Double-stranded RNA-activated protein kinase mediates induction of interleukin-8 expression by deoxynivalenol, Shiga toxin 1, and ricin in monocytes

Abstract

Translational inhibitors such as the trichothecene mycotoxin deoxynivalenol (DON) and ribosomal inhibitory proteins (RIPs) induce mitogen-activated protein kinase (MAPK)-driven chemokine and cytokine production by a mechanism known as the ribotoxic stress response (RSR). Double-stranded RNA-activated protein kinase (PKR) associates with the ribosome making it uniquely positioned to sense 28S ribosomal RNA damage and initiate the RSR. We have previously shown that PKR mediates DON-induced MAPK phosphorylation in macrophages and monocytes. The purpose of this study was to test the hypothesis that PKR is essential for induction of interleukin (IL)-8 expression in monocytes by DON and two prototypical RIPs, ricin, and Shiga toxin 1 (Stx1). Preincubation of human monocytic U937 cells with the PKR inhibitors C16 and 2-aminopurine (2-AP) blocked DON-induced expression of IL-8 protein and mRNA. Induction of IL-8 expression was similarly impaired in U937 cells stably transfected with a dominant negative PKR plasmid (UK9M) as compared with cells transfected with control plasmid (UK9C). Nuclear factor-kappa B binding, which has been previously shown to be a requisite for DON-induced IL-8 transcription, was markedly reduced in UK9M cells as compared with UK9C cells. As observed for DON, ricin-, and Stx1-induced IL-8 expression was suppressed by the PKR inhibitors C16 and 2-AP as well as impaired in UK9M cells. Taken together, these data indicate that PKR plays a common role in IL-8 induction by DON and the two RIPs, suggesting that this kinase might be a critical factor in RSR.

FIG. 1.

PKR inhibitors significantly suppress DON-induced IL-8 mRNA expression. U937 cells were pretreated with (A) C16 inhibitor (2.5μM) or a negative control inhibitor (2.5μM) for 45 min or with (B) 5mM 2-AP or vehicle (water) for 1 h before addition of DON at the concentrations indicated. RNA was isolated 6 h after addition of DON and IL-8 mRNA assessed by real-time PCR. Data are mean ± SEM (n = 3). Asterisk indicates significant difference (p < 0.05) from vehicle treatment. Results are representative of two independent experiments.

FIG. 2.

PKR inhibitors significantly suppress DON-induced IL-8 protein production. U937 cells were pretreated with the (A) C16 inhibitor or (B) 2-AP as described in Figure 1 legend before addition of DON (0 or 1.0 μg/ml). Culture supernatant was collected 12 h after addition of DON and IL-8 protein was assessed by ELISA. Data are mean ± SEM (n = 3). Asterisk indicates significant difference (p < 0.05) from vehicle treatment. Results are representative of three independent experiments.

FIG. 3.

Dominant negative PKR significantly suppresses DON-induced IL-8 mRNA expression. U937 cells, stably transfected with an empty vector (U9KC) or with a constitutively expressed nonfunctional form of PKR (U9KM), were treated with 0 or 1 μg/ml DON. RNA was isolated 6 h after addition of DON and IL-8 mRNA assessed by real-time PCR. Data are mean ± SEM (n = 3). Data are representative of two independent experiments. Asterisk indicates significant difference (p < 0.05) from vehicle treatment.

FIG. 4.

Dominant negative PKR reduces DON-induced p65 NF-κB binding. U937 cells stably transfected with an empty vector (U9KC) or with a constitutively expressed nonfunctional form of PKR (U9KM), were treated with 0 or 1 μg/ml DON for 3 h before isolation of nuclear protein and assessment of p65 NF-κB binding. Data are mean ± SEM of pooled results from two independent experiments (n = 4). Asterisk indicates significantly different from corresponding vehicle control (p < 0.05).

FIG. 5.

The p38 inhibitor SB203580 significantly reduces DON-induced p65 NF-κB binding. U937 cells were treated with 0 or 2.0μM SB203580 with 0 or 1 μg/ml DON for 3 h before isolation of nuclear protein and assessment of p65 NF-κB binding. Data are mean ± SEM of pooled results from two independent experiments (n = 3). Asterisk indicates significant difference from corresponding vehicle control (p < 0.05).

FIG. 6.

The C16 PKR inhibitor significantly suppresses Stx1- or ricin-induced IL-8 protein production. U937 cells were pretreated with C16 inhibitor (2.5μM) or a negative control inhibitor (2.5μM) for 45 min before addition of 0, 0.5, or 1 μg/ml (A) Stx1 or (B) ricin. Culture supernatant was collected 12 h after addition of toxin and IL-8 protein was assessed by ELISA. Data are mean ± SEM (n = 3). Asterisk indicated significantly different (p < 0.05) from vehicle control. Data are representative of two independent experiments.

FIG. 7.

The C16 PKR inhibitor significantly suppresses Stx1- or ricin-induced IL-8 mRNA expression. U937 cells were pretreated with C16 inhibitor (2.5μM) or a negative control inhibitor (2.5μM) for 45 min before addition of 0 or 1 μg/ml (A) Stx1 or (B) ricin. RNA was isolated 6 h after addition of toxin and IL-8 mRNA was assessed by real-time PCR. Data are mean ± SEM (n = 3). Asterisk indicates significant difference (p < 0.05) from vehicle control. Results are representative of two independent experiments.

FIG. 8.

2-AP significantly suppresses Stx1- or ricin-induced IL-8 mRNA. U937 cells were pretreated with 0 or 5mM 2-AP for 1 h before addition of 0 or 1 μg/ml (A) Stx1 or (B) ricin. RNA was isolated 6 h after addition of toxin and IL-8 mRNA assessed by real-time PCR. Data are mean ± SEM (n = 3). Asterisk indicates significant difference (p < 0.05) from vehicle control. Results are representative of two independent experiments.

FIG. 9.

Dominant negative PKR significantly suppresses Stx1- or ricin-induced IL-8 mRNA. U937 cells stably transfected with an empty vector (U9KC) or with a constitutively expressed nonfunctional form of PKR (U9KM) were treated with 0 or 1 μg/ml (A) Stx1 or (B) ricin. RNA was isolated 6 h after addition of DON and IL-8 mRNA assessed by real-time PCR. Data are mean ± SEM (n = 3). Asterisk indicates significant difference (p < 0.05) from vehicle control. Results are representative of two independent experiments.

FIG. 10.

Putative mechanism for DON-induced IL-8 expression. Results presented here and previously (Gray and Pestka, 2007; Islam et al., 2006; Zhou et al., 2003b) suggest that DON exposure sequentially induces PKR-dependent p38 phosphorylation and NF-κB activation both of which might drive IL-8 mRNA transcription and IL-8 protein translation.