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.