Hematopoietic cell kinase associates with the 40S ribosomal subunit and mediates the ribotoxic stress response to deoxynivalenol in mononuclear phagocytes

Abstract

The trichothecene deoxynivalenol (DON) binds to eukaryotic ribosomes and triggers p38-driven proinflammatory gene expression in the macrophage-a response that is dependent on both double-stranded RNA-activated protein kinase (PKR) and hematopoietic cell kinase (Hck). Here we elucidated critical linkages that exist among the ribosome and these kinases during the course of DON-induced ribotoxic stress in mononuclear phagocytes. Similar to PKR inhibitors, Hck inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2) suppressed p38 activation and p38-driven interleukin 8 (IL-8) expression in the U937 human monocyte cell line. U937 cells stably transfected with a PKR antisense vector (U9K-A1) displayed marked reduction of DON-induced p38 activation and IL-8 expression as compared to cells transfected with empty vector (U9K-C2), with both responses being completely ablated by PP2. Western analysis of sucrose density gradient fractions revealed that PKR and Hck interacted with the 40S ribosomal subunit in U9K-C2 but not U9K-A1 cells. Subsequent transfection and immunoprecipitation studies with HeLa cells indicated that Hck interacted with ribosomal protein S3. Consistent with U937 cells, DON induced p38 association with the ribosome and phosphorylation in peritoneal macrophages from wild-type but not PKR-deficient mice. DON-induced phosphorylation of ribosome-associated Hck in RAW 264.7 murine macrophages was also suppressed by 2-aminopurine (2-AP). Both 2-AP and PP2 inhibited DON-induced phosphorylation of p38 as well as two kinases, apoptosis signal-regulating kinase 1 and mitogen-activated protein kinase 3/6, known to be upstream of p38. Taken together, PKR and Hck were critical for DON-induced ribosomal recruitment of p38, its subsequent phosphorylation, and, ultimately, p38-driven proinflammatory cytokine expression.

FIG. 1.

Hck inhibition suppresses DON-induced IL-8 mRNA expression in U937 cells. Cells were pretreated with PP2 (2.5μM) or dimethyl sulfoxide vehicle (VEH) for 45 min before addition of 0 or 1000 ng/ml DON. IL-8 mRNA expression was measured by real-time PCR after a 6-h DON exposure. Data are mean ± SEM (n = 3). Asterisk indicates significantly different than VEH (p < 0.05). Representative of three independent experiments.

FIG. 2.

PKR and Hck inhibition suppresses DON-induced p38 phosphorylation in U937 cells. Cells were incubated for 45 min with (A) 2-AP (5.0mM) or water vehicle or (B) PP2 (0 or 2.5μM) or with dimethyl sulfoxide vehicle before treating with 0 or 500 ng/ml DON for 15 min. Protein in cell lysate was analyzed by Western blotting for p38 and phospho-p38. Representative of three independent experiments.

FIG. 3.

PKR antisense expression and Hck inhibition suppress DON-induced p38 phosphorylation and IL-8 production in U937 cells. U937 cells expressing control (U9K-C2) or PKR antisense vector (U9K-A1) were pretreated with PP2 (0.25–25μM) or dimethyl sulfoxide vehicle for 45 min before treating with 0 or 500 ng/ml DON. (A) Cells were lysed with SDS after 30 min DON treatment and proteins analyzed by Western blotting for phospho-p38. (B) Culture supernatant was collected after a 6-h DON treatment, and IL-8 protein was assessed by ELISA. Data are mean ± SEM (n = 3). Bars without same letter differ (p < 0.05). Representative of three independent experiments.

FIG. 4.

DON induces phosphorylation of ribosome-associated PKR and Hck in U937 cells. Cultures were treated with DON (0 or 500 ng/ml) for 1 min and subjected to sucrose density gradient fractionation. Fractions were analyzed by Western blotting. Lanes are aligned with density fractions depicted in above blot. Data are representative of three separate experiments.

FIG. 5.

PKR is required for Hck interaction with the ribosome in U937 cells. (A) U9K-C2 control and U9K-A1 PKR-antisense expressing cells were lysed with polysome extraction buffer, and cytoplasmic fractions were collected after centrifugation at 10,000 × g for 15 min. Protein was analyzed by Western blotting with PKR and Hck antibodies. (B) U9K-C2 or (C) U9K-A1 cells were treated with 0 or 500 ng/ml DON for 1 min. Ribosomal fractions were separated by sucrose gradient, and protein was analyzed by Western blotting. Lanes are aligned with density fraction profile shown above. Data are representative of three separate experiments.

FIG. 6.

Hck interacts with the RPS3. RPS3-GFP and Hck were overexpressed in HeLa cells. Lysates were immunoprecipitated with anti-GFP or Hck and analyzed by Western blotting with antibodies specific for Hck or RPS3. Results are representative of two separate experiments.

FIG. 7.

DON-induced MAPK interaction with the ribosome is suppressed in peritoneal macrophages from PKR-deficient mice. Peritoneal macrophages from WT and PKR KO mice were cultured with 0 or 500 ng/ml DON for 15 min. (A) Cells were lysed and analyzed by Western blotting. (B) Ribosomal proteins were separated by sucrose density gradient fractionation and pooled ribosomal fractions analyzed by Western blotting. Data are representative of three separate experiments.

FIG. 8.

PKR inhibition suppresses DON-induced Hck phosphorylation in ribosomal fractions of RAW 264.7 cells. Cells were treated with PKR inhibitor, 2-AP (5mM), or HCK inhibitor, PP2 (2.5μM), for 45 min before adding DON (250 ng/ml) for 5 min. Ribosomes were fractionated on sucrose gradient, and pooled ribosomal fractions analyzed by Western blotting. Data are representative of three separate experiments.

FIG. 9.

PKR and Hck phosphorylation precedes p38 interaction with the ribosome in RAW 264.7 cells. Cells were treated with 250 ng/ml DON for 0, 5, 15, or 30 min. Ribosomes were fractionated on sucrose gradient, and pooled ribosomal fractions analyzed by Western blotting with antibodies against phospho-PKR, phospho-Hck, p38, or RPL7. Data are representative of three separate experiments.

FIG. 10.

PKR and Hck inhibition suppresses DON-induced phosphorylation of ASK1, MKK3/6, and p38 in RAW 264.7 cells. Cells were incubated for 45 min with (A) 2-AP (5mM) or dimethyl sulfoxide vehicle or with (B) PP2 (2.5μM) or dimethyl sulfoxide vehicle prior to treating with 0 or 250 ng/ml DON for 15 min. Cell lysates were analyzed by Western blotting with specific antibodies. Data are representative of three separate experiments.

FIG. 11.

Ribosome functions as scaffold for PKR, Hck, and p38 in DON-induced ribotoxic stress response. DON-induced ribotoxic is proposed to involve: (1) rapid DON uptake and binding to ribosome, (2) activation of ribosomal-associated PKR and Hck, (3) interaction of p38 with the ribosome, (4) p38 phosphorylation, and (5) induction of proinflammatory genes such as IL-8.