Abstract
Bacterial infection sometimes impairs bone metabolism. In this study, we infected the osteoblastic cell line MC3T3-E1 with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and identified genes that were up-regulated in the BCG-infected cells by the suppression subtractive hybridization method. A gene encoding 4-1BB (CD137), a member of the tumor necrosis factor-alpha receptor family, was found to be one of the up-regulated genes. Up-regulation of 4-1BB was also observed by infection with Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and by treatment with lipopolysaccharides and heat-killed BCG. Bone marrow cells and the macrophage-like cell lines J774 and RAW264.7 were found to express 4-1BB ligand (4-1BBL). Recombinant 4-1BB (r4-1BB) that was immobilized on culture plates strongly inhibited macrophage colony stimulating factor (M-CSF)/receptor activator of nuclear factor-kappaB ligand (RANKL)-induced in vitro osteoclast formation from bone marrow cells. Anti-4-1BBL antibody also inhibited osteoclast formation to a lesser extent, indicating involvement of reverse signaling through 4-1BBL during inhibition of osteoclast formation. A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of r4-1BB on M-CSF/RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BBL reverse signaling. r4-1BB showed no effects on M-CSF- or RANKL-induced phosphorylation of I-kappaB, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway. r4-1BB also abolished RANKL-mediated induction of nuclear factor of activated T cells-2. This study may elucidate a novel role of 4-1BB in cell metabolism, especially osteoclastogenesis.
Highlights
A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of Recombinant 4-1BB (r4-1BB) on macrophage colony stimulating factor (M-CSF)/ RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BB ligand (4-1BBL) reverse signaling. r4-1BB showed no effects on M-CSF- or RANKLinduced phosphorylation of I-B, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway. r4-1BB abolished RANKL-mediated induction of nuclear factor of activated T cells-2
As TNF-␣ family proteins have recently been reported as important signaling molecules to regulate bone remodeling, especially bone resorption [16], we focused on the increased expression of 4-1BB of bacillus CalmetteGuerin (BCG)-infected MC3T3-E1 cells in this study
To determine what cells were regulated by 4-1BB, we examined mouse osteoblasts, the osteoblastic cell line MC3T3-E1, the fibroblast cell line NIH3T3, the macrophage-like cell lines J774 and RAW264.7, and G-10 column-eluted bone marrow cells for expression of 4-1BBL. 4-1BBL mRNA was detected in J774, RAW264.7, and G-10 column-eluted bone marrow cells, but not in mouse osteoblasts, MC3T3-E1, or NIH3T3 (Fig. 1C)
Summary
Antibodies and Chemicals—Recombinant human soluble RANKL was purchased from PeproTech EC Ltd. (London, England), and recombinant human M-CSF (Leukoprol) was purchased from Yoshitomi Pharmaceutical Co. (Osaka, Japan). G-10 column-eluted bone marrow cells were cultured at 6.5 ϫ 105 cells per well in 96-well plates for 3 or 5 days in culture medium consisting of ␣-MEM and 10% FBS at 37 °C in a humidified atmosphere of 5% CO2 in air. G-10 columneluted bone marrow cells were cultured in ␣-MEM with 10% FBS in the presence of 50 ng/ml M-CSF for 3 days. A, expression of osteoblastic markers in MC3T3-E1 cells with or without BCG infection. MC3T3-E1, NIH3T3, J774, RAW264.7 and G-10 column-eluted bone marrow (BM) cells were cultured for 3 days resulting in almost confluent cultures. Total RNA was extracted from these cells, and the expression of 4-1BBL was determined by RT-PCR using specific primers for 4-1BBL-encoding gene. Adherent cells were further cultured with osteoclast differentiation medium (␣-MEM with 10% FBS containing 10 ng/ml M-CSF and 20 ng/ml RANKL) for 3 days. After 12 h, the infected cells were rinsed three times with DPBS, followed by
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