Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.

Trichothecene mycotoxins and other translational inhibitors activate mitogen-activated protein kinase (MAPKs) by a mechanism called the "ribotoxic stress response," which drives both cytokine gene expression and apoptosis in macrophages. The purpose of this study was to identify upstream kinases involved in the ribotoxic stress response using the trichothecene deoxynivalenol (DON) and the RAW 264.7 macrophage as models. DON (100 to 1000 ng/ml) dose-dependently induced phosphorylation of c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs. MAPK phosphorylation in response to DON exposure occurred as early as 5 min, was maximal from 15 to 30 min, and lasted up to 8 h. Preincubation with inhibitors of protein kinase C, protein kinase A, or phospholipase C had no effect on DON-induced MAPK phosphorylation. In contrast, the Src family tyrosine kinase inhibitors, PP1 (4-amino-5-[4-methylphenyl)]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) and, PP2 (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) concentration-dependently impaired phosphorylation of all three MAPK families. PP1 suppressed DON-induced phosphorylation of the MAPK substrates c-jun, ATF-2, and p90(Rsk). MAPK phosphorylation by two other translational inhibitors, anisomycin and emetine, were similarly Src-dependent. PP1 reduced DON-induced increases in nuclear levels and binding activities of several transcription factors (NF-kappaB, AP-1, and C/EBP), which corresponded to decreases in TNF-alpha production, caspase-3 activation, and apoptosis. Tyrosine phosphorylation of hematopoeitic cell kinase (Hck), a Src found in macrophages, was detectable within 1 to 5 min after DON addition, and this was suppressed by PP1. Knockdown of Hck expression with siRNAs confirmed involvement of this Src in DON-induced TNF-alpha production and caspase activation. Taken together, activation of Hck and possibly other Src family tyrosine kinases are likely to be critical signals that precede both MAPK activation and induction of resultant downstream sequelae by DON and other ribotoxic stressors.

Exposure to the trichothecene mycotoxin deoxynivalenol (DON) alters immune functions in vitro and in vivo. To gain further insight into DON's immunotoxic effects, microarrays were used to determine how acute exposure to this mycotoxin modulates gene expression profiles in murine spleen. B6C3F1 mice were treated orally with 25mg/kg body weight DON, and 2h later spleens were collected for macroarray analysis. Following normalization using a local linear regression model, expression of 116 out of 1176 genes was significantly altered compared to average expression levels in all treatment groups. When genes were arranged into an ontology tree to facilitate comparison of expression profiles between treatment groups, DON was found primarily to modulate genes associated with immunity, inflammation, and chemotaxis. Real-time polymerase chain reaction was used to confirm modulation for selected genes. DON was found to induce the cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and IL-11. In analogous fashion, DON upregulated expression of the chemokines macrophage inhibitory protein-2 (MIP-2), cytokine-induced chemoattractant protein-1 (CINC-1), monocyte chemoattractant protein (MCP)-1, MCP-3, and cytokine-responsive gene-2 (CRG-2). c-Fos, Fra-, c-Jun, and JunB, components of the activator protein-1 (AP-1) transcription factor complex, were induced by DON as well as another transcription factor, NR4A1. Four hydrolases were found to be upregulated by DON, including mitogen-activated protein kinase phosphatase 1 (MKP1), catalytic subunit beta isoform (CnAbeta), protein tyrosine phosphatase receptor type J (Ptprj), and protein tyrosine phosphatase nonreceptor type 8 (Ptpn8), whereas three other hydrolases, microsomal epoxide hydrolase (Eph) 1, histidine triad nucleotide binding protein (Hint), and proteosome subunit beta type 8 (Psmb8) were significantly decreased by the toxin. Finally, cysteine-rich protein 61 (CRP61) and heat-shock protein 40 (Hsp40), genes associated with signaling, were increased, while Jun kinase 2 (JNK2) was decreased. Taken together, data suggest that DON upregulated the expression of multiple immediate early genes, many of which are likely to contribute to the complex immunological effects reported for this and other trichothecenes.

The purpose of this investigation was to evaluate the dose-dependent effects of docosahexaenoic acid (DHA) on deoxynivalenol (DON)-induced IgA nephropathy in mice and their relation to proinflammatory gene expression and mitogen-activated protein kinase (MAPK) activation. Consumption of a modified AIN-93G diet containing 1, 5, and 30 g/kg DHA resulted in dose-dependent increases of DHA in liver phospholipids with concomitant decreases in arachidonic acid compared with control diets. DHA dose dependently inhibited increases in serum IgA and IgA immune complexes (IC) as well as IgA deposition in the kidney in DON-fed mice; the 30 g/kg DHA diet had the earliest detectable effects and maximal efficacy. Both splenic interleukin-6 (IL-6) mRNA and heterogeneous nuclear RNA (hnRNA), an indicator of IL-6 transcription, were significantly reduced in DON-fed mice that consumed 5 and 30 g/kg DHA; a similar reduction was observed for cyclooxygenase (COX-2) mRNA. In a subsequent study, acute DON exposure (25 mg/kg body weight) induced splenic IL-6 mRNA and hnRNA as well as COX-2 mRNA in mice fed the control diet, whereas induction of both RNA species was significantly inhibited in mice fed 30 g/kg DHA. These latter inhibitory effects corresponded to a reduction in DON-induced phosphorylation of p38, extracellular-signal related kinase 1/2, and c-Jun N-terminal kinase 1/2 MAPKs in the spleen. Taken together, the results indicate that DHA dose-dependently inhibited DON-induced IgA dysregulation and nephropathy, and that impairment of MAPK activation and expression of COX-2 and IL-6 are potential critical upstream mechanisms.

Diets enriched in the (n-3) PUFAs, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and their precursor alpha-linolenic acid (ALA), were evaluated for efficacy in ameliorating the development of IgA nephropathy (IgAN) induced in mice by the mycotoxin deoxynivalenol (DON). The effects of DON were compared in mice that were fed for 18 wk with AIN-93G diets containing 1) 10 g/kg corn oil plus 60 g/kg oleic acid (control); 2) 10 g/kg corn oil plus 35 g/kg oleic acid and 25 g/kg DHA-enriched fish oil (DHA); 3) 10 g/kg corn oil plus 33 g/kg oleic acid and 27 g/kg EPA-enriched fish oil (EPA); and 4) 10 g/kg corn oil plus 37 g/kg oleic acid and 23 g/kg DHA + EPA (1:1) enriched fish oil (DHA + EPA). The DHA, EPA and DHA + EPA diets attenuated induction by dietary DON (10 mg/kg) of serum IgA and IgA immune complexes, kidney mesangial IgA deposition, and ex vivo IgA secretion by spleen cells. Consumption of the DHA + EPA diet for 8 wk significantly abrogated the DON-induced gene expression of interleukin (IL)-6, a requisite cytokine for DON-induced IgA nephropathy, in spleen and Peyer's patches. Finally, incorporation of ALA-containing flaxseed oil up to 60 g/kg in the AIN-93G diet did not affect DON-induced IgA dysregulation in mice. Taken together, both DHA and EPA, but not ALA, ameliorated the early stages of IgAN, and these effects might be related to a reduced capacity for IL-6 production.