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. 2006;8(5):R146.
doi: 10.1186/ar2038.

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Takashi Ando  1 , Jiro Ichikawa, Masanori Wako, Kyosuke Hatsushika, Yoshiyuki Watanabe, Michitomo Sakuma, Kachio Tasaka, Hideoki Ogawa, Yoshiki Hamada, Hideo Yagita, Atsuhito Nakao
Affiliations

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Takashi Ando et al. Arthritis Res Ther. 2006.

Abstract

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-kappaB (NF-kappaB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-kappaB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast- and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.

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Figures

Figure 1
Figure 1
MC3T3-E1 cells express functional Fn14. (a) Cell surface expression of Fn14 on MC3T3-E1 cells. MC3T3-E1 cells were stained with anti-Fn14 monoclonal antibody (open histogram) or control Ig (filled histogram) followed by phycoerythrin-labeled rabbit anti-mouse Ig antibody and analyzed by flow cytometry. (b) TWEAK phosphorylates the NF-κB p65 subunit in MC3T3-E1 cells. MC3T3-E1 cells were stimulated with 100 ng/ml TWEAK or 10 ng/ml TNF-α (as a positive control) in the presence or absence of 1 μg/ml mouse Fn14-Fc chimera (Fn14-Fc) or control mouse IgG for the indicated time periods. The cell lysates were then subjected to immunoblotting with antibody specific for the Ser536-phosphorylated NF-κB p65 subunit. Fn14, fibroblast growth factor-inducible 14; Ig, immunoglobulin; NF-κB, nuclear factor-κB; TNF-α, tumour necrosis factor-α; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.
Figure 2
Figure 2
TWEAK/Fn14 interaction induces RANTES production by MC3T3-E1 cells. (a) MC3T3-E1 cells were cultured in the presence or absence of 100 ng/ml TWEAK for 48 hours. The culture supernatants were then collected and subjected to a cytokine protein array. The table indicates the corresponding cytokines on the protein array membrane. (b) MC3T3-E1 cells were stimulated with the indicated doses of TWEAK for 48 hours in the presence or absence of the indicated doses (ng/ml) of mouse Fn14-Fc chimera or control mouse immunoglobulin G (mIgG). The culture supernatants were then collected, and the RANTES concentrations were measured by enzyme-linked immunosorbent assay. (c) MC3T3-E1 cells were stimulated with the indicated doses of TWEAK for 96 hours. Viable cell number was then measured by WST assay. The data were expressed as OD units. Values represent the mean ± standard deviation. *p < 0.05 compared with corresponding control. Similar results were obtained in at least three independent experiments. Fn14, fibroblast growth factor-inducible 14; G-CSF, granulocyte-cell-stimulating factor; GM-CSF, granulocyte macrophage-colony-stimulating factor; IFN-γ, interferon-γ; IL, interleukin; IP-10, inositol phosphate-10; M-CSF, macrophage-colony-stimulating factor; MIG, monokine induced by IFN-γ; MIP-1α, viral macrophage inflammatory protein-1α; OD, optical density; RANTES, regulated upon activation, healthy T cell expressed and secreted; TNF-α, tumour necrosis factor-α; TWEAK, tumour necrosis factor-like weak inducer of apoptosis; VEGF, vascular endothelial growth factor; WST, water-soluble tetrazolium.
Figure 3
Figure 3
TWEAK-induced RANTES production by MC3T3-E1 cells involves the PI3K-Akt pathway. (a) MC3T3-E1 cells were cultured in the presence or absence of 100 ng/ml TWEAK with or without 10 μM LY294002, 10 μM PD98059, or 3 μM Helenalin for 48 hours. The culture supernatants were then collected, and the RANTES concentrations were measured by enzyme-linked immunosorbent assay. (b) MC3T3-E1 cells were stimulated with 100 ng/ml TWEAK for the indicated time periods. The cell lysates were then subjected to immunoblotting with antibody specific for phosphorylated Akt, Akt, phosphorylated Erk p42/44, and Erk p42/44. Values represent the mean ± standard deviation. *p < 0.05 compared with corresponding control. Similar results were obtained in at least three independent experiments. RANTES, regulated upon activation, healthy T cell expressed and secreted; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.
Figure 4
Figure 4
TWEAK inhibits BMP2-induced osteoblastic differentiation in MC3T3-E1 cells. (a) MC3T3-E1 cells were stimulated with 100 ng/ml BMP-2 in the presence or absence of 100 ng/ml TWEAK for 5 days, and then ALP activity was determined using the TRACP & ALP double-stain kit. TWEAK inhibited BMP-2-induced ALP activity. Representative images are shown. (b) MC3T3-E1 cells were stimulated with 100 ng/ml BMP-2 with or without 100 ng/ml TWEAK or with 10 ng/ml TNF-α (as a positive control) for 48 hours. Osteocalcin mRNA expression was then evaluated by reverse transcription-polymerase chain reaction. (c) MC3T3-E1 cells were stimulated with 100 ng/ml BMP-2 in the presence or absence of 100 ng/ml TWEAK and/or 1 μg/ml Fn14-Fc chimera (Fn14-Fc) or control mouse IgG (mIgG) for 5 days, and then ALP activity was determined using the TRACP & ALP double-stain kit. Fn14-Fc chimera abrogated TWEAK inhibition of BMP-2-induced ALP activity. Representative images are shown. (d) MC3T3-E1 cells were stimulated with 100 ng/ml BMP-2 in the presence or absence of 100 ng/ml TWEAK and/or 1 μM PD98059 for 5 days, and then ALP activity was determined using the TRACP & ALP double-stain kit. PD98059 abrogated TWEAK inhibition of BMP-2-induced ALP activity. Representative images are shown. (e) MC3T3-E1 cells were cultured in the presence or absence of 100 ng/ml BMP-2 for 24 hours. The cells were then stained with anti-Fn14 monoclonal antibody (open histogram, unbroken line) or control Ig (filled histogram) followed by phycoerythrin-labeled rabbit anti-mouse Ig antibody and were analyzed by flow cytometry. Addition of BMP-2 did not affect surface Fn14 expression (open histogram, dotted line). Similar results were obtained in at least three independent experiments. ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; Fn14, fibroblast growth factor-inducible 14; Ig, immunoglobulin; TNF-α, tumour necrosis factor-α; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.
Figure 5
Figure 5
TWEAK/Fn14 interaction induces RANKL expression in MC3T3-E1 cells. (a) MC3T3-E1 cells were stimulated with 100 ng/ml TWEAK for the indicated times in the absence or presence of 1 μg/ml Fn14-Fc chimera, 10 μM LY2940002, or 10 μM PD98059. RNA was then extracted from the cells, and real-time PCR was performed using specific primers for RANKL and GAPDH (C, no treatment). The ratio of each gene to that of GAPDH was calculated, and the value of 1.0 was assigned to MC3T3-E1 cells that were incubated without TWEAK. (b) MC3T3-E1 cells were grown on slide chambers, stimulated with 100 ng/ml TWEAK for 24 and 48 hours, and then stained for RANKL as described in Materials and methods. RANKL-positive cells are shown in green. Goat IgG antibody was used as a negative control for the immunofluorescence staining. Fn14, fibroblast growth factor-inducible 14; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IgG, immunoglobulin G; RANKL, receptor activation of nuclear factor-κB ligand; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.
Figure 6
Figure 6
TWEAK expression in bone-related cell lines and the mouse bone tissue. (a) The indicated tissues were obtained from Balb/c mice, and RNA was extracted. Real-time PCR was then performed using specific primers for TWEAK and GAPDH. The ratio of each gene to that of GAPDH was calculated, and the value of 0.1 was assigned to the brain tissue. (b) RNA was extracted from MC3T3-E1, RAW264, ATDC5, and EL4 cells, and then real-time PCR was performed using specific primers for TWEAK and GAPDH. The ratio of each gene to that of GAPDH was calculated, and the value of 0.1 was assigned to MC3T3-E1 cells. (c) Immunohistochemical examination of the mouse bone tissue. Mouse tail bone tissues from Balb/c mice were stained with anti-TWEAK antibody. Representative images of weak enlargement (upper panel) and strong enlargement (lower panel) are shown. Positive staining is indicated as brown. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.
Figure 7
Figure 7
Human primary osteoblasts express Fn14 and induce RANTES and RANKL upon TWEAK stimulation. (a) Cell surface expression of Fn14 on human osteoblasts. Human osteoblasts were stained with anti-Fn14 monoclonal antibody (open histogram) or control Ig (filled histogram) followed by phycoerythrin-labeled rabbit anti-mouse Ig antibody and were analyzed by flow cytometry. (b) Human osteoblasts were stimulated with 100 ng/ml TWEAK for 48 hours in the absence or presence of 1 μg/ml Fn14-Fc chimera (Fn14-Fc). The culture supernatants were then collected, and the RANTES concentrations were measured by enzyme-linked immunosorbent assay. Values represent the mean ± standard deviation. *p < 0.05 compared with corresponding control. (c) Human osteoblasts were grown on slide chambers, stimulated with 100 ng/ml TWEAK for 48 hours, and then stained for RANKL as described in Materials and methods. RANKL-positive cells are shown in green. Goat IgG antibody was used as a negative control for the immunofluorescence staining, with negative results (data not shown). BMP, bone morphogenetic protein; Fn14, fibroblast growth factor-inducible 14; Ig, immunoglobulin; RANKL, receptor activation of nuclear factor-κB ligand; RANTES, regulated upon activation, healthy T cell expressed and secreted; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.
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