Deoxynivalenol induces p38 interaction with the ribosome in monocytes and macrophages

Abstract

Trichothecene mycotoxins rapidly induce p38-mediated gene expression and apoptosis in mononuclear phagocytes via a process known as the ribotoxic stress response. We hypothesized that the trichothecene deoxynivalenol (DON) induces interaction of p38 with the ribosome. Two models, U937 human monocytes and RAW 264.7 murine macrophages, were used to test this hypothesis based on their capacity to evoke rapid and robust p38 phosphorylation responses to DON. Following DON treatment of U937 cells, lysates were subjected to sucrose gradient fractionation and the resultant ribosomal fractions probed for p38 by Western blotting. p38 content in fractions containing ribosomal subunits and monosomes (RS + M) increased within 5 min of DON treatment and continued to increase up to 30 min. p38 appeared to be initially interact with the 40S subunit fraction and then subsequently with the 60S unit and monosome fractions. Although p38 phosphorylation was blocked by the inhibitor SB203580, interaction of the kinase with the ribosome was unaffected, suggesting that ribosomal binding and phosphorylation were dissociable events. In RAW 264.7 cells, radiolabeled DON uptake occurred within 15 min and this corresponded to sequential increases nonphosphorylated p38 and phosphorylated p38 in the RS + M fraction. As observed for p38, DON similarly induced both ribosomal interaction with two mitogen-activated protein kinases, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, and their subsequent phosphorylation in RAW 264.7 cells. Taken together, these data suggest that, in mononuclear phagocytes, DON induced p38 mobilization to the ribosome and its subsequent phosphorylation. The ribosome might thus play a central role as a scaffold in the ribotoxic stress response.

FIG. 1.

DON induces p38 phosphorylation in U937 monocytes. Cells were stimulated with 500 ng/ml of DON for 15 or 30 min. (A) Western analysis was conducted using antibodies specific for total p38 and phospho-p38. (B) Infrared fluorescence intensities of phospho-p38 band normalized against intensity of corresponding total p38 band. Data are representative of three separate experiments.

FIG. 2.

DON induces p38 association with the ribosome in U937 monocytes. (A) Representative sucrose gradient separation of ribosomal subunit, monosome and polysome fractions of U937 cells. Fractions containing small (40S) and large (60S) ribosomal subunits were detected by Western blotting with RPS6 and RPL7 antibodies, respectively. (B) Fractions were pooled and designated as RS + M or P and analyzed for p38 by Western blotting. Data are representative of three separate experiments.

FIG. 3.

DON induces sequential interaction of p38 with 40S and 60S ribosomal subunits in U937 monocytes. Cells were incubated with 500 ng/ml of DON for 5 or 15 min and p38 interactions within the ribosomal fractions compared with those for untreated cells. Data are representative of three independent experiments.

FIG. 4.

DON-induced p38 interaction with the ribosome does not require p38 phosphorylation in U937 monocytes. Cells were preincubated with the p38 kinase inhibitor SB203580 (2μM) for 45 min and treated with 500 ng/ml of DON for 30 min. Cell lysates and pooled RS + M were analyzed by Western blotting. Data are representative of two separate experiments.

FIG. 5.

Rapid [³H] DON uptake in RAW 264.7 macrophages corresponds with p38 phosphorylation and ribosome interaction. (A) Cells were incubated with [³H] DON (250 ng/ml) for various time intervals and uptake monitored. (B) Cells were stimulated with DON (250 ng/ml) for 15 or 30 min and p38 phosphorylation in lysates measured by in-cell Western assay. (C) Relative in-cell Western infrared intensities of phosphorylated p38 were normalized against intensities total p38. Bars without same letter differ (p < 0.05). (D) Pooled RS + M were analyzed for total and phosphorylated p38 by Western blotting. Data are representative of three separate experiments.

FIG. 6.

DON-induced p38 interaction with the ribosome does not require p38 phosphorylation in RAW 264.7 macrophages. Cells were preincubated with p38 kinase inhibitor (2μM) for 45 min and treated with 250 ng/ml of DON for 30 min. Cells lysates and pooled RS + M were analyzed by Western blotting. Data are representative of two separate experiments.

FIG. 7.

DON induces JNK and ERK ribosomal interaction and phosphorylation in RAW 264.7 macrophages. (A) Cells were stimulated with DON (250 ng/ml) for various time intervals and JNK and ERK phosphorylation in lysates measured by in-cell Western assay. (B) Relative in-cell Western infrared intensities of phosphorylated JNK and ERK were normalized against intensities of total JNK and ERK. Bars without same letter differ (p < 0.05). (C) Pooled RS + M were analyzed for total and phosphorylated JNK and ERK by Western blotting. Data are representative of three separate experiments.

FIG. 8.

Proposed scaffolding role of ribosome in DON-induced ribotoxic stress response. DON-induced ribotoxic stress response in mononuclear phagocytes is proposed to involve the following sequential steps: (1) rapid DON uptake and binding to ribosome, (2) interaction of p38 with the ribosome, (3) p38 phosphorylation, and (4) putative induction of p38-mediated sequelae including mRNA transcription,/stabilization, protein translation and activation of apoptotic effectors. A similar sequence of events is possible for JNK and ERK.