Summary Toll-like receptor (TLR) responses are regulated to avoid toxicity and achieve coordinated responses appropriate for the cell environment. We found that Notch and TLR pathways cooperated to activate canonical Notch target genes, including transcriptional repressors Hes1 and Hey1, and to increase production of canonical TLR-induced cytokines TNF, IL-6 and IL-12. Cooperation by these pathways to increase target gene expression was mediated the Notch pathway component and transcription factor RBP-J, which also contributed to lethality after endotoxin injection. TLR- and Notch-induced Hes1 and Hey1 attenuated IL-6 and IL-12 production. This Hes1- and Hey1-mediated feedback inhibitory loop was abrogated by interferon-γ (IFN-γ), which blocked TLR-induced activation of canonical Notch target genes by inhibiting Notch2 signaling and downstream transcription. These findings identify new immune functions for RBP-J, Hes and Hey proteins and provide insights into mechanisms by which Notch, TLR and IFN-γ signals are integrated to modulate specific effector functions in macrophages.

Dicer is essential for plant, Caenorhabditis elegans, and Drosophila antiviral responses because of its role in generating small interfering RNA (siRNA) from viral genomes. We show that because of impaired miRNA production, mice with a variant Dicer1 allele (Dicer1(d/d)) were more susceptible to vesicular stomatitis virus (VSV) infection. We did not detect VSV genome-derived siRNA in wild-type cells or any alteration of interferon-mediated antiviral responses by Dicer1 deficiency. Rather, we found that host miR24 and miR93 could target viral large protein (L protein) and phosphoprotein (P protein) genes, and a lack of miR24 and miR93 was responsible for increased VSV replication in Dicer1(d/d) cells. Our data suggest that host miRNA can play a role in host interactions with viruses.

In addition to caspase inhibition, X-linked inhibitor of apoptosis (XIAP) induces NF-kappaB and MAP kinase activation during TGF-b and BMP receptor signaling and upon overexpression. Here we show that the BIR1 domain of XIAP, which has no previously ascribed function, directly interacts with TAB1 to induce NF-kappaB activation. TAB1 is an upstream adaptor for the activation of the kinase TAK1, which in turn couples to the NF-kappaB pathway. We report the crystal structures of BIR1, TAB1, and the BIR1/TAB1 complex. The BIR1/TAB1 structure reveals a striking butterfly-shaped dimer and the detailed interaction between BIR1 and TAB1. Structure-based mutagenesis and knockdown of TAB1 show unambiguously that the BIR1/TAB1 interaction is crucial for XIAP-induced TAK1 and NF-kappaB activation. We show that although not interacting with BIR1, Smac, the antagonist for caspase inhibition by XIAP, also inhibits the XIAP/TAB1 interaction. Disruption of BIR1 dimerization abolishes XIAP-mediated NF-kappaB activation, implicating a proximity-induced mechanism for TAK1 activation.

The activation of p38α, a MAPK family member, is associated with macrophage activation by microbial pattern molecules, such as LPS. The requirement of p38α in inflammatory responses has been shown in a number of studies using chemical inhibitors, though the inhibitors also inhibit p38β and perhaps some other enzymes. In this study, we used conditional knockout of p38α in macrophages to address the role of p38α in macrophage activation. We found that p38α deficiency causes a significant inhibition in the production of LPS-induced TNF-α, IL-12, and IL-18, but it has little or no effect on IL-6 or IFN-β production. Knockout of p38α in macrophages did not affect LPS-induced activation of the other major signaling pathways (NF-κB, Jnk, and Erk), nor did it affect the transcriptional activity of NF-κB. It had little inhibitory effect on LPS-induced AP-1 activity, but it significantly inhibited LPS-induced C/EBP-β and CREB activation, indicating that the role of p38α in cytokine production in macrophages is at least in part through its regulation of C/EBP-β and CREB activation. In addition, we also confirmed that p38α is important for phagocytosis of bacteria by macrophages. Our in vivo studies with two murine models showed that p38α is involved in sepsis. Collectively, our data demonstrate that p38α is an important player in inflammatory responses.

α-fetoprotein (AFP) is not only a widely used biomarker in hepatocellular carcinoma (HCC) surveillance, but is also clinically recognized as linked with aggressive tumour behaviour. Here we show that deregulation of microRnA122, a liver-specific microRnA, is a cause of both AFP elevation and a more biologically aggressive phenotype in HCC. We identify CuX1, a direct target of microRnA122, as a common central mediator of these two effects. using liver tissues from transgenic mice in which microRnA122 is functionally silenced, an orthotopic xenograft tumour model, and human clinical samples, we further demonstrate that a microRnA122/ CuX1/microRnA214/ZBTB20 pathway regulates AFP expression. We also show that the microRnA122/CuX1/RhoA pathway regulates the aggressive characteristics of tumours. We conclude that microRnA122 and associated signalling proteins may represent viable therapeutic targets, and that serum AFP levels in HCC patients may be a surrogate marker for deregulated intracellular microRnA122 signalling pathways in HCC tissues.