FoxO proteins restrain osteoclastogenesis and bone resorption by attenuating H2O2 accumulation

Shoshana M Bartell, Ha-Neui Kim, Elena Ambrogini, Li Han, Srividhya Iyer, S Serra Ucer, Peter Rabinovitch, Robert L Jilka, Robert S Weinstein, Haibo Zhao, Charles A O'Brien, Stavros C Manolagas, Maria Almeida, Shoshana M Bartell, Ha-Neui Kim, Elena Ambrogini, Li Han, Srividhya Iyer, S Serra Ucer, Peter Rabinovitch, Robert L Jilka, Robert S Weinstein, Haibo Zhao, Charles A O'Brien, Stavros C Manolagas, Maria Almeida

Abstract

Besides their cell-damaging effects in the setting of oxidative stress, reactive oxygen species (ROS) play an important role in physiological intracellular signalling by triggering proliferation and survival. FoxO transcription factors counteract ROS generation by upregulating antioxidant enzymes. Here we show that intracellular H2O2 accumulation is a critical and purposeful adaptation for the differentiation and survival of osteoclasts, the bone cells responsible for the resorption of mineralized bone matrix. Using mice with conditional loss or gain of FoxO transcription factor function, or mitochondria-targeted catalase in osteoclasts, we demonstrate this is achieved, at least in part, by downregulating the H2O2-inactivating enzyme catalase. Catalase downregulation results from the repression of the transcriptional activity of FoxO1, 3 and 4 by RANKL, the indispensable signal for the generation of osteoclasts, via an Akt-mediated mechanism. Notably, mitochondria-targeted catalase prevented the loss of bone caused by loss of oestrogens, suggesting that decreasing H2O2 production in mitochondria may represent a rational pharmacotherapeutic approach to diseases with increased bone resorption.

Figures

Figure 1. RANKL decreases FoxO levels and…
Figure 1. RANKL decreases FoxO levels and activity.
(a) FoxO mRNA (triplicates) and (b) protein levels in BMMs, pre-osteoclasts (pOC) and mature (m) osteoclast cultures. (c) Luciferase activity in RAW264.7 cells transfected with a FoxO-luc reporter construct and incubated with the indicated doses of RANKL in the presence or absence of fetal bovine serum (FBS) for 16 h (triplicates). RLU, relative luminescence units. *P<0.05 versus respective untreated cells, #P<0.05 versus untreated cells in 10% serum. (d) Luciferase activity as in c in cells co-transfected with a control plasmid (pcDNA) or with plasmids expressing FoxO1, 3 or 4 and treated with 60 ng ml−1 RANKL. *P<0.05 versus respective vehicle (veh)-treated cells, #P<0.05 versus veh-treated cells with pcDNA. (e) Protein levels of FoxOs and the nuclear protein lamin A in nuclear extract of BMMs incubated with or without RANKL in the absence or presence of 10% FBS. (f) Catalase mRNA levels in cells described in a. *P<0.05. (g) RANKL-induced Akt, Erk, FoxO1 and FoxO3 phosphorylation in BMMs. (h) Luciferase activity as in d in cells treated with veh, 50 nM PD98059 and 10 μM LY294002. *P<0.05 versus respective veh-treated cells, #P<0.05 versus similar treated cells with pcDNA. (i) Akt, Erk and FoxO1 phosphorylation in BMMs induced by RANKL for 15 min in the presence or absence of 10 μM LY294002 or 0.2 μM wortmannin. (j) Protein levels of FoxOs in cells treated similar to that in i for 2 days. (k,l) BMM incubated with or without RANKL for 6 h (k) or 3 h (l) in the presence or absence of 20 mM MG132. *P<0.05 versus respective veh-treated cells, #P<0.05 versus RANKL alone. Bars represent mean and s.d.; Student’s t-test.
Figure 2. Deletion of FoxOs in osteoclasts…
Figure 2. Deletion of FoxOs in osteoclasts increases bone resorption.
(a) FoxOs mRNA levels in BMM cultures (triplicates). (b) FoxOs mRNA (right) and protein (left) in mature osteoclast cultures. (c) Representative μCT images of distal femur of 6-week-old male mice. Scale bar, 1 mm. (d) Cancellous BMD, bone volume relative to the total volume (BV/TV) and trabecular (Tb) number, thickness and spacing in distal femur of 6-week-old male FoxO1,3,4f/f (n=10) and FoxO1,3,4f/f;LysM-Cre (n=8) littermates. (e) Cortical bone measurements determined using micro-CT in femoral diaphysis of the samples described in d. Pm, perimeter. (f) Osteoclasts in cancellous bone of vertebra from FoxO1,3,4f/f (n=8) and FoxO1,3,4f/f;LysM-Cre (n=11) mice described in d. (g) Histological sections of bone, described in f, stained for TRAP activity (osteoclasts stain red). Scale bar, 20 μm. (h) Serum concentration of a collagen degradation product (CTx) in mice described in d (n=8 mice per group). (i) Cathepsin K (CatK), TRAP, calcitonin receptor (calcr) and osteocalcin (OCN) mRNA levels in vertebrae of mice described in d (n=6 mice per group). Bars represent mean and s.d. *P<0.05 versus FoxO1,3,4f/f by Student’s t-test.
Figure 3. Overexpression of FoxO3 in osteoclasts…
Figure 3. Overexpression of FoxO3 in osteoclasts decreases bone resorption.
(a) FoxO3 transgene mRNA and (b) protein in mature osteoclast cultures (triplicates); ND, not detected. (c) Representative μCT images of distal femur of 12-week-old male mice. Scale bar, 1 mm. (d) Cancellous BMD and bone volume relative to the total volume (BV/TV) of 12-week-old male LysM-Cre (n=12) and FoxO3C;LysM-Cre (n=13) littermates. (e) Osteoclasts in cancellous bone of vertebra from mice described in d (n=6 mice per group). (f) Histological sections of bone, described in e, stained for TRAP activity (osteoclasts stain red). Scale bar, 20 μm. Bars represent mean and s.d. *P<0.05 by Student’s t-test.
Figure 4. FoxOs attenuate osteoclast progenitor proliferation…
Figure 4. FoxOs attenuate osteoclast progenitor proliferation and decrease mature osteoclast lifespan.
(a) Representative pictures (left) and number of TRAP-positive multinucleated cells (MNCs) generated from BMMs (right, triplicates). Scale bar, 500 μm. (b) Cathepsin K (CatK), TRAP and calcitonin receptor (calcr) mRNA levels in osteoclast cultures (triplicates). (c) Representative pictures of BrdU (green) and propidium iodide (red) staining (left) and percentage of BrdU-positive cells in BMMs cultures (right; triplicates). Scale bar, 20 μm. (d–f) Same as a–d using cells from FoxO3C;LysM-Cre and LysM-Cre littermates. (g) Caspase-3 activity in mature osteoclast cultures (triplicates). AFU, arbitrary fluorescent units. (h) Cyclin D1 mRNA and (i) protein levels in BMM cell cultures (triplicate). Bars represent mean and s.d.; *P<0.05 versus respective FoxO1,3,4f/f or LysM-Cre cells by Student’s t-test.
Figure 5. FoxOs attenuate bone resorption by…
Figure 5. FoxOs attenuate bone resorption by decreasing H2O2.
(a) Catalase mRNA levels in BMM and in mature osteoclast (mOC) cultures (triplicates). (b) Catalase protein levels in mature osteoclast. (c) MnSOD mRNA (triplicates). (d) H2O2 levels using Amplex Red in osteoclast progenitors (triplicates). (e) Proliferation by BrdU labelling; and (f) cyclin D1 mRNA levels in BMM cultures treated with vehicle (veh) or 75 Units ml−1 of pegylated catalase (triplicates). (g) Cyclin D1 mRNA levels in BMM silenced for catalase (triplicates). Sh, short hairpin. (h) Four-week-old male mice were administered veh or pegylated catalase (CAT) for 6 weeks. Bone volume relative to the total volume (BV/TV), and trabecular (Tb) number and spacing in vertebrae; and (i) cortical thickness at the midshaft of femur (n=10–11 per group). Bars represent mean and s.d. *P<0.05 by Student’s t-test. #P<0.05 by two-way analysis of variance (ANOVA).
Figure 6. Mitochondria-derived H 2 O 2…
Figure 6. Mitochondria-derived H2O2 promotes osteoclast generation and bone resorption.
(a) Catalase activity in soft tissues and osteoclast (Oc) cultures from LysM-Cre (n=4) and MitoCat-FS;LysM-Cre (n=4) littermates. RLU, relative light units. (b) Representative μCT images of distal femur of 12-week-old male mice. Scale bar, 1 mm. (c) Cancellous BMD and bone volume relative to the total volume (BV/TV) of 12-week-old male LysM-Cre (n=12) and MitoCat-FS;LysM-Cre (n=10) littermates. (d) Cortical thickness determined by μCT in femoral diaphysis of the samples described in c. (e) Osteoclasts in cancellous bone of vertebra from mice described in c (n=6 mice per group). (f) Histological sections of bones, described in e, stained for TRAP activity (osteoclasts stain red). Scale bar, 20 μm. (g) Number of TRAP-positive MNCs generated from BMMs (triplicates). (h) Cathepsin K (CatK), TRAP and calcitonin receptor (calcr) mRNA levels in osteoclast cultures (triplicates). (i) Proliferation by BrdU labelling in BMM cultures (triplicates). (j) Caspase-3 activity in mature osteoclast cultures (triplicates). AFU, arbitrary fluorescent units. (k) Twelve-week-old mice were sham-operated or OVX and killed 6 weeks later. BMD by DEXA as % change from the initial BMD in LysM-Cre sham (n=10), LysM-Cre OVX (n=9), MitoCat-FS;LysM-Cre sham (n=7) and MitoCat-FS;LysM-Cre OVX (n=7) mice. (l) Serum concentration of a collagen degradation product (CTx), in mice described in k, at the time of killing. Bars represent mean and s.d. *P<0.05 by Student’s t-test. #P<0.05 by two-way ANOVA.
Figure 7. Effects of FoxOs on the…
Figure 7. Effects of FoxOs on the birth and death of osteoblasts and osteoclasts.
Aberrant cell number is the culprit of most metabolic bone diseases, including osteoporosis. Earlier work, with targeted loss or gain of FoxO function at different stages of osteoblast differentiation in mice, revealed a restraining effect of FoxOs on Wnt signalling in mesenchymal progenitors. This effect leads to a decrease in the number of osteoblasts and is independent of the antioxidant properties of FoxOs. On the other hand, FoxOs promote the survival of mature osteoblasts. Previous work has also elucidated that FoxOs reduce ROS and promote stemness in haematopoietic stem cells. The present report shows that FoxOs prevent the accumulation of H2O2 in osteoclasts and their progenitors by increasing the expression of catalase and, thereby, promote cell cycle arrest and apoptosis.

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