Prostaglandin E2 stimulates β1-integrin expression in hepatocellular carcinoma through the EP1 receptor/PKC/NF-κB pathway

Abstract

Prostaglandin E2 (PGE2) has been implicated in cell invasion in hepatocellular carcinoma (HCC), via increased β1-integrin expression and cell migration; however, the mechanism remains unclear. PGE2 exerts its effects via four subtypes of the E prostanoid receptor (EP receptor 1-4). The present study investigated the effect of EP1 receptor activation on β1-integrin expression and cell migration in HCC. Cell migration increased by 60% in cells treated with 17-PT-PGE2 (EP1 agonist), which was suppressed by pretreatment with a β1-integrin polyclonal antibody. PGE2 increased β1-integrin expression by approximately 2-fold. EP1 receptor transfection or treatment with 17-PT-PGE2 mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. 17-PT-PGE2 treatment induced PKC and NF-κB activation; PKC and NF-κB inhibitors suppressed 17-PT-PGE2-mediated β1-integrin expression. FoxC2, a β1-integrin transcription factor, was also upregulated by 17-PT-PGE2. NF-κB inhibitor suppressed 17-PT-PGE2-mediated FoxC2 upregulation. Immunohistochemistry showed p65, FoxC2, EP1 receptor and β1-integrin were all highly expressed in the HCC cases. This study suggested that PGE2 upregulates β1-integrin expression and cell migration in HCC cells by activating the PKC/NF-κB signaling pathway. Targeting PGE2/EP1/PKC/NF-κB/FoxC2/β1-integrin pathway may represent a new therapeutic strategy for the prevention and treatment of this cancer.

Figure 1. EP1 receptor activation promoted β1-integrin expression in hepatocellular carcinoma cells.

(A). Effects of EP agonists on β1-integrin expression in Huh-7 cells. Huh-7 cells were exposed to 5 μM EP1 agonist (17-PT-PGE₂), EP2 agonist (butaprost), EP3 agonist (sulprostone) and EP4 agonist (PGE1 alcohol) for 24 h, respectively. The cropped gels are used and full-length gels are presented in Supplementary Figure S1 and S2. (B). Effects of EP antagonists on PGE₂-mediated β1-integrin expression in Huh-7 cells. Huh-7 cells were pretreated with various EP antagonists for 1 h, followed by PGE₂ for 24 h (EP1 antagonist sc19220, EP2 antagonist AH6809 and EP3 antagonist L-798106, EP4 antagonist AH23848). The cropped gels are used and full-length gels are presented in Supplementary Figure S3 and S4. (C). Effects of expression of the EP1 receptor on PGE₂-mediated β1-integrin regulation in HEK293 cells. HEK293 cells (3 × 10⁵ cells) were transfected with EP1R-pcDNA3 plasmid or empty pcDNA3 plasmid as a control. After transfection, cells expressing the EP1 receptor were selected by G418. EP1 receptor-transfected HEK293 cells were exposed to PGE₂ for 24 h, with or without sc19220 pre-treatment. Results are presented as the mean ± SD from three different experiments. *P < 0.05, compared to control cells; #P < 0.05, compared with PGE₂-treated cells. (D). RNA interference targeting the EP1 receptor suppressed PGE₂-mediated β1-integrin upregulation in Huh-7 cells. Huh-7 cells were transfected with an EP1R-siRNA. After 72 h, the cells were exposed to PGE₂ for 24 h. The cropped gels are used and full-length gels are presented in Supplementary Figure S5 and S6. Results are shown as the mean ± SD from three different experiments. ** indicates a significant difference at P < 0.01 compared with the cells without PGE₂ treatment; # indicates a significant difference at P < 0.05 compared with the siRNA negative control cells. $$ indicates a significant difference at P < 0.01 compared with the siRNA negative control cells after PGE₂ treatment. (E). Effect of anti-β1-integrin antibody on 17-PT-PGE₂-mediated cell migration in Huh-7 cells. The cell migration assay was performed in a12-well transwell. Huh-7 cells were pretreated with an anti-β1-integrin antibody for 30 min, followed by stimulation with PGE₂. The in vitro migration activity was measured after 24 h. Results are presented as the mean ± SD from three different experiments. *P < 0.05, compared with control cells; #P < 0.05, compared with 17-PT-PGE₂ –treated group. The gels have been run under the same experimental conditions.

Figure 2. The expression of EP1 receptor and β1-integrin in liver cancer tissues.

(a). Representative immunohistochemical images of human hepatocellular carcinoma tissue stained with the anti-human EP1 receptor antibody. (b). Representative immunohistochemical images of human hepatocellular carcinoma tissue stained with the anti-human β1-integrin antibody. (c). Representative immunohistochemical images of normal human liver tissue stained with the anti- EP1 receptor antibody. (d). Representative immunohistochemical images of normal human liver tissue stained with the anti-β1-integrin antibody. (Magnification: ×400).

Figure 3. PKC is involved in EP1 receptor-mediated β1-integrin upregulation in hepatocellular carcinoma cells.

(A). PKC activity assay. Huh-7 cells were treated with 5 μM 17-PT-PGE₂ for 0, 5, 10, 15, 20, 25 or 30 min. Equal amounts of total proteins (30 μg) were added to microcentrifuge tubes and assayed for PKC levels using a direct human PKC enzyme activity assay kit. (B). Effect of a PKC activator on β1-integrin expression in Huh-7 cells. Huh-7 cells were treated with 100 nM PMA for 24 h. Total protein was isolated and visualized with an anti-β1-integrin antibody. Levels of β-actin served as a loading control. (C). Effect of a PKC inhibitor on 17-PT-PGE₂-mediated β1-integrin expression in Huh-7 cells. Huh-7 cells were treated with 17-PT-PGE₂ for 24 h, with or without pre-treatment of 5 μM rottlerin for 1 h. Total protein was isolated and visualized with an anti-β1-integrin antibody. Levels of β-actin served as a loading control. The cropped gels are used and full-length gels are presented in Supplementary Figure S7. Results are presented as the mean ± SD from three different experiments. **P < 0.01, compared with control cells; ##P < 0.01, compared with 17-PT-PGE₂ –treated group. (D). Effect of a PKC inhibitor on 17-PT-PGE₂-mediated β1-integrin expression in EP1 receptor-expressed HEK293 cells. Stable EP1 receptor-expressed HEK293 cells were treated with 17-PT-PGE₂ for 24 h, with or without pre-treatment of rottlerin for 1 h. Total protein was isolated and visualized with an anti-β1-integrin antibody. Levels of β-actin served as a loading control. Results are presented as the mean ± SD from three different experiments. **P < 0.01, compared with control cells; ##P < 0.01, compared with 17-PT-PGE₂ –treated group. (E). Effect of a PKC inhibitor on 17-PT-PGE₂-mediated cell migration in Huh-7 cells. The cell migration assay was performed in a12-well transwell plate. Huh-7 cells were treated with 17-PT-PGE₂ for 12 h, with or without pre-treatment of 5 μM rottlerin for 1 h. Cells on the lower surface were stained with 0.1% crystal violet, solubilized with acetic acid solution and quantified by measuring their absorbance at 570 nm. Results are presented as the mean ± SD from three different experiments. **P < 0.01, compared with control cells; ##P < 0.01, compared with 17-PT-PGE₂-treated cells. The gels have been run under the same experimental conditions.

Figure 4. NF-κB is involved in EP1 receptor-mediated β1-integrin upregulation in hepatocellular carcinoma cells.

(A). Effects of 17-PT-PGE₂ on NF-κB and IκB phosphorylation in Huh-7 cells. Huh-7 cells were treated with 5 μM 17-P-T-PGE₂ for 0, 30, 60, 120 min. Equal amounts of total proteins were separated by SDS-PAGE. Relative levels of phosphorylated and total IκBα or p65 were determined using specific antibodies. The cropped gels are used and full-length gels are presented in Supplementary Figure S8 and S9. (B). Effects of PGE₂ on NF-κB and IκB phosphorylation in EP1 receptor-expressed HEK293 cells. EP1 receptor-expressed HEK293 cells were treated with PGE₂ for 0, 30, 60, 120 min. (C). Effects of 17-PT-PGE₂ on NF-κB translocation in Huh-7 cells. Huh-7 cells were treated with 5 μM 17-PT-PGE₂ for 0, 30, 60, 120 min; the cells were then fixed with ice-cold methanol. The p65 protein was detected by immunofluorescence and activated p65 was translocated into the nuclie (arrow). All pictures were taken at 400× magnification. (D). Effect of NF-κB inhibitor on 17-PT-PGE₂-mediated β1-integrin expression in Huh-7 cells. Huh-7 cells were treated with 17-PT-PGE₂ for 24 h, with or without pre-treatment of PDTC for 24 h. Total protein was isolated and visualized with an anti-β1-integrin antibody. Levels of β-actin served as a loading control. The cropped gels are used and full-length gels are presented in Supplementary Figure S10 and S11. Densitometric quantitation of the above blots is shown. Results are presented as the mean ± SD from three different experiments. **P < 0.01, compared with control cells; ##P < 0.01, compared with 17-PT-PGE₂-treated cells. (E). Effect of NF-κB inhibitor on 17-PT-PGE₂-mediated cell migration in Huh-7 cells. The cell migration assay was performed in a 12-well transwell plate. Huh-7 cells were treated with 17-PT-PGE₂ for 12 h, with or without pre-treatment of 10 μM PDTC for 24 h. Results are presented as the mean ± SD from three different experiments. **P < 0.01, compared with control cells; ##P < 0.01, compared with 17-PT-PGE₂-treated cells. The gels have been run under the same experimental conditions.

Figure 5. FoxC2 is involved in EP1 receptor/NF-κB-mediated β1-integrin upregulation in hepatocellular carcinoma.

(A). Effect of NF-κB in EP1 receptor-mediated FoxC2 upregulation in hepatocellular carcinoma cells. Huh-7 cells were treated with 17-PT-PGE₂ for 24 h, with or without pre-treatment of PDTC for 24 h. Total protein was isolated and visualized with an anti-FoxC2 antibody. Levels of β-actin served as a loading control. The cropped gels are used and full-length gels are presented in Supplementary Figure S12. Densitometric quantitation of the above blots is shown. Results are presented as the mean ± SD from three different experiments. *P < 0.05, compared with control cells; #P < 0.05, compared with 17-PT-PGE₂-treated cells. The gels have been run under the same experimental conditions. (B). Co-expression of EP1 receptor, p65 and FoxC2 in liver cancer tissues. (a). Representative immunohistochemical images of human hepatocellular carcinoma tissue stained with the anti-human EP1 receptor antibody. (b). Representative immunohistochemical images of hepatocellular carcinoma tissue stained with the anti-p65 antibody. (c). Representative immunohistochemical images of hepatocellular carcinoma tissue stained with the anti-FoxC2 antibody. (Magnification: ×400).

Figure 6. Proposed mechanisms for PGE₂/EP1 receptor-mediated hepatocellular carcinoma cell migration.

Our data showed that the EP1 receptor played a key role in PGE₂-mediated hepatocellular carcinoma cell migration. EP1 receptor may upregulate β1-integrin expression to improve cell migration. PKC/NF-κB/FOXC2 signaling pathways were involved in EP1 receptor-mediated β1-integrin expression.