Redox-dependent control of FOXO/DAF-16 by transportin-1.

Forkhead box O (FOXO; DAF-16 in worms) transcription factors, which are of vital importance in cell-cycle control, stress resistance, tumor suppression, and organismal lifespan, are largely regulated through nucleo-cytoplasmic shuttling. Insulin signaling keeps FOXO/DAF-16 cytoplasmic, and hence transcriptionally inactive. Conversely, as in loss of insulin signaling, reactive oxygen species (ROS) can activate FOXO/DAF-16 through nuclear accumulation. How ROS regulate the nuclear translocation of FOXO/DAF-16 is largely unknown. Cysteine oxidation can stabilize protein-protein interactions through the formation of disulfide-bridges when cells encounter ROS. Using a proteome-wide screen that identifies ROS-induced mixed disulfide-dependent complexes, we discovered several interaction partners of FOXO4, one of which is the nuclear import receptor transportin-1. We show that disulfide formation with transportin-1 is required for nuclear localization and the activation of FOXO4/DAF-16 induced by ROS, but not by the loss of insulin signaling. This molecular mechanism for nuclear shuttling is conserved in C. elegans and directly connects redox signaling to the longevity protein FOXO/DAF-16.

FOXO target gene CTDSP2 regulates cell cycle progression through Ras and p21(Cip1/Waf1).

Activity of FOXO (forkhead box O) transcription factors is inhibited by growth factor-PI3K (phosphoinositide 3-kinase)-PKB (protein kinase B)/Akt signalling to control a variety of cellular processes including cell cycle progression. Through comparative analysis of a number of microarray datasets we identified a set of genes commonly regulated by FOXO proteins and PI3K-PKB/Akt, which includes CTDSP2 (C-terminal domain small phosphatase 2). We validated CTDSP2 as a genuine FOXO target gene and show that ectopic CTDSP2 can induce cell cycle arrest. We analysed transcriptional regulation after CTDSP2 expression and identified extensive regulation of genes involved in cell cycle progression, which depends on the phosphatase activity of CTDSP2. The most notably regulated gene is the CDK (cyclin-dependent kinase) inhibitor p21(Cip1/Waf1) and in the present study we show that p21(Cip1/Waf1) is partially responsible for the cell cycle arrest through decreasing cyclin-CDK activity. Our data suggest that CTDSP2 induces p21(Cip1/Waf1) through increasing the activity of Ras. As has been described previously, Ras induces p21(Cip1/Waf1) through p53-dependent and p53-independent pathways and indeed both p53 and MEK inhibition can mitigate the CTDSP2-induced p21(Cip1/Waf1) mRNA up-regulation. In support of Ras activation by CTDSP2, depletion of endogenous CTDSP2 results in reduced Ras activity and thus CTDSP2 seems to be part of a larger set of genes regulated by FOXO proteins, which increase growth factor signalling upon FOXO activation.

The small GTPase RALA controls c-Jun N-terminal kinase-mediated FOXO activation by regulation of a JIP1 scaffold complex.

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

Proteolytic and Opportunistic Breaching of the Basement Membrane Zone by Immune Cells during Tumor Initiation.

Cancer-related inflammation impacts significantly on cancer development and progression. From early stages, neutrophils and macrophages are drawn to pre-neoplastic cells in the epidermis, but before directly interacting, they must first breach the underlying extracellular matrix barrier layer that includes the basement membrane. Using several different skin cancer models and a collagen I-GFP transgenic zebrafish line, we have undertaken correlative light and electron microscopy (CLEM) to capture the moments when immune cells traverse the basement membrane. We show evidence both for active proteolytic burrowing and for the opportunistic use of pre-existing weak spots in the matrix layer. We show that these small holes, as well as much larger, cancer cell-generated or wound-triggered gaps in the matrix barrier, provide portals for immune cells to access cancer cells in the epidermis and thus are rate limiting in cancer progression.

Sex-dependent resistance to the pathogenic fungus Cryptococcus neoformans.

Sex differences occur in most species and affect a variety of biological traits including morphology, behavior, and life history. The nematode Caenorhabditis elegans exists as a population of self-fertile hermaphrodites with occasional males, which differ anatomically and behaviorally from hermaphrodites. Here we show that male C. elegans also differ from hermaphrodites in their susceptibility to a fungal pathogen, Cryptococcus neoformans. Wild-type males show greater resistance than hermaphrodite animals to killing by this pathogen and this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioral changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to C. neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results hint at an overlap between the pathways controlling immunity and longevity and raise the possibility that differential regulation of these pathways may contribute to sex-dependent and species-dependent variation.

CCM1 and the second life of proteins in adhesion complexes.

It is well recognized that a number of proteins present within adhesion complexes perform discrete signaling functions outside these adhesion complexes, including transcriptional control. In this respect, β-catenin is a well-known example of an adhesion protein present both in cadherin complexes and in the nucleus where it regulates the TCF transcription factor. Here we discuss nuclear functions of adhesion complex proteins with a special focus on the CCM-1/KRIT-1 protein, which may turn out to be yet another adhesion complex protein with a second life.

A two-gene balance regulates Salmonella typhimurium tolerance in the nematode Caenorhabditis elegans.

Lysozymes are antimicrobial enzymes that perform a critical role in resisting infection in a wide-range of eukaryotes. However, using the nematode Caenorhabditis elegans as a model host we now demonstrate that deletion of the protist type lysozyme LYS-7 renders animals susceptible to killing by the fatal fungal human pathogen Cryptococcus neoformans, but, remarkably, enhances tolerance to the enteric bacteria Salmonella Typhimurium. This trade-off in immunological susceptibility in C. elegans is further mediated by the reciprocal activity of lys-7 and the tyrosine kinase abl-1. Together this implies a greater complexity in C. elegans innate immune function than previously thought.

Integrating opposing signals toward Forkhead box O.

Transcription factors are the common convergence points of signal transduction pathways to affect gene transcription. Signal transduction activity results in posttranslational modification (PTM) of transcription factors and the sum of these modifications at any given time point will determine the action of the transcription factor. It has been suggested that these PTMs provide a transcription factor code analogous to the histone code. However, the number and variety of these modifications and the lack of knowledge in general of their dynamics precludes at present a concise view of how combinations of PTMs affect transcription factor function. Also, a single type of PTM such as phosphorylation can have opposing effects on transcription factor activity. Transcription factors of the Forkhead box O (FOXO) class are predominantly regulated through signaling, by phosphoinositide 3-kinase/protein kinase B (also known as AKT) pathway and a reactive oxygen species/c-Jun N-terminal kinase pathway. Both pathways result in increased FOXO phosphorylation yet with opposing result. Whereas PKB-mediated phosphorylation inactivates FOXO, c-Jun N-terminal kinase-mediated phosphorylation results in activation of FOXO. Here we discuss regulation of FOXO transcription factors by phosphorylation as an example for understanding integration of signal transduction at the level of transcription activity.

The soluble leukocyte-associated Ig-like receptor (LAIR)-2 antagonizes the collagen/LAIR-1 inhibitory immune interaction.

Leukocyte-associated Ig-like receptor (LAIR)-1 is a collagen-receptor that inhibits immune cell function upon collagen binding. Next to LAIR-1, the human genome encodes LAIR-2, a putative soluble homolog. In this study we show, for the first time, that the LAIR-2 gene is broadly transcribed in human PBMC, mirroring the expression profile of LAIR-1. LAIR-2 protein is expressed as a soluble receptor exhibiting high affinity for various collagen molecules to which it binds in a hydroxyproline-dependent manner. In vitro stimulation of PBMC induces secretion of LAIR-2. We detect high amounts of LAIR-2 in urine of pregnant women, indicating that the soluble receptor is indeed produced in vivo and can be cleared from the body via urine. Furthermore, LAIR-2 levels are increased in synovial fluid of patients with rheumatoid arthritis as compared with osteoarthritis patients. We hypothesize that soluble LAIR-2 may function as a natural competitor for LAIR-1, thereby regulating its inhibitory potential. Indeed, LAIR-2 prevents binding of human LAIR-1 to collagens and LAIR-1 cross-linking in vitro, suggesting that the protein has an immunoregulatory function in vivo. Hence, we reveal a novel mechanism of immune regulation by a soluble LAIR receptor regulating the inhibitory potential of the membrane-bound LAIR-1 via competition for ligands.