The hemoglobin receptor protein of porphyromonas gingivalis inhibits receptor activator NF-kappaB ligand-induced osteoclastogenesis from bone marrow macrophages

Abstract

Extracellular proteinaceous factors of Porphyromonas gingivalis, a periodontal pathogen, that influence receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL)-induced osteoclastogenesis from bone marrow macrophages were investigated. The culture supernatant of P. gingivalis had the ability to inhibit RANKL-induced in vitro osteoclastogenesis. A major protein of the culture supernatant, hemoglobin receptor protein (HbR), suppressed RANKL-induced osteoclastogenesis in a dose-dependent fashion. HbR markedly inhibited RANKL-induced osteoclastogenesis when present in the culture for the first 24 h after addition of RANKL, whereas no significant inhibition was observed when HbR was added after 24 h or later, implying that HbR might interfere with only the initial stage of RANKL-mediated differentiation. HbR tightly bound to bone marrow macrophages and had the ability to induce phosphorylation of ERK, p38, NF-kappaB, and Akt. RANKL-induced phosphorylation of ERK, p38, and NF-kappaB was not suppressed by HbR, but that of Akt was markedly suppressed. HbR inhibited RANKL-mediated induction of c-Fos and NFATc1. HbR could induce beta interferon (IFN-beta) from bone marrow macrophages, but the induction level of IFN-beta might not be sufficient to suppress RANKL-mediated osteoclastogenesis, implying presence of an IFN-beta-independent pathway in HbR-mediated inhibition of osteoclastogenesis. Since rapid and extensive destruction of the alveolar bone causes tooth loss, resulting in loss of the gingival crevice that is an anatomical niche for periodontal pathogens such as P. gingivalis, the suppressive effect of HbR on osteoclastogenesis may help the microorganism exist long in the niche.

FIG. 1.

Inhibitory effect of P. gingivalis culture supernatant on RANKL-induced osteoclastogenesis from bone marrow macrophages. Bone marrow macrophages (10⁴ cells) were incubated in the culture medium containing M-CSF and RANKL with or without P. gingivalis culture supernatants (0.1, 1, and 10 μg/ml) or LPS (1 ng/ml) in the presence or absence of polymyxin B (0.1 μg/ml) for 5 days. Cells were fixed and stained for TRAP, and the TRAP-positive multinuclear cells were counted. The results are from one experiment and are representative of three separate experiments. ✽, P < 0.05; ✽✽, P < 0.01 (compared to the experiment with RANKL and M-CSF).

FIG. 2.

Two-dimensional gel analysis of P. gingivalis culture supernatant. Crude protein extracts of the supernatant of P. gingivalis 33277 culture for 7 days were subjected to two-dimensional gel analysis. The arrows indicate HbR.

FIG. 3.

Inhibitory effect of HbR on RANKL-induced osteoclastogenesis from bone marrow macrophages and no inhibitory effect of HbR on the viability of bone marrow macrophages. (A) HbR was purified from HbR-overexpressing E. coli by DEAE-Sepharose column chromatography. Lanes: 1, before IPTG induction; 2, after IPTG induction; 3, precipitates with ammonium sulfate; 4, after DEAE-Sepharose. (B to E) Bone marrow macrophages were incubated in the culture medium with M-CSF and RANKL in the presence of the indicated concentrations of HbR for 5 days. Cells were stained for TRAP (B), the numbers of TRAP-positive multinuclear cells were counted (C), and the TRAP intensity was determined (D). Cell viability was determined by using a cell counting kit (E). The results are from one experiment and are representative of three separate experiments. ✽✽, P < 0.01 (compared to the experiment with RANKL and M-CSF).

FIG. 4.

HbR-mediated inhibition in the initial stage of osteoclast differentiation. Bone marrow macrophages were incubated in the culture medium with M-CSF, RANKL, and HbR (10 μg/ml). After 1, 2, 3, or 4 days of incubation, the culture medium was changed to that with M-CSF and RANKL to remove HbR. On the other hand, bone marrow macrophages were incubated in the culture medium with M-CSF and RANKL. After 1, 2, 3, or 4 days of incubation, HbR (10 μg/ml) was added to the culture, and the incubation was continued for 4 days, 3 days, 2 days, or 1 day, respectively. Cells were stained for TRAP (A), the numbers of TRAP-positive multinuclear cells were counted (B), and the TRAP intensity was determined (C). The results are from one experiment and are representative of three separate experiments.

FIG. 5.

Fluorescence microscopy. Bone marrow macrophages were treated with (B and D) or without (A and C) HbR (10 μg/ml) for 24 h. HbR was then visualized by immunostaining with anti-HbR and Alexa 488-conjugated anti-rabbit IgG antibodies. Cells were observed by light microscopy (A and B) and fluorescence microscopy (C and D).

FIG. 6.

Effects of HbR on RANKL-induced signaling. (A) Bone marrow macrophages were treated with RANKL alone, HbR (10 μg/ml) alone, or RANKL plus HbR for the indicated times. Proteins in the cells were separated by SDS-PAGE, transferred to PVDF membranes, and then immunostained with anti-phospho-ERK1/2 (Tyr²⁰⁴), anti-ERK2, anti-phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²), anti-p38, anti-phopho-NF-κB p65 (Ser⁵³⁶), anti-β-actin, anti-phospho-Akt (Ser⁴⁷³), and anti-Akt1/2/3. (B) Bone marrow macrophages were incubated in the culture medium with M-CSF alone, M-CSF plus RANKL, or a combination of M-CSF, RANKL, and HbR (10 μg/ml) for the indicated times. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and then immunostained with anti-NFATc1, anti-TRAF6, anti-c-Fos, and anti-β-actin.

FIG. 7.

HbR-mediated induction of IFN-β and the effect of anti-IFN-β antibody on HbR- or IFN-β-mediated inhibition of osteoclastogenesis. (A) HbR-mediated induction of IFN-β. Bone marrow macrophages were incubated in the culture medium with M-CSF alone, M-CSF plus RANKL, M-CSF plus HbR (10 μg/ml), or a combination of M-CSF, RANKL, and HbR for 24 or 48 h. The amounts of IFN-β in culture supernatants were determined. (B) Effect of anti-IFN-β antibody on HbR- or IFN-β-mediated inhibition of osteoclastogenesis. Bone marrow macrophages were incubated in the culture medium with M-CSF, RANKL, and anti-IFN-β antibody (0, 10, 100, or 1,000 U/ml) in the presence of IFN-β (0, 1, 10, or 100 U/ml) or HbR (10 μg/ml) for 5 days. The TRAP-positive multinuclear cells were counted. “αIFN-β” indicates anti-IFN-β antibody. The results are from one experiment and are representative of three separate experiments. ✽✽, P < 0.01 (compared to the experiment with RANKL and M-CSF).