PKD prevents H2O2-induced apoptosis via NF-kappaB and p38 MAPK in RIE-1 cells - PubMed (original) (raw)

PKD prevents H2O2-induced apoptosis via NF-kappaB and p38 MAPK in RIE-1 cells

Jun Song et al. Biochem Biophys Res Commun. 2009.

Abstract

Previously, we demonstrated that protein kinase D (PKD) plays a protective role during H(2)O(2)-induced intestinal cell death. Here, we sought to determine whether this effect is mediated by nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPKs). Treatment with H(2)O(2) activated NF-kappaB in RIE-1 cells; H(2)O(2) also induced the translocation of NF-kappaB p65 as well as phosphorylation of IkappaB-alpha. PKD1 siRNA inhibited H(2)O(2)-induced activation, translocation of NF-kappaB, and phosphorylation of IkappaB-alpha. We also found that overexpression of wild type PKD1 attenuated H(2)O(2)-induced phosphorylation of p38 MAPK and its upstream activator, MAPK kinase (MKK) 3/6, whereas the phosphorylation was increased by PKD1 siRNA or kinase-dead PKD1. Phosphorylation of neither extracellular signal-regulated kinases (ERK) 1/2 nor c-Jun N-terminal kinases (JNK) was altered by PKD1 plasmids or siRNA. Our findings suggest that PKD protects intestinal cells through up-regulation of NF-kappaB and down-regulation of p38 MAPK.

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Figures

Fig. 1

Fig. 1. H2O2 induces the activation of NF-κB, translocation of NF-κB p65 and IκBα phosphorylation in RIE-1 cells

(A) Cells were transfected with 1 μg of a plasmid containing the NF-κB promoter fragment. After 42 h incubation, the transfected cells were treated with H2O2 (100 μM) for 6 h and luciferase activity measured in the cell lysates. The results were normalized for transfection efficiency using the pRL-Tk-luc plasmid [*=p< 0.05 vs. control (−)]. (B) Cells were grown on chamber slides for 2 days then treated with H2O2 (500 μM) or vehicle for 1, 5, 15 min. A representative photomicrograph is shown demonstrating NF-κB p65 localization in using immunofluorescent imaging. (C) After cells were treated with H2O2 (500 μM) over a time course, IκBα phosphorylation was detected by Western blotting using anti-phospho-IκBα (Ser32) antibody. IκBα was used as a loading control.

Fig. 2

Fig. 2. PKD1 is required for H2O2-stimulated NF-κB activation, translocation and IκBα phosphorylation

(A) RIE-1 cells were co-transfected with a NF-κB plasmid and control siRNA or PKD1 siRNA. After 42 h incubation, the cells were treated with H2O2 for 6 h, and harvested and luciferase activity measured in the cell lysates [*=p< 0.05 vs. control siRNA (−); †=p< 0.05 vs. control siRNA transfected-cells with H2O2 treatment (upper panel)]. PKD expression was examined using anti-PKD antibody (lower panel). (B) RIE-1 cells were transfected with control or PKD1 siRNA and grown on chamber slides for 2 days prior to treatment with H2O2 (500 μM) for 15 min. A representative photomicrograph is shown demonstrating NF-κB localization. (C). Cells were transfected with control or PKD1 siRNA. After 3 days, cells were treated with H2O2 (500 μM) for 15 min and Western blotting was performed. PKD expression was examined using anti-PKD antibody (top row). Phosphorylation of IκBα was detected (middle row) and IκBα was used as a loading control (bottom row).

Fig. 3

Fig. 3. PKD1 affects H2O2-induced p38 MAPK phosphorylation but not ERK1/2 and JNK>

(A) RIE-1 cells were transfected with PKD1WT, PKD1KD. At 48 h after transfection, cells were treated with H2O2 (500 μM) for 30 min, and protein was extracted for Western blot analysis. PKD overexpression was demonstrated using anti-GST antibody. Phosphorylation of ERK1/2, JNK and p38 MAPK was determined with anti-phospho-ERK1/2, JNK and p38 antibodies. (B) RIE-1 cells were transfected with control or PKD1 siRNA. After 3 days, cells were treated with H2O2 (500 μM) for 30 min, and protein was extracted for Western blotting. Inhibition of PKD expression by PKD1 siRNA was shown with anti-PKD antibody (top row). Phosphorylation of ERK1/2, JNK and p38 MAPK was determined with anti-phospho-ERK1/2, JNK and p38 antibodies. β-actin was used as a loading control.

Fig. 4

Fig. 4. Phosphorylation of p38 MAPK induced by H2O2 mediates through MKKs

(A) RIE-1 cells were treated with H2O2 (500 μM) in normal growth medium over a time course; phosphorylation of MKK3/6 was detected by Western blotting (top panel). β-actin was used as a loading control. (B) RIE-1 cells were transfected with PKD1WT, PKD1KD. At 48 h after transfection, cells were treated with H2O2 (500 μM) for 30 min, and protein was extracted for Western blotting. PKD overexpression was demonstrated using anti-PKD antibody (top panel). Phosphorylation of MKK3/6 was determined with anti-phospho-MKK3/6 antibody (second panel). (C) RIE-1 cells were transfected with PKD1 or control siRNA. After 3 days, cells were treated with H2O2 (500 μM) for 30 min, and protein was extracted for Western blotting. Inhibition of PKD expression by PKD1 siRNA was determined using anti-PKD antibody (top panel). Phosphorylation of MKK3/6 was determined with anti-phospho-MKK3/6 antibody (second panel). β-actin was used as a loading control.

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