Identification of LRRc17 as a negative regulator of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation

Abstract

Osteoblasts are the primary cells responsible for bone formation. They also support osteoclast formation from bone marrow precursors in response to osteotropic factors by inducing receptor activator of NF-kappaB ligand (RANKL) expression and down-regulating osteoprotegerin (OPG) production. In addition to the RANKL-RANK-OPG signaling axis, other factors produced by osteoblasts/stromal cells are involved in osteoclastogenesis. Here, we describe the identification and characterization of leucine-rich repeat-containing 17 (LRRc17), a member of the LRR superfamily that acts as a negative regulator of RANKL-induced osteoclast differentiation. Osteoblasts showed high levels of LRRc17 expression, which was down-regulated in response to the pro-osteoclastogenic factor 1,25-dihydroxyvitamin D(3). Recombinant LRRc17 protein inhibited RANKL-induced osteoclast differentiation from bone marrow precursors, whereas it did not affect the differentiation or activation of macrophages and dendritic cells. These results suggest that among the cell types derived from common myeloid precursors, LRRc17 specifically regulates osteoclasts. Further analysis revealed that LRRc17 attenuated RANKL-induced expression of NFATc1 by blocking phospholipase C-gamma signaling, which, in turn, inhibited RANKL-mediated osteoclast differentiation. Taken together, our results demonstrated a novel inhibitory activity of LRRc17 in RANKL-induced osteoclastogenesis.

FIGURE 1.

LRRc17 mRNA expression. A, Northern blot analysis of NIH3T3 cells, osteoblasts (OB), and osteoblasts treated with 1,25(OH)₂D₃ for 2 days (OB/1,25(OH)₂D₃). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, Northern blot analysis of LRRc17 in various mouse tissues, osteoblasts, and bone marrow-derived osteoclasts. C, Northern blot analysis was performed using total RNA from primary calvarial osteoblasts stimulated with 1,25(OH)₂D₃ for the indicated periods of time. Sequences specific for LRRc17, OPG, and RANKL were used as probes.

FIGURE 2.

Role of LRRc17 in osteoclast differentiation. A, mouse bone marrow cells and primary calvarial osteoblasts were cocultured for 6 days with 1,25(OH)₂D₃ (1 × 10^-8 m) or PTH (1 × 10^-8 m)/prostaglandin E₂ (PGE₂, 5 × 10^-8 m) in the presence of 3 μg/ml heat-inactivated recombinant murine LRRc17-Fc or native LRRc17-Fc. Cells were fixed and stained for TRAP (left). The TRAP⁺ MNCs were counted as osteoclasts (right). B, BMMs were cultured for 4 days with M-CSF (30 ng/ml) and RANKL (150 ng/ml) in the presence of various concentrations of heat-inactivated recombinant murine LRRc17-Fc or native LRRc17-Fc as indicated. Cells were fixed and stained for TRAP (left). TRAP⁺ MNCs were counted as osteoclasts (right).

FIGURE 3.

Effect of LRRc17 on dendritic cell differentiation and the phagocytic activity of macrophages. A, BMMs were cultured for 3 days with M-CSF alone or M-CSF and RANKL in the presence of 1 μg/ml murine LRRc17-Fc or control IgG. Cultured cells were incubated with fluorescein-conjugated zymosan particles for 1 h and washed with phosphate-buffered saline. Cells were fixed and observed with UV illumination under a microscope. Fluorescein-conjugated zymosan particles incorporated by the cells appear as bright dots in the dark field. B and C, BMMs were cultured for 4 days with GM-CSF to generate dendritic cells in the presence of 1 μg/ml murine LRRc17-Fc or control IgG. B, the cells were harvested and stained for FACS analysis with anti-CD11c antibodies (dotted lines) or control IgG (solid lines). C, LPS (1 μg/ml) was added to cultures to induce dendritic cell maturation. The cells were harvested the next day and stained for FACS analysis with anti-CD86 or anti-I-A^b antibodies (solid lines, without LPS; dotted lines, with LPS).

FIGURE 4.

Effect of LRRc17 on osteoblast differentiation. A, Northern blot analysis was performed using total RNA from primary calvarial osteoblasts stimulated with ascorbic acid and β-glycerophosphate for the indicated periods of time. Sequences specific for LRRc17, OPG, ALP, and bone sialoprotein (BSP) were used as probes. B and C, stromal cells derived from bone marrow were cultured with ascorbic acid, β-glycerophosphate, and BMP-2 in the presence of various concentrations of murine LRRc17-Fc or control IgG as indicated. B, on days 6 and 12 of culture, ALP activity was measured at 570 nm. C, on day 12, cells were fixed and stained for nodules. W/O OB medium, without osteogenic medium; W/OB medium, with osteogenic medium.

FIGURE 5.

LRRc17 does not affect immediate RANKL-induced signaling. BMMs were stimulated with 500 ng/ml RANKL for the indicated periods of time in the presence of 1 μg/ml human IgG (hIgG), heat-inactivated murine LRRc17-Fc, or murine LRRc17-Fc. Whole-cell extracts were subjected to Western blot analysis with specific antibodies as indicated. p-JNK, phospho-JNK,; p-ERK, phospho-ERK; p-p38, phospho-p38; p-IkB, phospho-IκB.

FIGURE 6.

LRRc17 blocks PLCγ2 signaling and the induction of NFATc1 expression during RANKL-mediated osteoclastogenesis. A, real-time quantitative PCR analysis of RANK, MITF, c-Fos, NFATc1, OSCAR, TRAP, and CSF1R. RNA was isolated on the indicated days after stimulation with M-CSF and RANKL. Day 0 indicates the day RANKL was added to the BMM cultures. B, BMMs were cultured for 2 days with M-CSF and RANKL. The cultured cells were starved in 0.5% FBS for 6 h and then stimulated with 500 ng/ml RANKL (left panel) or anti-OSCAR antibodies cross-linked with secondary anti-rat IgG antibodies (right panel) for the indicated period time in the presence of 1 μg/ml heat-inactivated murine LRRc17 (mLRRc17) or murine LRRc17. Whole-cell extracts were subjected to Western blot analysis with specific antibodies as indicated. The numbers below the lanes indicate the fold induction of PLCγ2 phosphorylation (pPLCγ2) relative to control samples.

FIGURE 7.

Overexpression of NFATc1 overcomes the inhibitory effect of LRRc17 on RANKL-induced osteoclastogenesis. BMMs were transduced with pMX-IRES-EGFP (pMX, control) or retrovirus containing sequence for a constitutively active form of NFATc1 (CaNFATc1), where IRES indicates internal ribosomal entry site and EGFP indicates enhanced green fluorescent protein. BMMs were cultured for 4 days with M-CSF and RANKL in the presence of various concentrations of murine LRRc17-Fc or control IgG as indicated. A, cultured cells were fixed and stained for TRAP. B, TRAP⁺ MNCs with more than three nuclei were counted as osteoclasts.