Inhibition of hypoxia/reoxygenation-induced apoptosis by an antisense oligonucleotide targeted to JNK1 in human kidney cells

Abstract

Listen

Translate article

The present study explored the relationship between activation of c-Jun N-terminal kinase (JNK) and apoptosis following exposure of primary human kidney cells to hypoxia/reoxygenation. Apoptosis induction was apparent only after prolonged exposure of cells to hypoxia (>48 hr), when a 2-fold increase in DNA fragmentation was observed. In contrast, 15 hr of reoxygenation following either 4 or 8 hr of hypoxia was associated with a pronounced (>17-fold) increase in DNA fragmentation. Fluorescence microscopy, using DNA binding dyes, demonstrated that cell death following hypoxia/reoxygenation was due predominantly to apoptosis and not necrosis. Furthermore, reoxygenation, but not hypoxia alone, caused a time-dependent increase in the activation of JNK as monitored by western blot analysis using a phospho-specific JNK antibody. In contrast, p38 mitogen-activated protein kinase was activated following hypoxia, but this activation was not augmented during reoxygenation. Exposure of human kidney cells to a 2′-methoxyethyl mixed backbone antisense oligonucleotide directed against human JNK1 (JNK1 AS) resulted in a potent suppression of JNK mRNA and protein expression, whereas a 6-base mismatch control oligonucleotide was without effect. Moreover, a significant diminution of reoxygenation-induced apoptosis was observed in cells exposed to JNK1 AS but not to the mismatch control oligonucleotide. Taken together, these results strongly indicate that activation of the JNK signaling cascade is a major mechanism whereby hypoxia/reoxygenation induces apoptosis.