Signal transduction pathways involved in oxidative stress-induced intestinal epithelial cell apoptosis

Abstract

Necrotizing enterocolitis (NEC) is a devastating inflammatory condition of the gut that occurs in premature infants. Ischemia-reperfusion gut injury with production of reactive oxygen species (ROS) is thought to contribute to NEC; the exact cellular mechanisms involved are largely unknown. The purpose of this study was to determine the intracellular signaling transduction pathways involved in oxidative stress-induced intestinal epithelial cell apoptosis. H2O2 treatment resulted in rat intestinal epithelial cell apoptosis in a dose- and time-dependent manner; the caspase inhibitor, zVAD-fmk, blocked this response. Western blotting was performed to determine phosphorylation of kinases and ELISA was used to assess DNA fragmentation, as a measure of apoptosis. A rapid increase in phosphorylation of extracellular signal-related kinase (ERK)1/2, c-Jun N-terminal kinase (JNK)1/2, and Akt was noted. Inhibition of ERK and JNK decreased H2O2-induced apoptosis. Additionally, inhibition of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) attenuated and enhanced H2O2-mediated apoptosis and mitochondrial membrane potential decrease, respectively. Furthermore, activation of PKC reduced the Akt phosphorylation, whereas inhibition of PKC attenuated H2O2-mediated activation of caspase-3 and enhanced the H2O2-induced Akt phosphorylation. This study shows that activation of multiple signaling transduction pathways occurs during oxidative stress-induced intestinal epithelial cell injury. In contrast to ERK, JNK, and PKC, PI3-K/Akt may play an important role as a protective cellular signaling pathway during this process.

Fig. 1. H₂O₂ induces RIE-1 cell apoptosis

(A) RIE-1 cells were plated for 24 h prior to treatment with H₂O₂ for 3 h or (B) pretreated with zVAD-fmk for 30 min prior to treatment with H₂O₂. Apoptosis was estimated by a DNA fragmentation ELISA (Data represent triplicate determinations ± SEM; * = P < 0.05 vs. control; ^† = P < 0.05 vs. H₂O₂ alone). Experiments were repeated twice. (C) RIE-1 cells were treated with H₂O₂ for various timepoints. A representative Western blot from three separate experiments is shown here.

Fig. 2. Activation of signal transduction pathways by H₂O₂

RIE-1 cells were treated with H₂O₂ over a time course for Western blot analysis. A representative blot from three separate experiments is shown here.

Fig. 3. Effects of kinase inhibitors on H₂O₂-induced RIE-1 cell death

(A, C) RIE-1 cells were pretreated with kinase inhibitors [JNK inhibitor SP600125, the MEK1 inhibitor U0126 or combination (A) and PI3-K inhibitor wortmannin or PKC inhibitor GF109203× (C)] for 30 min prior to treatment with H₂O₂ for 3 hours. Apoptosis was estimated by a DNA fragmentation ELISA (Data represent triplicate determinations ± SEM; * = P < 0.05 vs. control; ^† = P < 0.05 vs. H₂O₂ alone). Experiments were repeated twice. (B) RIE-1 cells were pretreated with the U0126 for 30 min prior to treatment with H₂O₂ for 30 min and Western blotting performed. (D) RIE-1 cells were pretreated with the wortmannin for 30 min prior to treatment with H₂O₂ for 3 hours and Western blotting performed. Representative blots from three separate experiments are shown as (B) and (D).

Fig. 4. Inhibition of PKC results in the activation of Akt and blocks H₂O₂-induced caspase-3 cleavage

(A) RIE-1 cells were pretreated with the PI3-K inhibitor wortmannin or the PKC inhibitor GF109203× for 30 min prior to treatment with H₂O₂ for 3 h for Western blot analysis. (B, C) RIE-1 cells were pretreated with the GF109203× for 30 min prior to treatment with H₂O₂ (B) or PMA (C) for Western blot analysis. Representative blots from three separate experiments are shown here.

Fig. 5. Inhibition of PKC attenuates, while inhibition of PI3-K enhances, H₂O₂-induced mitochondrial depolarization

RIE-1 cells were pretreated with the PI3-K inhibitor wortmannin or the PKC inhibitor GF109203× for 30 min prior to treatment with H₂O₂. for 3 h. Cells were labeled with 2 μM JC-1 for 15 min at room temperature, and intensities of FL-1 and FL-2 fluorescence were measured. JC-1 fluorescence in the FL-1 channel increases as the mitochondrial membrane potential drops while its fluorescence in FL-2 channel decreases. Percentage numbers in Q1 and Q2, Q4 indicate proportion of cells with normal and depolarized mitochondria, respectively. Representative data from three separate experiments are shown here.