RANKL increases the level of Mcl-1 in osteoclasts and reduces bisphosphonate-induced osteoclast apoptosis in vitro

Abstract

Introduction: Bisphosphonates are the most widely used class of drug for inhibiting osteoclast-mediated bone loss, but their effectiveness at preventing joint destruction in rheumatoid arthritis has generally been disappointing. We examined whether the ability of bisphosphonates to induce osteoclast apoptosis and inhibit bone resorption in vitro is influenced by the cytokine receptor activator of nuclear factor-kappa B ligand (RANKL), an important mediator of inflammation-induced bone loss.

Methods: Rabbit osteoclasts were treated with the bisphosphonates clodronate or alendronate for up to 48 hours in the absence or presence of RANKL. Changes in cell morphology and induction of apoptosis were examined by scanning electron microscopy, whilst resorptive activity was determined by measuring the area of resorption cavities. Changes in the level of anti-apoptotic proteins, including Mcl-1, Bcl-2, and Bcl-x>L, were determined in rabbit osteoclasts and in cytokine-starved mouse osteoclasts by Western blotting.

Results: RANKL significantly attenuated the ability of both clodronate and alendronate to induce osteoclast apoptosis and inhibit bone resorption. Treatment of rabbit osteoclasts with RANKL was associated with an increase in the anti-apoptotic protein Mcl-1 but not Bcl-2. A role for Mcl-1 in osteoclast survival was suggested using osteoclasts generated from mouse bone marrow macrophages in the presence of RANKL + macrophage colony-stimulating factor (M-CSF) since cytokine deprivation of mouse osteoclasts caused a rapid loss of Mcl-1 (but not Bcl-2 or Bcl-xL), which preceded the biochemical and morphological changes associated with apoptosis. Loss of Mcl-1 from mouse osteoclasts could be prevented by factors known to promote osteoclast survival (RANKL, M-CSF, tumour necrosis factor-alpha [TNF-alpha], or lipopolysaccharide [LPS]).

Conclusions: RANKL protects osteoclasts from the apoptosis-inducing and anti-resorptive effects of bisphosphonates in vitro. The ability of RANKL (and other pro-inflammatory factors such as TNF-alpha and LPS) to increase the level of Mcl-1 in osteoclasts may explain the lack of effectiveness of some bisphosphonates in preventing inflammation-induced bone loss.

Figure 1

RANKL attenuates the effect of bisphosphonates on osteoclast number, apoptosis, and bone resorption in vitro. Cultures of mature osteoclasts from rabbit bones were treated with 100 μM ALN or CLO, ± 50 ng/mL RANKL for 48 hours. Cells were then fixed, stained for tartrate-resistant acid phosphatase (TRAP), and counterstained with DAPI. (a) Results are the mean number of TRAP-positive multinucleated osteoclasts (more than three nuclei per cell) ± standard error of the mean (SEM) (n = 3) or (b) the percentage of non-apoptotic and apoptotic osteoclasts. Data are expressed as the mean ± SEM (n = 3 replicates). **P = 0.01, ***P = 0.001 compared with ALN or CLO alone (analysis of variance). ^#Treatment with ALN or CLO alone caused a significant decrease in osteoclast number compared with control (CTL) cultures (P = 0.01) and a significant increase in osteoclast apoptosis compared with control cultures (P = 0.001). (c) Values of resorption area are the mean resorbed area (mm²) per slice ± SEM (n = 6 slices). ***P = 0.001 compared with CLO alone and **P = 0.01 compared with ALN alone (analysis of variance). ^#Treatment with ALN or CLO alone caused a significant decrease in osteoclastic bone resorption compared with control cultures (P = 0.001). The data shown are representative of three independent experiments. ALN, 4-amino-1-hydroxy-butylidene-1,1-bisphosphonate (alendronate); CLO, dichloromethylene-1,1-bisphosphonate (clodronate); DAPI, 4,6-diamidino-2-phenylindole; RANKL, receptor activator of nuclear factor-kappa-B ligand.

Figure 2

Scanning EM analysis of the effect of CLO, ALN, and RANKL on the morphology of mature osteoclasts cultured in vitro. Rabbit osteoclasts were cultured on ivory discs for 24 hours with 50 μM CLO or 50 μM ALN, ± 100 ng/mL RANKL. (a) Control. (b) CLO. (c) CLO + RANKL. (d) ALN. (e) ALN + RANKL. Osteoclasts were fixed and processed for scanning EM analysis. Bars = 10 μm. ALN, 4-amino-1-hydroxy-butylidene-1,1-bisphosphonate (alendronate); CLO, dichloromethylene-1,1-bisphosphonate (clodronate); RANKL, receptor activator of nuclear factor-kappa-B ligand.

Figure 3

RANKL prevents activation of caspase-9 and increases Mcl-1 in osteoclasts but does not prevent inhibition of protein prenylation. Cultures of mature osteoclasts from rabbit bones were treated with 100 μM ALN ± 100 ng/mL RANKL for 48 hours and stained using an Apofluor Green Caspase-9 Activity Assay kit and Hoechst 33342. (a) A representative non-apoptotic and apoptotic osteoclast. (b) Quantification of caspase-9-positive osteoclasts after treatment with alendronate ± RANKL for 48 hours. *P ≤ 0.05 compared with ALN alone or ^#P ≤ 0.05 compared with control (CTL) (analysis of variance). Values are the mean ± standard error of the mean (n = 3 replicates) (100 to 150 cells counted per well). The data shown are representative of three independent experiments. (c) Purified rabbit osteoclasts were treated for 48 hours with 100 μM ALN ± 100 ng/mL RANKL or with RANKL alone. Cell lysates were then analysed by Western blotting for the unprenylated form of Rap1A and for β-actin. (d) Purified rabbit osteoclasts were treated for 48 hours with 100 μM ALN ± 100 ng/mL RANKL or with 100 ng/mL RANKL alone. Cell lysates were then analysed by Western blotting for Mcl-1 and Bcl-2. The level of Mcl-2 or Bcl-2 was quantified by densitometric analysis and expressed as a ratio of the level in control cells. Data shown are representative of three independent experiments. ALN, 4-amino-1-hydroxy-butylidene-1,1-bisphosphonate (alendronate); RANKL, receptor activator of nuclear factor-kappa-B ligand.

Figure 4

Mcl-1 levels decrease rapidly in mouse osteoclasts following cytokine starvation but are restored by pro-survival factors. Mouse osteoclasts were generated by culturing bone marrow macrophages for 5 days with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappa-B ligand (RANKL). (a) Osteoclasts were starved of M-CSF and RANKL for 8 hours and then fixed and processed for scanning EM analysis. Representative osteoclasts from non-starved (control) or starved cultures are shown. Bars = 10 μm. (b) Mouse osteoclasts were starved of M-CSF and RANKL for 2 to 12 hours. Western blot analysis was then used to determine the level of Mcl-1, cleaved caspase-3, Bcl-2, and Bcl-x_L at each time point. (c) After osteoclasts were generated, the medium was replaced with normal medium (control [Ctrl]: M-CSF + RANKL), with medium lacking cytokines (starved), or with medium containing recombinant M-CSF, RANKL, tumour necrosis factor-alpha (TNF-α), or lipopolysaccharide (LPS). After 12 hours, Western blotting was used to determine the level of Mcl-1 in 40 μg of cell lysate. Data shown are representative of three independent experiments.