Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells.

Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-ß (TGF-ß) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-ß-driven tolerance in noninflammatory contexts.

HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis.

Apoptotic signalling by p53 occurs at both transcriptional and non-transcriptional levels, as p53 may act as a direct apoptogenic stimulus via activation of the intrinsic mitochondrial pathway. HOPS is a highly conserved, ubiquitously expressed shuttling protein with an ubiquitin-like domain. We generated Hops-/- mice and observed that they are viable with no apparent phenotypic defects. However, when treated with chemotherapeutic agents, Hops-/- mice display a significant reduction in apoptosis, suggesting an impaired ability to respond to genotoxic stressors. We show that HOPS acts as a regulator of cytoplasmic p53 levels and function. By binding p53, HOPS inhibits p53 proteasomal degradation and favours p53 recruitment to mitochondria and apoptosis induction. By interfering with importin α, HOPS further increases p53 cytoplasmic levels. Thus, HOPS promotes the p53-dependent mitochondrial apoptosis pathway by preserving cytoplasmic p53 from both degradation and nuclear uptake.

Aryl hydrocarbon receptor control of a disease tolerance defence pathway.

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

INSL4 as prognostic marker for proliferation and invasiveness in Non-Small-Cell Lung Cancer.

Non-small-cell-lung cancer accounts for 80-85% of all forms of lung cancer as leading cause of cancer-related death in human. Despite remarkable advances in the diagnosis and therapy of lung cancer, no significant improvements have thus far been achieved in terms of patients' prognosis. Here, we investigated the role of INSL4 - a member of the relaxin-family - in NSCLC. We overexpressed INSL4 in NSCLC cells to analyse in vitro the growth rate and the tumourigenic features. We investigated the signalling pathways engaged in INSL4 overexpressing cells and the tumour growth ability by studying the tumour development in a patient derived tumour xenograft mouse model. We found an INSL4 cell growth promoting effect in vitro in H1299 cells and in vivo in NOD/SCID mice. Surprisingly, in NSCLC-A549 cells, INSL4 overexpression has not similar effect, despite huge basal INSL4-mRNA expression respect to H1299. The INSL4-mRNA analysis of eight different NSCLC-derived cell lines, revealed highly difference in the INSL4-mRNA amount. Transfection of NSCLC lines with INSL4-Myc showed huge level of INSL4-mRNA with a very low amount of protein expressed. Notably, similar discrepancy has been observed in NSCLC patients. However, in a cohort of NSCLC patients analysing a database, we found a significant inverse correlation between INSL4 expression and Overall Survival. By combining the in vitro and in vivo results, suggest that in patients whose NSCLC adenocarcinoma spontaneously expressed high levels of INSL4 post-transcriptional modifications affecting INSL4 do not allow to assess precision therapy in selected patients without consider protein INSL4 amount.

Identification and characterization of a novel peptide interacting with cAMP-responsive elements binding and cAMP-responsive elements modulator in mouse liver.

BACKGROUND/AIMS: Transcription factors coupled to cyclic adenosine mono phosphate (cAMP) signalling in the cAMP-responsive elements binding (CREB)/ATF family constitute a family of activators or repressors that bind to cAMP-responsive promoter elements (CREs) in the regulatory regions of cAMP-inducible genes. A role for CREB/ATF family has been advocated in the control of hepatocellular carcinoma progression. CREB appears to be activated by the X protein of hepatitis B virus, which links to the unphosphorylated form of CREB and activates transcription, thus obviating an otherwise indispensable Ser-133 phosphorylation. Identification of factors capable of triggering transcription via cAMP-responsive elements modulator (CREM)/CREB signalling in the absence of Ser phosphorylation will improve our knowledge of the molecular mechanism of liver cell proliferation. METHODS: To isolate and study proteins binding and activating CREB and/or CREM in the liver, we performed the screening of a mouse liver cDNA library using the Two-Hybrid System. RESULTS: We report the identification and characterization of a novel peptide, VTIP-peptide (VTIP-P), which binds and enhances the activation of CREM/CREB, obviating the need for transcription factor phosphorylation. We demonstrated that VTIP-P physically interacts with the activation domain (AD) of the transcription factors CREB/CREM and activates transcription by modifying their phosphorylation pattern in hepatoma cells. The data allowed the conclusion that VTIP-P binds the AD of CREB and CREM by stabilizing their phosphorylation. CONCLUSION: The characterization of molecules capable of interfering in the liver with an important pathway such as CREB could be significant in designing and/or developing new therapeutic approaches to the control of liver cell proliferation.

Different functions of HOPS isoforms in the cell: HOPS shuttling isoform is determined by RIP cleavage system.

Hepatocyte odd protein shuttling (HOPS) moves between nucleus and cytoplasm. HOPS overexpression leads to cell cycle arrest in G 0/G 1, and HOPS knockdown causes centrosome alterations, with subsequent abnormal cell division. Recently, we demonstrated that HOPS acts as a functional bridge in NPM-p19(Arf) interactions. Here we show that HOPS is present in 3 different isoforms that play distinct intracellular functions. Although HOPS is a transmembrane ubiquitin, an isoform with intermediate molecular weight is cleaved from the membrane and released into the cytosol, to act as the shuttling protein. We identified a signal peptide peptidase structure in N-terminal membrane-bound HOPS that allows the regulated intramembrane proteolysis (RIP) system to control the relative amounts of the released, shuttling isoform capable of binding NPM. These results argue for distinct, isoform-specific functions of HOPS in the nucleolus, nucleus, and cytoplasm and provide insight into the dynamics of HOPS association with NPM, whose mutation and subsequent delocalization is found in 30% of acute myeloid leukemia patients.

NEDD4 controls the expression of GUCD1, a protein upregulated in proliferating liver cells.

Liver regeneration is a unique means of studying cell proliferation in vivo. Screening of a large cDNA library from regenerating liver has previously allowed us to identify and characterize a cluster of genes encoding proteins with important roles in proliferative processes. Here, by examining different rat and human tissues as well as cell lines, we characterized a highly conserved gene, guanylyl cyclase domain containing 1 (GUCD1), whose modulation occurs in liver regeneration and cell cycle progression in vitro. High-level expression of GUCD1 transcripts was observed in livers from patients with hepatocellular carcinoma. A yeast two-hybrid interaction assay, aimed at identifying any relevant interaction partners of GUCD1, revealed direct interactions with NEDD4-1 (E3 ubiquitin protein ligase neural precursor cell expressed, developmentally downregulated gene 4), resulting in control of GUCD1 stability. Thus, we have characterized expression and function of a ubiquitous protein, GUCD1, which might have a role in regulating normal and abnormal cell growth in the liver.

HOPS is an essential constituent of centrosome assembly.

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, gamma-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.