Caspase-2 maintains bone homeostasis by inducing apoptosis of oxidatively-damaged osteoclasts.

Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging

Loss of caspase-2 results in altered biomechanical properties of bone.

<p>Parameters measured using micro-indentation analyses (n = 3) include (A) Total indentation distance (TID) (B) Indentation distance increase (IDI), (C) Energy dissipated (ED), (D) Average Loading Slope (Avg LS) and (E) Average Unloading Slope (Avg US). Three-point bending tests (n = 3) were used to measure (F) Load to Yield and (G) Yield Stress. Data are represented as mean ± SEM. <i>*</i>, <i>p</i><0.05.</p

Caspase-2 regulates osteoclast numbers via apoptosis.

<p>(A) Primary cultures of <i>in vitro</i> differentiated <i>Casp2^+/+</i> and <i>Casp2^−/−</i> osteoclasts grown in 96-well plates were TRAP-stained. (B) TRAP-positive, multinucleated osteoclasts were counted (<i>*</i>, <i>p</i><0.05). (C) TRAP activity of in vitro cultured primary osteoclasts from <i>Casp2^+/+</i> and <i>Casp2^−/−</i> mice was measured and normalized to cell viability as determined by MTT assay (<i>*</i>, <i>p</i><0.0005). (D) Osteoclasts were cultured in the presence of a caspase-2-selective inhibitor, zVDVAD and, (E) Intact TRAP-positive osteoclasts were counted (<i>*</i>, <i>p</i><0.0001). (F) Osteoclasts cultured with or without zVDVAD were stained with TUNEL and counted (<i>*</i>, <i>p</i><0.001). (G) Overexpression of wild type caspase-2 but not its enzymatic mutant (Casp2-m) in the osteoclast precursor cell line, RAW264.7, significantly reduced TRAP activity (<i>*</i>, <i>p</i>< 0.005). Scale bar, 100 μm.</p

Loss of caspase-2 increases oxidative stress <i>in vivo</i>.

<p>(A) Bone sections from 27-month old mice (n = 3) were immunostained for 4-HNE; <i>Casp2^−/−</i> section was used as a no-primary control to determine antibody specificity. Data shown is representative of two experiments. Scale bar, 100 μm. (B) Percentage of cells stained (<i>*</i>, <i>p</i> = 0.0007) and relative intensities of staining were measured using Image J and plotted (<i>*</i>, <i>p</i><0.0001). (C) Representative western blots of osteoclast lysates probed for the anti-oxidant enzyme, SOD2; GAPDH was used as a loading control.</p

<i>Casp2^−/−</i> osteoclasts exhibit increased resorption.

<p>(A) Representative images of resorption pits (arrows) from <i>Casp2^+/+</i> and <i>Casp2^−/−</i> mice. Three wells per mouse and three mice per genotype were analyzed for (B) number of resorption pits, (C) area of resorption pit (<i>*</i>, <i>p</i><0.005) and (D) intensity of resorption pit (<i>*</i>, <i>p</i><0.005). RLU =  Relative Light Units.</p

Ablation of caspase-2 results in bone loss.

<p>(A) Dual X-ray energy absorptiometry (DXA) analysis indicated a significant decrease in total (n = 10; <i>*</i>, <i>p</i> = 0.0017) and femoral (n = 10; <i>*</i>, <i>p</i> = 0.0016) bone mineral density (BMD) in 27-month old male <i>Casp2^−/−</i> mice as compared to <i>Casp2^+/+</i> mice. Data represent mean ± SEM. (B) 2D (panels a & b) and 3D (panels c-h) μCT images of 27-month old male <i>Casp2^+/+</i> and <i>Casp2^−/</i> mice showing midshaft femur (panels c & d), lateral view of distal femur (panels e & f) and axial view of distal femur (panels g & h). A higher endocortical porosity and a wider (medio-lateral) metaphyseal compartment are observed (n = 8). (C) Parameters evaluated using μCT (n = 8;<i>*</i>, <i>p</i><0.05); data are represented as mean ± SEM. (D) X-ray (panels a & c) and H&E staining (panels b & d) indicated thinner inter-lacing trabecular bone, and increased cortical bone porosity in <i>Casp2^−/−</i> mice. Arrow indicates growth plate.</p

Caspase-2 is induced in osteoclasts on treatment with oxidative stress-inducing agents.

<p>(A) Primary cultures of <i>Casp2^+/+</i> osteoclasts treated with 500 μM H₂O₂ for the indicated time points (n = 3), were fixed and probed for caspase-2 (green); nuclei were stained blue. <i>Casp2^−/−</i> osteoclasts treated with 500 μM H₂O₂ for 4 h served as a negative control. Scale bar, 50 μm. (B) RAW 264.7 macrophages were differentiated into osteoclasts, treated with 500 μM H₂O₂ for the indicated time periods, lysed and probed for caspase-2 (n = 3). Stripped blot was probed for total actin as loading control.</p