LPS-induced apoptosis in Transformed Bovine Brain Endothelial Cells (TBBEC) and Human Dermal Microvessel Endothelial Cells (HMEC): the role of JNK

摘要

Stimulation of transformed bovine brain endothelial cells (TBBEC) with lipopolysaccharide (LPS) leads to apoptosis while human microvessel endothelial cells (HMEC) needs the presence of cycloheximide (CHX) with LPS to induce apoptosis. To investigate the molecular mechanism of LPS-induced apoptosis in HMEC or TBBEC, we analyzed the involvement of mitogen activated protein kinases (MAPKs) and PI3K in TBBEC and HMEC. LPS-induced apoptosis in TBBEC was hallmarked by the activation caspase-3, -6, and -8 after the stimulation of LPS, followed by poly-ADP ribose polymerase (PARP) cleavage and lactate dehydrogenase (LDH) release. We also observed DNA cleavage determined by terminal deoxy transferase (TdT)-mediated dUTP nick and labeling [TUNEL] staining in TBBEC treated with LPS. Herbimycin A, a tyrosine kinase inhibitor, and SP600125, a c-jun N-terminal kinase (JNK) inhibitor, suppressed the activation of caspases and LDH release. Moreover, a phosphatidyl-inositol 3-phosphate (PI3K) inhibitor () suppressed activation of caspases, and combined treatment with both SP600125 and completely inhibited the activation of caspases. These results suggest that JNK signaling pathway through the tyrosine kinase and PI3K pathways are involved in the induction of apoptosis in LPS-treated TBBEC. On the other hand, we observed sustained JNK activation in HMEC treated with LPS and CHX, and neither Erk1/2 nor AKT were activated. The addition of SP600125 suppressed phosphorylation of JNK and the activation of caspase-3 in HMEC treated with LPS and CHX. These results suggest that JNK plays an important role in the induction of apoptosis in endothelial cells.