteoclast differentiation (Lee et al., 2005), we found that NAC treatment 24 hr after RANKL application (i.e., after early osteoclast precursor commitment), did not lower the steady-state levels of osteoclastogenic differentiation factors (NFATc1 and cFOS) or the transcripts of two fusion-related proteins (La or AnxA5) (Figure 5c). We then explored the effects of suppressing ROS signaling on osteoclast formation and the trafficking of La, as in Cao and Picklo, 2014; Kim et al., 2019, we found that NAC-mediated inhibition of ROS signaling suppresses osteoclast multinucleation in a dose-dependent manner (Figure 5d). Suppressing ROS signaling with NAC also inhibited the transition from reduced to oxidized, cytoplasmic La species, as evidenced by decrease in staining of permeabilized osteoclasts with α-oxidized La antibody and a complementary increase in staining of permeabilized osteoclasts with α-reduced La antibody (Figure 5—figure supplement 1a–d). Most importantly, NAC application inhibited the cell surface delivery of La in human osteoclasts (Figure 5e). These findings suggest that NAC inhibits La function in osteoclasts by suppressing ROS-induced oxidation of La and its delivery to the surface of osteoclasts. Moreover, our findings also indicate that the transition from reduced to oxidized La species occurs inside the osteoclast cytoplasm during their commitment program rather than being a consequence of La arriving at the oxidizing environment of the extracellular milieu.

While most of La in human cells is phosphorylated at Ser 366 (Schwartz et al., 2004), at the time of fusion, La is mostly dephosphorylated, as evidenced by a loss of the cell staining with antibodies specific for La phosphorylated at Ser366 (α-p366 La Ab) (Whitlock et al., 2023). We find that while little phosphorylated La is typically observed in the nuclei of differentiating osteoclasts during fusion timepoints, that NAC suppression of transient ROS signaling greatly increases phosphorylated La in osteoclasts (Figure 6a, b). These data indicate that inhibition of intracellular ROS generation prevents La dephosphorylation and the loss of nuclear localization in differentiating osteoclast precursors. These findings substantiate the hypothesis that the redox signaling, which triggers a shift in La functional properties, is important for osteoclast fusion and takes place inside the cell.

We found that NAC application both inhibits osteoclast multinucleation and suppresses delivery of oxidized La species to the cell surface of fusing osteoclasts (Figure 5d, e). These findings motivated us to explore whether the suppressed osteoclast multinucleation caused by NAC treatment could be the result of deficient La delivery to the cell surface. If the suppressed fusion observed following NAC treatment is a consequence of suppressing an ROS triggered ‘La trafficking switch’, then perhaps simply adding La to the medium bathing NAC-treated cells can rescue La surface pools and fusion? Indeed, we found that application of La 194–408 to NAC-treated cells rescued osteoclast fusion inhibition (Figure 6c, d). However, the ability of La 194–408 to rescue NAC-inhibited osteoclast fusion is at l=east partially dependent on Cys 232 and Cys 245, as mutation of these residues to Ala greatly diminished the ability of La 194–408 to rescue NAC effects on fusion. Finding that the NAC-mediated ROS suppression of surface trafficking and fusion can be compensated for by the application of exogenous La strongly supports the conclusion that ROS signaling plays a vital role in triggering La’s delivery to the surface of osteoclasts and the promotion of their multinucleation. Moreover, the loss of La’s ability to rescue NAC suppression of osteoclast multinucleation when Cys 232 and Cys 245 are mutated to Ala also suggests that these residues are vital for oxidized La’s ability to promote osteoclast multinucleation and resorptive function.

In summary, ROS signaling downstream of osteoclast commitment contributes to the cell fusion stage of osteoclast formation by promoting intracellular oxidation of La and its delivery to the surface of fusion-committed cells, where La promotes multinucleation and subsequent resorptive activity in human osteoclasts (Figure 7).

Human M-CSF and RANKL were purchased from Cell Sciences (Catalogue # CRM146B and CRR100B, respectively). LPC (1-lauroyl-2- hydroxy-sn-glycero-3-phosphocholine, # 855475); PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, # 850375 C) was purchased from Avanti Polar Lipids. Bone Resorption Assay Kits were purchased from Cosmo Bio Co (Catalogue # CSR-BRA-24KIT) and used according to the manufacturer’s instructions. Hoechst 33342 and phalloidin-Alexa 555 were purchased from Invitrogen (# H3570 and A30106, respectively). TCEP was purchased from Thermo Fisher Scientific (Pierce TCEP-HCl; Catalogue # A35349). NAC (N-acetyl-L-cysteine) and sodium iodoacetamide were purchased from Sigma-Aldrich (Catalogue # A9165 and GERPN6302).

Elutriated monocytes from healthy donors were obtained through the Department of Transfusion Medicine at National Institutes of Health under protocol 99-CC-0168 approved by the National Institutes of Health Institutional Review Board. Research blood donors provided written informed consent and blood samples were de-identified prior to distribution, Clinical Trials Number: NCT00001846. We also used elutriated monocytes from healthy donors obtained through Elutriation Core Facility, University of Nebraska Medical Center, informed consent was obtained under an Institutional Review Board approved protocol for human subject research 0417-22-FB. Research blood donors provided informed consent and samples were de-identified prior to distribution. Primary human osteoclasts were derived as described previously (Whitlock et al., 2023). Briefly, elutriated monocytes were added to complete media [α-minimal essential media (α-MEM) (Gibco) + 10% fetal bovine serum (FBS) (Gibco) + 1× penicillin/streptomycin/glutamate (Gibco)] supplemented with 100 ng/ml recombinant M-CSF and plated at 1 × 10⁶ cells/ml for 6 days (refreshing media at day 3). Next, the cells were placed into complete medium supplemented with 100 ng/ml recombinant M-CSF and 100 ng/ml recombinant RANKL to induce osteoclastogenesis 3–4 days to obtain multinucleated, resorption competent human osteoclasts.

Murine monoclonal α-La antibodies that specifically recognize oxidized and reduced species of La or both species of La (7B6, 312B, and 5B9, respectively Berndt et al., 2021a), referred to as oxidized La Ab, reduced La Ab, and pan α-La Ab were described and characterized by the laboratory of Dr. Michael Bachmann. The antibodies were produced recombinantly as described in Berndt et al., 2021a.

We also used rabbit anti-La Phospho-Ser366 antibody (Abcam, 61800), referred to as α-p366 La Ab that recognizes phosphorylated human La (phosphoSer366). We also used an additional monoclonal murine α-La antibody (α-La mAb; Abcam, Catalogue # 75927) that we found to recognize both oxidized and reduced forms of La. A α-6xhis murine monoclonal antibody (Abcam, ab18184) was used to recognize the 6xhis tag covalently modifying our recombinantly produced La protein fragments and α-Cyclophilin B (Cell Signaling Technology, D1VdJ Rabbit mAb #43603) as a loading control.

Constructs encoding recombinant La 194–408 and the cysteine mutants used in this manuscript were previously described, characterized, and provided by the Bachmann Lab. Each was transformed into BL2 (DE3) chemically competent Escherichia coli (Thermo Fisher Scientific) and recombinant protein production was induced via isopropyl-beta-D-thiogalactoside induction (Sigma). Cells were lysed with BugBuster HT (Millipore) supplemented with protease inhibitors (Complete, Pierce), 6xHis-La proteins were purified using HisPur Cobalt Spin columns (Thermo Fisher Scientific), and endotoxin was removed via Pierce high-capacity endotoxin removal columns (Thermo Fisher Scientific), each according to the manufacturer’s instructions. Proteins were sterile filtered, aliquoted, and kept at −80°C. Some La 194–408 was irreversibly modified via 45-min incubation with 10 mM TCEP followed by a 45-min incubation with 10 mM iodoacetamide. Modified protein was then subsequently exchanged into phosphate buffered saline (PBS) (Gibco) using 10 K MW concentrators according to the manufacturer’s instructions (Amicon). Control La 194–408 for these experiments was treated identically, except for the omission of TCEP and iodoacetamide.

For high-resolution immunofluorescence analysis of protein localization, we washed cells with PBS and then rapidly fixed with warm, freshly prepared 4% formaldehyde in PBS at 37°C. The cells were subsequently washed with PBS. To permeabilize cells, we incubated them for 5 min in 0.1% Triton X-100 in PBS. The cells were subsequently stained in PBS supplemented with 10% FBS for 10 min at room temperature to suppress non-specific binding. Then, cells were incubated with primary antibodies for 1 hr in PBS supplemented with 10% FBS. After five washes in PBS, we incubated the cells with fluorescent secondary FAB fragments raised to the species corresponding to the primary antibody for 1 hr in PBS supplemented with 10% FBS (either Anti-rabbit IgG Fab2 Alexa Fluor 555 or Anti-mouse IgG Fab2 Alexa Flour 488, both Cell Signaling Technology, Catalogue # 647 4414 S and 4408 S, respectively, in 1:500 dilution) and then washed five times with PBS prior to imaging. For non-permeabilized conditions, we followed the same protocol, but omitted all use of detergents.

Images were captured on a Zeiss LSM 800, confocal microscope using a C-Apochromat 63×/1.2 water immersion objective lens.

For basic immunofluorescence analysis of cell fusion and morphology, we stained osteoclast cytoskeletal boundaries with Phallodin-Alexa Flour 488 (Thermo Fisher Scientific, 1:2000) and nuclei with Hoechst 33342 (Thermo Fisher Scientific, 1:5000). Cells were fixed as described above, washed with PBS, and stained with toxin/dyes for 1 hr in complete staining buffer (PBS + 5% FBS + 0.1% TX100) before a final PBS wash prior to imaging. Ten selected fields of view were imaged on a grid from the center of each well/dish in automated fashion using Alexa 488, Hoechst and phase contrast compatible filter cubes (BioTek) on a Lionheart FX microscope using a 10×/0.3 NA Plan Fluorite WD objective lens (BioTek) using Gen3.10 software (BioTek). Each image was separated by approximately 1800 µm in both x and y parameters.

All image data were evaluated using Fiji/ImageJ’s open-source image processing package v.2.1.0/1.53c.

Osteoclast fusion efficiency was evaluated as the number of fusion events between cells in 10 images. In brief, since regardless of the sequence of fusion events, the number of cell-to-cell fusion events required to generate syncytium with N nuclei is always equal to N − 1, we calculated the fusion number index as Σ(Ni − 1) = Ntotal − Nsyn, where Ni = the number of nuclei in individual syncytia and Nsyn = the total number of syncytia. We normalized the number of fusion events to the total number of nuclei (including unfused cells) to control for small variations in cell density from dish to dish and image to image. In contrast to traditional fusion index measurements, this approach gives equal consideration to fusion between two mononucleated cells, one mononucleated cell and one multinucleated cell and two multinucleated cells. In traditional fusion index calculations, fusion between two multinucleated cells does not change the percentage of nuclei in syncytia. If instead one counts the number of syncytia, a fusion event between two multinucleated is not just missed but decreases the number of syncytia. In contrast, the fusion number index is inclusive of all fusion events.

Osteoclast fusion was synchronized as described in Verma et al., 2014. Briefly, osteoclast media was refreshed with media supplemented with 100 ng/ml M-CSF, 100 ng/ml RANKL, and 350 μM lauroyl-LPC 72 hr post-RANKL treatment. Following 16 hr, LPC was removed via five washes with fresh media and cells were allowed to fuse in the presence or absence of antibody treatment or recombinant La at the concentrations described in the figure legends for 90 min.

For real-time polymerase chain reaction (PCR), total RNA was collected from cell lysates using PureLink RNA kit following the manufacturer’s instructions (Invitrogen # 12183018 A). cDNA was generated from total RNA via reverse transcription reactions using a High-Capacity RNA-to-cDNA kit according to the manufacturer’s instructions (Applied Biosystems, # 4387406). cDNA was then amplified using the iQ SYBR Green Supermix (Bio-Rad). All primers were predesigned KiCqStart SYBR Green primers with the highest rank score specific for the gene of interest or glyceraldehyde 3-phosphate dehydrogenase (GAPHD) control and were used according to the manufacturer’s instructions (Sigma). All real-time PCR reactions were performed and analyzed on a CFX96 real-time system (Bio-Rad), using GAPDH as an internal control. Fold-change of gene expression was determined using the ΔΔCt method. Three to four independent experiments were performed, and each was analyzed in duplicate.

Mineral resorption was evaluated using mineral resorption assay kits from Cosmo Bio USA according to the manufacturer’s instructions. In short, fluoresceinamine-labeled chondroitin sulfate was used to label 24-well, calcium phosphate-coated plates. Human, monocyte-derived osteoclasts were differentiated as described above, using α-MEM without phenol red. Media were collected at 4–5 days post-RANKL addition, and fluorescence intensity within the media was evaluated as recommended by the manufacturer. Data were normalized to the level of fluorescence released by control cells where RANKL was not added.

Statistical analyses were performed using Prism software (GraphPad Prism version 8.0.0). Unless stated in the legend, differences between groups were observed in each experiment, cells from each donor were paired across the conditions described, and statistical significance was assessed via Student’s t-test. Due to the inherent variability in the derivation of primary human monocytes to osteoclast, we analyzed statistical significance using a ratio paired t-test, where the raw values for the assay are logarithmically transformed and then assessed, when the precise time course of osteoclast differentiation and baseline extents of fusion varied considerably from donor to donor. The quantitated results presented all represent the mean ± the standard error of the mean. While the p values for each statistical comparison are defined in the legends of each figure, we graphically represented our statistical evaluations using the following symbols: ns, p = >0.05; *p = <0.05; **p = <0.01; ***p = <0.001.