Raf kinase inhibitor protein regulates oxygen-glucose deprivation-induced PC12 cells apoptosis through the NF-κB and ERK pathways

Abstract

Raf-1 kinase inhibitory protein (RKIP) is a critical molecule for cellular responses to stimuli. In this study, we investigated whether RKIP is responsible for neural cell apoptosis induced by oxygen-glucose deprivation (OGD) and explored the role of NF-κB and ERK pathways regulated by RKIP under OGD stimuli. RKIP was overexpressed or knocked down using lentivirus in PC12 cells, which were then challenged by OGD. RKIP overexpression significantly increased the cell viability of OGD cells, and attenuated apoptosis, cell cycle arrest, and reactive oxygen species generation. RKIP knockdown induced reverse effects. Moreover, we found that RKIP interacted with TAK1, NIK, IKK, and Raf-1 and negatively regulated the NF-κB and ERK pathways. RKIP overexpression significantly inhibited IKK, IκBα, and P65 phosphorylation in NF-κB pathway and MEK, ERK, and CREB phosphorylation in ERK pathway, respectively. RKIP knockdown induced reverse effects. Furthermore, a NF-κB inhibitor BAY 11-7082 and a MEK inhibitor U0126 blocked the changes caused by RKIP down-regulation after OGD. In conclusion, these results demonstrate that RKIP plays a key role in neural cell apoptosis caused by OGD partly via regulating NF-κB and ERK pathways. The present study may provide new insights into the role of RKIP in ischemic stroke.

Keywords: ERK pathway; NF-κB pathway; OGD; PC12 cells; RKIP.

Fig. 1

The expression of RKIP in PC12 cells was determined by real-time PCR (A) and western blot (B) after OGD. GAPDH acted as the internal control. For real-time PCR and western blot, the data was from three independent experiments (n = 3), and quantitative comparisons (C) were made by densitometry relative to GAPDH. *p<0.01 vs control and ^#p<0.01 vs OGD by one-way ANOVA analysis of variance with Tukey’s HSD post hoc test.

Fig. 2

RKIP regulated OGD-induced PC12 cells apoptosis. (A) The effect of RKIP overexpression and knockdown on the cell viability of normal and OGD-induced PC12 cells. (B, C) Apoptosis assay of RKIP overexpression and knockdown to OGD-induced PC12 cells by flow cytometry. (D) Cell cycle assay of RKIP overexpression and knockdown to OGD-induced PC12 cells by flow cytometry. (E, F) Effect of RKIP overexpression and knockdown on intracellular accumulation of ROS in OGD-induced PC12 cells. (G) Quantitative comparisons were made by densitometry relative to GAPDH. *p<0.05 and **p<0.01 vs control and ^#p<0.01 vs OGD by one-way ANOVA analysis of variance with Tukey’s HSD post hoc test.

Fig. 3

RKIP regulates PC12 cells survival from OGD injury partly via ERK Pathway. (A) The effect of RKIP overexpression, knockdown and U0126 on the cell viability of normal and OGD-induced PC12 cells. (B) Raf-1 phosphorylation induced by OGD. (C) The interaction between RKIP and Raf-1 was detected by co-immunoprecipitation. (D) The expression of total MEK, ERK, and CREB and their phosphorylation was measured by western blot. (E) Quantitative comparisons of protein bands were analyzed by densitometry normalized to GAPDH. Data shown are the results of three different experiments. *p<0.01 vs OGD by one-way ANOVA analysis of variance with Tukey’s HSD post hoc test.

Fig. 4

RKIP regulates PC12 cells survival induced by OGD partly via NF-κB Pathway. (A) The effect of RKIP overexpression, knockdown and BAY 11-7082 on the cell viability of normal and OGD-induced PC12 cells. (B) TAK1 and NIK phosphorylation induced by OGD. (C) The interaction between RKIP, TAK1, IKK, and NIK was detected by co-immunoprecipitation. (D) The phosphorylation expression of IKKβ, IκBα, and P65 were measured by western blot. (E) Quantitative comparisons of protein bands were analyzed by densitometry normalized to GAPDH. Data shown are the results of three different experiments. *p<0.01 vs OGD by one-way ANOVA analysis of variance with Tukey’s HSD post hoc test.

Fig. 5

Schematic form of the proposed mechanisms for RKIP regulated OGD-induced PC12 cells apoptosis. RKIP binds to Raf-1, TAK1, IKK, and NIK, inhibiting activation of ERK and NF-κB pathways.