Caveolin-1 Scaffolding Domain Peptide Regulates Colon Endothelial Cell Survival through JNK Pathway

Figures

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  Figure 1. AP-Cav inhibits mouse colon endothelial cell proliferation. (a) AP peptide pretreatment shows no effect on colon endothelial cell proliferation. (b) AP-Cav inhibits endothelial cell proliferation induced by VEGF. Cells were treated with peptide at indicated concentrations for 30 minutes before being treated with VEGF (50 ng/ml) for 4 hours. Data are mean ± s.e.m. from three independent experiments ∗p < 0.05.

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  Figure 2. AP-Cav inhibits ERK1/2 phosphorylation. (a) After overnight low serum starvation, the sample was treated for 30 minutes with the AP-Cav peptide and then was stimulated with 50 ng VEGF/ml for 10 minutes. (b) After overnight low serum starvation, 30 minutes AP-Cav peptide treatment, and stimulation with 50 ng VEGF/ml for 2 hours, the ERK1/2 phosphorylation level was determined. Cells were treated with peptide for 30 minutes and then stimulated with 50 ng/ml of VEGF at the indicated times. At the end of the stimulation, cells were immediately lysed in RIPA buffer. The lysates were subjected to SDS-PAGE and western blot analysis with specific antibodies for phospho-ERK 1/2 and total ERK 1/2. Bar graph represents the phospho-ERK 1/2 to total ERK 1/2 ratio normalized to beta-actin levels. The data are mean ± s.e.m. from three independent experiments, ∗p < 0.05.

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  Figure 3. Effect of AP-Cav on cell cycle protein level of p27kip1, Rb in mouse colon endothelial cells. (a) After overnight low serum starvation, 30 minute AP-Cav peptide treatment, and stimulation with 50 ng VEGF/ml for 0, 2, and 4 hours, cells were subjected to SDS-PAGE and western blot analysis with specific antibodies for p27kip1 or Rb. Beta-actin was used as a loading control. (b) Bar graph represents relative Rb level normalized to beta-actin level. (c) Bar graph represents relative level of p27kip1 normalized to beta-actin level. Data are mean ± s.e.m. from three independent experiments, ∗p < 0.05.

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  Figure 4. AP-Cav peptide upregulates caspase-3 activity and JNK activity in mouse colon endothelial cells. (a) Mouse colon endothelial cells were starved in media containing 1% FBS overnight, treated with the peptide for 30 minutes, and stimulated with 50 ng/ml of VEGF for 0, 2, and 4 hours. At the end of the stimulation, cells were lysed in boiling SDS-sample buffer, and cell lysates were subjected to SDS-PAGE and western blot analysis with specific antibodies for the activated form of caspase-3 and phospho-JNK. The protein loading was probed with antibody for beta-actin. (b) Bar graph represents relative cleaved form of caspase-3 normalized to beta-actin level. (c) Bar graph represents phospho-JNK level normalized to beta-actin level. Data are mean ± s.e.m. from three independent experiments, ∗p < 0.05.

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  Figure 5. JNK inhibition reverses the effect of AP-Cav on mouse colon endothelial cell apoptosis. Mouse colon endothelial cells were starved in media containing 1% FBS overnight, treated with JNK inhibitor for 1 hour, incubated for 30 minutes with AP or AP-Cav peptide, and stimulated with 50 ng/ml of VEGF for 4 hours. (a) At the end of the treatment, the cells were lysed in boiling SDS-sample buffer followed by western blot analysis with antibody for activated form of caspase-3. (b) Bar graph represents relative cleaved form of caspase-3 level normalized to beta-actin level. (c) At the end of the treatment, the cells were fixed and analyzed using fluorescein FragEL DNA fragmentation detection kit and then evaluated for apoptotic cells under a fluorescence microscope. The TUNEL positive cell number per field under 10x objective was counted. Data are mean ± s.e.m. from three independent experiments, ∗p < 0.05.