Mutation in Osteoactivin Promotes Receptor Activator of NFκB Ligand (RANKL)-mediated Osteoclast Differentiation and Survival but Inhibits Osteoclast Function

Abstract

We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in the Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb(+)). In these D2J mice, micro-computed tomography and histomorphometric analyses revealed increased cortical thickness, whereas total porosity and eroded surface were significantly reduced in D2J mice compared with wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts and that survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3β pathway supports this observation. Furthermore, this was abrogated by the addition of recombinant OA to cultures, which restored osteoclastogenesis to wild-type levels. Moreover, mix and match co-cultures demonstrated an induction of osteoclastogenesis in D2J osteoblasts co-cultured with osteoclasts of D2J or wild-type. Last, in functional osteo-assays, we show that bone resorption activity of D2J osteoclasts is dramatically reduced, and these osteoclasts present an abnormal ruffled border over the bone surface. Collectively, these data support a model whereby OA/Gpnmb acts as a negative regulator of osteoclast differentiation and survival but not function by inhibiting the ERK/AKT signaling pathways.

Keywords: Akt; MAP kinases (MAPKs); bone; bone marrow; osteoactivin; osteoclast; osteopetrosis.

FIGURE 1.

Cortical bone mass is increased in D2J mice. Shown is μCT analysis of femoral diaphyses of 16-week-old OA/Gpnmb mutant (D2J) and wild-type (D2J/Gpnmb⁺) mice. A, representative two-dimensional μCT sagittal section of femoral diaphysis showing decreased width of marrow medullary cavity. Dotted line, 1000 μm of the diaphysis subjected to μCT analysis. B, representative μCT images of cross-sections of femoral diaphysis from 16-week D2J and D2J/Gpnmb⁺ showing decreased width of medullary cavity (double-headed arrows), increased cortical width (opposite arrowheads), and decreased cortical porosity (small arrows) in D2J compared with D2J/Gpnmb⁺ mice. C, representative three-dimensional μCT reconstructed images of the axial plane of femoral diaphysis at two different angles showing decreased resorption pits and tracks on the endosteal surface in D2J mice (black arrows). D–N, μCT parameters (femur total cross sectional area (Tt.Ar) (D), cortical bone area (Ct.Ar) (E), cortical area fraction (Ct.Ar/Ct.Ar) (F), cortical bone thickness (Ct.Th) (G), marrow area (Ma.Ar) (H), cortical porosity (Ct.Po) (I), pore number (Po.N) (J), total pore volume (Po.V) (K), and average pore volume (avg. Po.V) (L)) were calculated in D2J and D2J/Gpnmb⁺ (n ≥ 4). M and N, μCT parameters in 4-, 8-, and 16-week D2J and D2J/Gpnmb⁺. D2J samples overall show an increased trend over time in cortical thickness (M) and decreased cortical porosity (N) compared with D2J/Gpnmb⁺. The mean value is represented in all graphs. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Error bars, S.E.

FIGURE 2.

Bone resorption markers are decreased in D2J mice. Serum ELISA was measured in 4- and 8-week-old mice for the osteoclast formation marker RANKL (A), the osteoclast-impeding marker OPG (B), and the ratio of RANKL/OPG (C), which was not different between D2J and D2J/Gpnmb (n ≥ 5). Serum ELISA of osteoclast marker TRAP5b (D), bone resorption marker CTX-1 (E), and the ratio of CTX-1/TRAP5b (F) were measured and showed significant reduction in total CTX-1 and CTX-1/TRAP5b in D2J compared with D2J/Gpnmb⁺ mice (n = 6). The mean value is represented in all graphs. *, p < 0.05; **, p < 0.01; ****, p < 0.0001. Error bars, S.E.

FIGURE 3.

OA/Gpnmb mutation inhibited D2J osteoclast function in vivo. A and B, undecalcified sagittal sections of femurs were prepared from 8-week-old D2J and wild-type mice. Sections were stained for TRAP and counterstained with toluidine blue. TRAP-positive osteoclasts appear red. A, low magnification images show the width of cortical diaphysis (opposite arrows). B, high magnification images show TRAP-positive osteoclasts facing the trabecular surface (arrows) in D2J. C, high magnification of TRAP-positive osteoclasts on cortical bone surface. Note the smooth surface in D2J compared with bony erosions in D2J/Gpnmb⁺. D–I, histomorphometric analysis of the trabecular and cortical osteoclasts (total number of TRAP-positive osteoclasts (N.Oc)/bone perimeter (B.Pm) (D), number of multinucleated osteoclasts (N.mOc)/bone perimeter (B.Pm) (E), size of multinucleated osteoclasts × 10³ (mm²) (F), percentage of eroded surface (ES)/bone surface (BS) (G), number of resorption pits (N.Pit)/bone perimeter (H), and average depth of resorption pit (μm) (I)) were performed in D2J and D2J/Gpnmb⁺ (n ≥ 4). The mean value is represented in all graphs. *, p < 0.05; **, p < 0.01. Scale bars, 200 μm (A) and 10 μm (B and C). Error bars, S.E.

FIGURE 4.

Enhanced differentiation of D2J osteoclasts ex vivo. A–E, OCP from D2J and D2J/Gpnmb⁺ were differentiated into osteoclasts with RANKL (1, 5, 10, 20, and 50 ng/ml) and TRAP-stained. A, microscopic images show hyperresponsiveness to RANKL of TRAP-positive osteoclasts (arrows) and their size (double-headed arrows) in D2J compared with D2J/Gpnmb⁺. Parameters of osteoclast differentiation that include TRAP activity (B), count of TRAP-positive osteoclasts (≥3 nuclei) (C), size of TRAP-positive osteoclasts (D), and differential count of osteoclasts at 20 ng/ml RANKL (E) are significantly increased in D2J compared with wild type. F–J, OCP cells from D2J and D2J/Gpnmb⁺ were differentiated into osteoclasts with 20 ng/ml RANKL for 2–4 days. F, microscopic images show a temporal increase of TRAP-positive osteoclasts and their size in D2J compared with wild type. Parameters of osteoclast differentiation that include TRAP activity (G), count of TRAP-positive osteoclasts (3–19 nuclei) (H), osteoclast count (≥20 nuclei) (I), and osteoclast size (J) are significantly increased in D2J compared with D2J/Gpnmb⁺. K and L, hematopoietic stem cells from D2J and D2J/Gpnmb⁺ mice were immunostained for CD11b, CD3, and CD45R and subjected to flow cytometry. The data are presented in pseudocolor density plots, whereas the green box represents CD11b⁺CD3⁻CD45R⁻ cells and the red box represents CD11b⁻CD3⁻CD45R⁻ cells (K). Data were quantified (TN, triple negative) (L). M–P, OCP from D2J and D2J/Gpnmb⁺ were differentiated into osteoclasts with RANKL and MCSF in the presence or absence of rOA protein (10 and 100 ng/ml). M, microscopic images show decreased number and size of D2J osteoclasts in RANKL and rOA compared with D2J with no rOA treatment. TRAP activity (N), count of TRAP-positive osteoclasts (O), and size of osteoclasts (P) were reduced in D2J osteoclasts differentiated with RANKL and rOA compared with D2J without rOA. Data presented in all graphs represent mean ± S.E. (error bars) in six replicates per condition. The experiment was repeated six times and showed similar patterns. The mean value is represented in all graphs ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Scale bars, 200 μm.

FIGURE 5.

D2J osteoblasts stimulate osteoclastogenesis in a mix and match experiment. A, osteogenesis array of tibia showing increased RANKL expression in the calvaria of D2J compared with D2J/Gpnmb⁺ in 8- and 16-week-old mice. B, quantitative PCR analysis showing increased RANKL in D2J tibia of 8-week-old mice. C–F, co-culture of D2J and D2J/Gpnmb⁺ osteoblasts and BM cells, in mix and match, was treated with vitamin D₃ (10⁻⁸ m) and PGE₂ (10⁻⁶ m) for 7 days to stimulate osteoclast differentiation. C, microscopic pictures show increased TRAP-positive osteoclasts (arrows) and their size (double-headed arrows) in D2J osteoblasts co-cultured with D2J or wild-type BM. Osteoclast differentiation parameters that include TRAP activity (D), count of TRAP-positive osteoclasts (E), and osteoclast size (F) were exponentially increased in D2J osteoblast co-cultures compared with D2J/Gpnmb⁺. G and H, comparative quantitative PCR analysis of calvarial osteoblasts showing increased MCSF (G) and RANKL (H) in D2J osteoblasts compared with D2J/Gpnmb⁺. I, ELISA showing increased RANKL/OPG ratios in D2J osteoblasts of neonatal calvaria and adult long bones compared with wild type. J, Western blot and densitometry (K) of calvarial osteoblasts showing increased production of RANKL in D2J compared with D2J/Gpnmb⁺. Experiments were repeated at least three times and showed similar results. The mean value is represented in all graphs ± S.E. (error bars). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Scale bars, 200 μm.

FIGURE 6.

Temporal expression of osteoclast markers in D2J osteoclasts. A–L, comparative quantitative RT-PCR analyses of mRNA collected from D2J and D2J/Gpnmb⁺ BM cells, OCP, and 2- and 4-day RANKL-differentiated osteoclasts, were performed for the following genes: osteoactivin (OA/Gpnmb) (A), PU.1 (B), MITF (C), TRAP (D), DC-STAMP (E); cFMS (F), RANK receptors (G), c-Fos (H), IKKβ (I), NFκB (J), RelA/p65 (K), and NFATc1 (L). Experiments were repeated three times and showed similar results. Data presented in all graphs represent mean ± S.E. (error bars) in duplicate per sample. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

The MAPK pathway is enhanced in D2J differentiated osteoclasts. A, ELISA of OA in the cell lysates and conditioned medium of BM cells, OCP, and 2- and 4-day RANKL-differentiated osteoclasts in D2J and D2J/Gpnmb⁺. B–S, OCP from D2J and D2J/Gpnmb⁺ were differentiated into osteoclasts for 2 and 4 days and serum-starved for 1 h before treatment with RANKL (20 ng/ml) at the indicated time points. Cell lysates were collected for Western blot analysis. Blots were probed with anti-RANK (RANK) (B), anti-phospho-NFκB (p-NFκB) (D), anti-NFATc1 (NFATc1) (F), anti-phospho-ERK (p-ERK) (H and N), anti-phospho-P38 (p-P38) (J and P), and anti-phospho-JNK (p-JNK) (L and R). The blots were stripped and reprobed with the respective proteins to determine loading in the bottom panels. To correct for experimental variability, the amount of total proteins in individual bands was quantified, and the ratios of phosphorylated proteins to total proteins were calculated. C, RANK/actin; E, phospho-NFκB/NFκB; G, NFATc1/actin; I and O, phospho-ERK/ERK; K and Q, phospho-P38/P38; M and S, phospho-JNK/JNK. Note that D2J osteoclasts overall have significantly increased levels of RANK and NFATc1 and phosphorylation of ERK, P38, and JNK compared with wild type at both 2 and 4 days; however, no significant increase was seen for phospho-NFκB. A representative Western blot of three experiments is shown. Data presented in C, E, G, I, O, K, Q, M, and S were quantitated from three independent blots. Data presented are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01.

FIGURE 8.

Enhanced survival of D2J osteoclasts ex vivo. A and B, comparative quantitative RT-PCR analyses of RNA collected from BM cells, OCP, and 2- and 4-day RANKL-differentiated osteoclasts, showing decreased BCL2 expression (survival) (A) and increased BAX pro-apoptotic (B) transcription factors in D2J compared with D2J/Gpnmb⁺. C–F, mature osteoclasts from D2J and D2J/Gpnmb⁺ were serum-starved for 1 h prior to treatment with RANKL (20 ng/ml) at the indicated time points. Blots were probed with anti-phospho-AKT (p-AKT) (C) and anti-phospho-GSK-3β (p-GSK-3β) (E) in the top panel. The blots were stripped and reprobed with the respective total AKT and GSK-3β proteins to determine loading (bottom panels). The ratio of phosphorylated proteins to total proteins was measured to correct for experimental variability: phospho-AKT/AKT (D) and phospho-GSK-3β/GSK-3β (F). G–I, mature osteoclasts from D2J and D2J/Gpnmb⁺ were treated with AKT inhibitor LY2940 (1–5 μm) with/without RANKL (20 ng/ml) for 48 h. G, microscopic image shows the surviving osteoclasts. Parameters of osteoclast differentiation that include TRAP activity of surviving osteoclasts (H) and count of TRAP-positive surviving osteoclasts (I) were increased in D2J compared with D2J/Gpnmb⁺. Graphs represent the data as a percentage of the initial count of plated mature osteoclasts surviving after 48 h. J–M, mature osteoclasts are serum-starved for 1 h before treatment with LY2940 and/or RANKL (20 ng/ml) for 16 h. Cell lysates were collected for Western blot analysis. Blots were probed with anti-phospho-AKT (J) and anti-Bim apoptotic marker (L). Experimental variability was corrected by quantifying total proteins and measuring the ratio of phospho-AKT/AKT (K) and Bim/actin (L). N and O, mature osteoclasts from D2J and D2J/Gpnmb⁺ were treated with RANKL in the presence/absence of rOA. Proteins from cell lysates were subjected to SDS-PAGE and probed for phospho- and total AKT (N) and quantified by densitometry (O). Caspase-Glo 3/7 activity of mature osteoclasts without MCSF for 16 h is decreased in D2J compared with D2J/Gpnmb⁺ (P). Experiments were repeated at least three times and showed similar results. Data presented in all graphs represent mean ± S.E. (error bars) in six replicates per condition. The experiment was repeated three times and showed similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 9.

Decreased resorptive activity of D2J osteoclasts. A–C, OCP were differentiated into osteoclasts over osteo-assay discs with RANKL for 4 days. A, microscopic images show larger rounded D2J osteoclasts, surrounded by a minimal clear zone, compared with wild type. B, microscopic images show less resorbed tracks (representative analyses are shown within the blue dotted circle) in D2J compared with D2J/Gpnmb⁺. Scale bars, 200 μm. C, percentage area fraction of the clear resorbed area over non-resorbed area in D2J and D2J/Gpnmb⁺ osteoclasts in the presence or absence of rOA (100 ng/ml). Note the decrease in resorption in D2J compared with wild type. D, CTX-1 values in the conditioned medium of D2J and wild-type osteoclasts, differentiated over a human osteo-assay for 2 and 4 days. E–J, OCP were differentiated on collagen-precoated 6-well plates. Mature osteoclasts were seeded with RANKL on cortical bone slices for 72 h. E, microscopic images show TRAP-stained large D2J osteoclasts compared with D2J/Gpnmb⁺ (yellow arrows). F, microscopic images of resorption pits after removal of osteoclasts and staining with toluidine blue. Note the smaller resorption pit in D2J (yellow arrows). G, average resorption area per osteoclast with a significant reduction in D2J compared with D2J/Gpnmb⁺. H, CTX-1 values in the conditioned media of D2J and D2J/Gpnmb⁺ osteoclasts. I, microscopic images of the F-actin ring in osteoclasts (yellow arrows). Note the abnormal localization of F-actin in D2J compared with wild type. J, scanning electron micrographs of D2J and D2J/Gpnmb⁺ osteoclasts on bone slices. Note the significant resorption pit (yellow arrow) made by wild-type osteoclast compared with D2J. K, transmission electron microscopy of D2J osteoclast in vivo showing the larger osteoclast setting on the bone surface (no lacuna) with large intracellular vesicles (V), absence of secretory domain (SD), and few short ruffled borders (inset) compared with the D2J/Gpnmb⁺ osteoclast with the normal appearance of a ruffled border (RB) and the presence of a secretory domain. Experiments were repeated three times and showed similar results. Data presented represent mean ± S.E. (error bars) in triplicate per strain. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 10.

Schematic diagram of osteoactivin effects on osteoclast differentiation, survival, and function. Mutation of Gpnmb (XGpnmbX) promotes osteoclast differentiation mediated by MAPK phosphorylation and up-regulation of osteoclastogenic factors: PU.1, RANK receptors, DC-STAMP, and NFATc1. Mutation of Gpnmb also promotes osteoclastogenesis by stimulating RANKL production of osteoblasts. Moreover, mutation of Gpnmb enhances osteoclast survival, perhaps by phosphorylation of the AKT-GSK3β pathway and regulated expression of BCL2 and Bim. Finally, mutation of Gpnmb inhibits osteoclast activity in bone resorption by altering the cytoskeletal organization through an unknown mechanism yet to be determined.