JNK/AP-1 pathway is involved in tumor necrosis factor-alpha induced expression of vascular endothelial growth factor in MCF7 cells

Abstract

Vascular endothelial growth factor (VEGF) has been implicated in breast tumor angiogenesis. And tumor necrosis factor-alpha (TNF-alpha) is a positive regulator of VEGF. This study was aimed to identify the signalling pathway of TNF-alpha in VEGF expression regulation in breast cancer cell line MCF7. Using luciferase reporter assays, we demonstrated that TNF-alpha significantly increased activator protein-1 (AP-1) transcriptional activity in the MCF7 cells. The expression of the AP-1 family members c-Jun, c-Fos and JunB and phosphorylation levels of c-Jun were upregulated by TNF-alpha, whereas other AP-1 family members Fra-1, Fra-2, and JunD were unaffected. The activation of AP-1 was associated with the formation of p-c-Jun-c-Jun and p-c-Jun-JunB homodimers. Furthermore, the phosphorylation levels of c-Jun N-terminal kinase (JNK) but not P38 and ERK were elevated by TNF-alpha in MCF7 cells. TNF-alpha potently upregulated the mRNA and protein levels of VEGF, which were significantly reversed by JNK inhibitor SP600125. Finally using chromatin immunoprecipitation (CHIP) assays, we found that p-c-Jun bound to the VEGF promoter and regulated VEGF transcription directly. These data suggest that the pro-inflammatory cytokine TNF-alpha is a critical regulator of VEGF expression in breast cancer cells, at least partially via a JNK and AP-1 dependent pathway.

Fig. 1

TNF-α elevates AP-1 luciferase reporter activity. MCF7 cells were transiently cotransfected with the firefly luciferase reporter construct AP-1-LUC and β-galactosidase plasmid (as an internal control) plus pCMV-TAM-67 or pCMV-neo vector. Eight hours after transfection, cells were serum starved for 24 h, and then treated with TNF-α (20 ng/ml) or vehicle (Control) for the indicated periods of time. Then cells were harvested for luciferase reporter assays. The relative values of AP-1 luciferase activity to β-galactosidase are shown as means ± SEM of three independent experiments. *P < 0.05 vs. levels in cells cotransfected with pCMV-neo vector and treated with vehicle.

Fig. 2

TNF-α induces the expression of c-Jun, JunB and c-Fos, but not other Jun or Fos proteins. MCF7 cells were serum starved for 24 h and treated with TNF-α (20 ng/ml) for indicated periods of time. Nuclear protein samples were harvested for Western blot analyses to determine the protein levels of c-Jun, JunB, c-Fos (A), Fra-1, Fra-2, JunD (B) and Ser⁷³-phosphorylated-c-Jun (p-c-Jun; C). The protein levels of β-actin were assessed simultaneously in each group as a loading control. Immunoblots are representative of three separate experiments.

Fig. 3

AP-1 complex is composed of p-c-Jun-c-Jun and p-c-Jun-JunB homodimers after TNF-α treatment. MCF7 cells were serum starved for 24 h and treated without (Control) or with TNF-α (20 ng/ml) for 3 h or 24 h. Cell nuclear lysates were subjected to immunoprecipitation (IP) with an antibody against p-c-Jun followed by immunoblotting (IB) with antibodies against c-Jun, c-Fos and JunB. Representative Immunoblots of three separate experiments are shown.

Fig. 4

Effects of TNF-α on the activities of MAPK pathways. MCF7 cells were serum starved for 24 h and treated with TNF-α (20 ng/ml) for the indicated periods of time, and JNK (p-JNK), P38 (p-p38) and ERK (p-ERK) activation was assessed using phosphorylation specific antibodies. The levels of β-actin protein in each group were used as the references. Representative immunoblots of three separate experiments are shown.

Fig. 5

TNF-α induces VEGF luciferase reporter activity. MCF7 cells were transiently transfected with VEGF-LUC construct and β-galactosidase plasmid (as an internal control). Eight hours after transfection, cells were serum starved for 24 h, and then treated with TNF-α (20 ng/ml) or vehicle (Control) for the indicated periods of time. Then cells were collected for luciferase reporter assays. The relative values of LXRE luciferase activity to β-galactosidase are shown as means ± SEM of three independent experiments. *P < 0.05 vs. control.

Fig. 6

TNF-α stimulates VEGF mRNA transcription in MCF7 cells. MCF7 cells were treated with 20 ng/ml of TNF-α for the indicated time periods after 24 h serum starvation. Total RNA was isolated and 1 µg RNA was used for RT-PCR analyses of VEGF and GAPDH (as a reference) mRNA levels. PCR products were revealed by ethidium bromide staining under UV after agarose gel electrophoresis. Representative graphs of three separate experiments are shown.

Fig. 7

TNF-α increases VEGF protein levels in MCF7 cells. After serum starvation for 24 h, MCF7 cells were pretreated without or with SP600125 for 30 min, and treated with TNF-α (20 ng/ml) for the indicated time periods. Cell lysates were collected for Western blot analyses with antibodies against VEGF or β-actin (as a loading control). Representative Immunoblots of three separate experiments are shown.

Fig. 8

p-c-Jun is recruited to the VEGF promoter following TNF-α treatment. After serum starvation for 24 h, MCF7 cells were treated without (Control) or with TNF-α (20 ng/ml) for the indicated time periods, and chromatin protein-DNA complexes were cross-linked using formaldehyde. The purified nucleoprotein complexes were immunoprecipitated with Ser⁷³-phosphorylated-c-Jun antibody (p-c-Jun) or nonimmune IgG and DNA samples recovered were amplified by PCR as detailed in Materials and Methods. Representative graphs of three separate experiments are shown.