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

Abstract

It has been reported that pathological angiogenesis contributes to both experimental colitis and inflammatory bowel disease. Recently, we demonstrated that endothelial caveolin-1 plays a key role in the pathological angiogenesis of dextran sodium sulfate (DSS) colitis. However, the molecular mechanism of caveolin-1 regulation of endothelial function is unknown. In this study, we examined how the antennapedia- (AP-) conjugated caveolin-1 scaffolding domain (AP-Cav) modulates vascular endothelial growth factor- (VEGF-) dependent colon endothelial cell angiogenic responses, as seen during colitis. We used mouse colon endothelial cells and found that AP-Cav significantly inhibited VEGF-mediated bromodeoxyuridine (BrdU) incorporation into colon microvascular endothelial cells. AP-Cav significantly blunted VEGF-dependent extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation at 10 minutes and 2 hours after stimulation, compared with the AP control peptide. AP-Cav + VEGF-A treatment also significantly increased c-Jun N-terminal kinase (JNK) phosphorylation at 2 hours. AP-Cav + VEGF-A treatment significantly downregulated retinoblastoma (Rb) protein levels, upregulated cleaved caspase-3 protein levels at 4 hours, and induced apoptosis. Thus, our study suggests that disruption of endothelial caveolin-1 function via the AP-Cav diverts VEGF signaling responses away from endothelial cell proliferation and toward apoptosis through the inhibition of mitogen-activated protein (MAP) kinase signaling and the induction of JNK-associated apoptosis.

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.

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.

Figure 3

Effect of AP-Cav on cell cycle protein level of p27^kip1, 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 p27^kip1 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 p27^kip1 normalized to beta-actin level. Data are mean ± s.e.m. from three independent experiments, ^∗p < 0.05.

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.

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.