MicroRNA-29b Enhances Osteoclast Survival by Targeting BCL-2-Modifying Factor after Lipopolysaccharide Stimulation

Abstract

Recent findings suggest that microRNAs (miRs) play a critical role in osteoclastogenesis, which regulates bone loss. We hypothesized that inflammation induces miR-29b, which increases the survival rate in osteoclasts (OCs), leading to bone loss. The expression level of miR-29b increased in OC stimulated by lipopolysaccharide (LPS) in an in vitro system which correlated with its increase in tibiae from mice that received LPS injections compared with those that received vehicle treatment. An miR-29b mimic increased OC survival rate without any change in OC differentiation, and furthermore, the inhibition of endogenous miR-29b induced by LPS decreased OC survival rate. Increased OC survival rate after overexpression of miR-29b was associated with antiapoptotic activity, as shown by staining annexin V-positive cells. We found that a target gene of miR-29b is BCL-2-modifying factor (Bmf), which acts as a proapoptotic factor, and that miR-29b binds to the 3'-UTR of Bmf. Our data demonstrate that LPS-induced miR-29b increases the number of OC by enhancing OC survival through decreased BMF.

Figure 1

LPS upregulates the expression of miR-29b. Tibiae from LPS-treated or vehicle-treated (V, PBS) mice were analyzed by qPCR to quantify the expression of miR-29b (a) and TNF-α, IL-1β, and IL-6 (d) (5 mg/kg/d LPS: n = 8; V: n = 8). BMMs were prepared and incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h, washed thoroughly, incubated further with LPS (50 ng/ml) in the presence of M-CSF (30 ng/ml) for the indicated times, and then analyzed by qPCR to quantify the expression of miR-29b (b), TNF-α, IL-1β, and IL-6 (e). (c) To determine TRAP-positive OCs in vivo, mouse femora were excised free of a soft tissue and decalcified in EDTA. Representative histological sections of the distal femoral metaphysis of mice from each of the two groups were stained for TRAP to identify OCs (indicated by the arrowhead) to calculate OC.N/BS. Scale bar: 50 μm in the representative photos. (f) BMMs were prepared and incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h, washed thoroughly, incubated further with LPS (50 ng/ml) in the presence of M-CSF (30 ng/ml) and each of neutralizing Abs (anti-TNF-α Ab, 0.5 μg/ml; anti-IL-1β Ab, 2 μg/ml; and anti-IL-6 Ab, 2 μg/ml) or its corresponding IgG (mouse IgG) for the indicated times, and then analyzed by qPCR to quantify the expression of miR-29b. (g) Primary osteoblasts were treated with LPS (50 ng/ml) for the indicated time points and analyzed by qPCR to quantify miR-29b expression. The Ct values of the genes were widely distributed between 17.15 and 28.41 ^∗∗p < 0.01; ^∗∗∗p < 0.001 compared with each corresponding V-treated group. Similar results were obtained in three independent experiments.

Figure 2

Overexpression of miR-29b increases the number of OCs by increasing the survival rate. BMMs were prepared and incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h and washed thoroughly. (a, b) Cells were transfected with 30 nM of miR-29b mimic or con mimic in the presence of M-CSF (30 ng/ml) with or without LPS (50 ng/ml). After the indicated times, total RNA was analyzed by qPCR to quantify the expression of miR-29b. Expression levels with con mimic treatment were set at 1 (a). Cells were incubated for 24 h to quantify the expression of TRAP, calcitonin receptor, ATP6v0d2, and DC-STAMP and for 48 h to count TRAP-positive MNCs per well (b). (c) Cells were transfected with miR-29b mimic or con mimic in the presence of M-CSF and LPS. Cells were washed with medium and analyzed after 6 h to measure TRAP-positive MNCs and annexin V-positive cells. (d, e) Cells were transfected with 30 nM of anti-miR-29b or con inh in the presence of LPS (50 ng/ml) and M-CSF (30 ng/ml). After the indicated times, total RNA was analyzed by qPCR to quantify the expression of miR-29b. Expression levels with the con inh treatment were set at 100 (d). Cells were washed with medium, stimulated with LPS for 6 h, and analyzed to measure TRAP-positive MNCs and annexin V-positive cells. Representative photos of OCs. Scale bar: 200 μm. The Ct values of the genes were within 17.82 and 28.65 cycles. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 compared with each corresponding control. Similar results were obtained in three independent experiments.

Figure 3

Identification of target for LPS-induced miR-29b in OCs. BMMs were incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h, washed thoroughly, and incubated further with LPS (50 ng/ml) in the presence of M-CSF (30 ng/ml) for 48 h. (a) Cells were transfected with 30 nM of miR-29b mimic or con mimic in the presence of M-CSF (30 ng/ml) for 6 h. Total RNA was analyzed by qPCR to quantify the expression of BMF, PUMA, BAK1, BIM, and HRK. Expression levels with con mimic treatment were set at 1. Cell lysates were subjected to Western blot analysis with antibodies against BMF, PUMA, BAK1, BIM, HRK, and BCL-2. Antibodies against β-actin were used for normalization. (b) Without transfection, total RNA was analyzed by qPCR to quantify the expression of BMF, and cell lysates were subjected to Western blot analysis with anti-BMF Ab. (c) Total RNA and tissue lysate of tibiae from LPS-treated or vehicle-treated (V, PBS) mice were analyzed by qPCR to quantify the expression of BMF and were subjected to Western blot analysis with antibodies against BMF. (d) Cells were thoroughly washed, transfected with 30 nM of anti-miR-29b or con inh, and stimulated with LPS (50 ng/ml) in the presence of M-CSF. After 48 h, total RNA was analyzed by qPCR to quantify BMF expression. Expression levels with con inh treatment were set at 1.0. Cell lysates were subjected to Western blot analysis with anti-BMF and anti-BCL-2 Ab. (e) Cells were thoroughly washed, transfected with 50 nM of scRNA or siBMF, stimulated with LPS (50 ng/ml) in the presence of M-CSF for 48 h, and analyzed to measure TRAP-positive MNCs and annexin V-positive cells. siRNA-mediated silencing of BMF was confirmed by RT-PCR and qPCR. The Ct values of the genes were widely distributed between 17.33 and 30.92. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 compared with each corresponding control. Similar results were obtained from three independent experiments.

Figure 4

miR-29b directly interacts with Bmf 3′-UTRs. (a) The target site of miR-29b in Bmf 3′-UTR is conserved in humans and rodents (marked by the star). (b) Bmf 3′-UTR mutant was generated by deleting the miR-29 binding site (indicated by the line) to disrupt pairing with mature miR-29b (mmu-miR-29b). (c) Mouse Bmf wild-type 3′-UTR containing miR-29b binding sites or its mutated counterpart was cloned into a luciferase reporter vector and transfected into RAW264.7 cells with miR-29b mimic or anti-miR-29b with its corresponding control. Luciferase assays were performed using a dual-luciferase reporter assay system. ^∗∗∗p < 0.001 compared to its corresponding control. Similar results were obtained in three independent experiments.