Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation.

Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis.

Brain glucose sensing and counterregulatory response to hypoglycaemia.

An important obstacle to achieve optimal glycaemic control in diabetics on intensive insulin therapy is the frequent occurrence of insulin induced hypoglycaemic events. In healthy subjects and in diabetics without autonomic neuropathy hypoglycaemia activates the sympathetic nervous system, resulting in epinephrine and glucagon release. Both hormones increase hepatic glucose production and this counterregulatory response is of key importance of glucose homeostasis. Recent research shed light on the fact that antecedent hypoglycaemic episodes play pivotal role in hypoglycaemia associated autonomic failure (HAAF). In this condition the sympatho-adrenal response to decreased blood glucose level is blunted. The existence of HAAF clearly indicates that the nervous system contributes to glucose homeostasis in a substantial manner. This review outlines the mechanisms of both peripheral and central neuronal glucose sensing and of neural pathways involved in the counterregulatory response.

Better cardiorespiratory fitness associated with favourable metabolic control and health-related quality of life in youths with type 1 diabetes mellitus.

OBJECTIVE: to explore the relationship among health-related quality of life (HRQoL), clinical variables, anthropometric measures, physical activity and cardiorespiratory fitness in children and adolescents with type 1 diabetes.Furthermore, we aimed to find predictors of HRQoL and metabolic control. METHODS: A total of 106 patients (sex ratio: 1:1) with mean HbA1c of 8.55 (± 1.44) % and diabetes duration of 5.15 (± 3.24) years were assessed. The average age was 13.22 (± 3.08) years. RESULTS: We observed statistically significant negative medium correlation between HbA1c and VO2max (r = ­ 0.343; p = 0.000). There was statistically significant small positive correlation between the HRQoL and the maximal oxygen consumption (r = 0.208; p = 0.032). We found no significant correlation between the HbA1c and the patients' HRQoL. In the multiple linear regression analysis both the better metabolic control and the HRQoL was predicted by the VO2max, other variables had no effect. Physical activity level did not explain the HRQoL. Boys had significantly better HRQoL and less skinfold thickness than girls. CONCLUSION: Better cardiorespiratory fitness associated with both favourable metabolic control and better HRQoL of diabetic youths.Regular aerobic exercise improves the young patients' physical fitness and overall health status, and perception of health-related quality of life, respectively.

The Tribbles-1 protein in humans: roles and functions in health and disease.

This review describes the key role of the serine-threonine kinase like protein Tribbles-1 in health as well as in diverse human pathologies. Tribbles-1 is a homolog protein of the Drosophila Tribbles. In Drosophila, the Tribbles protein is involved in the cell-cycle progression during mitosis and in mammals initial data showed TRIB1 to be involved in cell proliferation. In mammals, TRIB1 lacks a catalytic domain and thus acts as an adaptor protein by interacting with several partners. The activity of TRIB1 seems to be very specific to the environment and the cells type in which it is expressed, and a role for this molecule has been mainly described in several pathological states including various cancers such as acute myeloid leukemia and ovarian cancer. Further evidence has also linked TRIB1 to the control of plasmalipid homeostasis thus indicating the role of this molecule as a risk factor for myocardial infarction. Finally, TRIB1 is shown to be up-regulated during inflammatory events such as chronic inflammation of atherosclerotic arteries or chronic antibody-mediated rejection of transplanted organs. Here we provide a review of the current state of the scientific literature for TRIB1, highlighting its role in diverse pathologies and inflammatory states. A better understanding of the role of this protein as both a target as well as a biological marker in diseases should drive the development of new therapeutic strategies.

Polymorphisms in the interleukin-1 receptor antagonist and interleukin-6 genes affect risk of osteolysis in patients with total hip arthroplasty.

OBJECTIVE: To determine whether osteolysis after total hip arthroplasty (THA) is associated with common polymorphisms within the genes encoding the interleukin-1 (IL-1) family and IL-6, and to determine whether polymorphisms that are associated with osteolysis affect in vitro messenger RNA (mRNA) expression in human peripheral blood mononuclear cells (PBMCs) in response to wear particles. METHODS: Unrelated white subjects of North European descent (n=612) were recruited a mean of 11 years after cemented THA for primary osteoarthritis. Of these subjects, 272 had previous osteolysis and 340 had no radiographic evidence of osteolysis (control group). Genomic DNA was genotyped for the following single-nucleotide polymorphisms (SNPs): IL1A +4845, IL1B +3954, IL1B -3737, IL1B -511, IL1RA +2018, IL6 -174, IL6 -572, and IL6 -597. In a subset of 60 subjects, PBMCs were extracted and stimulated with titanium particles and/or endotoxin, and cytokine mRNA expression was measured using quantitative real-time reverse transcriptase-polymerase chain reaction. RESULTS: The odds ratio (OR) for osteolysis associated with carriage of the IL1RA +2018C allele was 0.66 (95% confidence interval [95% CI] 0.48-0.91) (P=0.012). The remaining SNPs were not individually associated with osteolysis. The uncommon IL6 haplotype -174G/-572G/-597A (osteolysis group frequency 2.4%, control group frequency 0.8%) was associated with osteolysis (P=0.02, calculated using Haploview software). The IL1RA +2018CC genotype was associated with increased mRNA expression compared with the +2018TT genotype in both unstimulated and stimulated PBMCs (P=0.01 by analysis of variance, after Bonferroni correction). CONCLUSION: The IL1RA +2018C allele is associated with a decreased risk of osteolysis after THA and with increased IL-1 receptor antagonist mRNA expression in vitro. An uncommon haplotype within the promoter region of the gene for IL-6 is positively associated with osteolysis.

Tribbles: a family of kinase-like proteins with potent signalling regulatory function.

The recent identification of tribbles as regulators of signal processing systems and physiological processes, including development, together with their potential involvement in diabetes and cancer, has generated considerable interest in these proteins. Tribbles have been reported to regulate activation of a number of intracellular signalling pathways with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. The current review summarises our current understanding of interactions between tribbles and various other proteins. Since our understanding on the molecular basis of tribbles function is far from complete, we also describe a bioinformatic analysis of various segments of tribbles proteins, which has revealed a number of highly conserved peptide motifs with potentially important functional roles.

Tribbles: novel regulators of cell function; evolutionary aspects.

Identification of rate-limiting steps or components of intracellular second messenger systems holds promise to effectively interfere with these pathways under pathological conditions. The emerging literature on a recently identified family of signalling regulator proteins, called tribbles gives interesting clues for how these proteins seem to link several 'independent' signal processing systems together. Via their unique way of action, tribbles co-ordinate the activation and suppression of the various interacting signalling pathways and therefore appear to be key in determining cell fate while responding to environmental challenges. This review summarises our current understanding of tribbles function and also provides an evolutionary perspective on the various tribbles genes.

Regulation of expression and signalling modulator function of mammalian tribbles is cell-type specific.

The constant need to respond to changes in the environment is a common feature for all life forms. During evolution, a number of intracellular signal processing systems have evolved to fulfill this requirement. One of the most ancient such systems is the mitogen activated protein kinase (MAPK) signalling network, shared by all eukaryotes. Activation of MAPKs is key to regulation of mitosis and in cellular responses to stress or hormones, for instance. In addition, activity of this signalling system is essential during embryonic development. However, many aspects of MAPK mediated responses are strongly cell-type specific. A family of proteins, called tribbles have recently been described as novel regulators of MAPK function. Our group has previously shown that alterations in tribbles levels lead to profound changes in the activation of the various MAPKs. However, little is known about the cell-type specific aspects of regulation of tribbles expression. Here, we report that expression of all three members of the human tribbles family is dynamically controlled in response to inflammatory stimulation. This regulation, however, is strongly cell-type dependent. Our observations suggest regulation of tribbles expression may play an important role in the cell-type specific cellular responses, mediated by the MAPK network.

Functional mapping of Toll/interleukin-1 signalling networks by expression cloning.

Multiple cellular proteins have been identified as participating in Toll/interleukin-1 receptor-mediated inflammatory gene expression. The continuing isolation of novel components, based on sequence similarities, protein-protein interactions and protein purification, suggests that many elements of this signalling network remain to be identified. We report here the development of a high-throughput functional screening platform and its application for the identification of components of inflammatory signalling networks. Our results enable us to estimate that 100-150 gene products are involved in controlling the transcription of the human interleukin 8 gene. The approach, which is simple and robust, constitutes a general method for mapping signal transduction systems and for rapid isolation of a large number of signalling components based on the control of pathways leading to regulation of gene expression.

Rapid secretion of interleukin-1beta by microvesicle shedding.

The proinflammatory cytokine interleukin-1beta (IL-1beta) is a secreted protein that lacks a signal peptide and does not follow currently known pathways of secretion. Its efficient release from activated immune cells requires a secondary stimulus such as extracellular ATP acting on P2X(7) receptors. We show that human THP-1 monocytes shed microvesicles from their plasma membrane within 2-5 s of activation of P2X(7) receptors. Two minutes after such stimulation, the released microvesicles contained bioactive IL-1beta, which only later appeared in the vesicle-free supernatant. We conclude that microvesicle shedding is a major secretory pathway for rapid IL-1beta release from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins.

Ras controls tumor necrosis factor receptor-associated factor (TRAF)6-dependent induction of nuclear factor-kappa b. Selective regulation through receptor signaling components.

In the present study, we show that Ras activity differentially controls interleukin (IL)-1 induced transcription factor activation by selective regulation of responses mediated by receptor complex components. Initial experiments revealed that stimulation with IL-1 caused a rapid, matrix-dependent activation of Ras. The effect was transient, peaking at 5 min and returning to base levels after 30 min. Activation correlated with pronounced changes in cell shape in EGFPH-Ras transfected cells. Transfection with the dominant negative mutant, Ras(Asn-17), inhibited IL-1 induced activation of the IL-8 promoter as well as of NF-kappa B and AP-1 synthetic promoters in transient transfection assays. Furthermore, overexpression of the IL-1 signaling proteins TRAF6 or MyD88 gave characteristic activation of IL-8, which was accentuated in the presence of IL-1. Co-transfection with Ras(Asn-17) gave a dose-dependent inhibition of TRAF6-induced responses in the presence and absence of IL-1, but had no effect on MyD88 mediated activity. Similarly, induction of NF-kappa B was abolished by Ras(Asn-17) only in TRAF6-transfected cells. In contrast, inhibiting Ras activity limited AP-1-mediated responses through both receptor complex proteins. Constitutively active Ras(Val-12) increased the TRAF6 induced activity of the NF-kappa B pathway similar to the effect induced by IL-1, while the Ras(Val-12) induced activity was not inhibited by co-transfection with a dominant negative TRAF6. Our data show that activation of the Ras GTPase is an early, matrix-dependent response in IL-1 signaling which participates in structural regulation of IL-1-induced genes. In addition, they show that the Ras induced effect selectively regulates TRAF6-mediated activation of the NF-kappa B pathway, suggesting that Ras GTPase represents a convergence point in structural and cytokine responses, with distinct effects on a subset of downstream signaling events.

Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses.

Members of the Toll-like receptor (TLR) family are components of the mammalian anti-microbial response, signaling with a domain closely related to that of IL-1 receptors. In this report the expression and function of TLR1, a TLR of unknown function, are examined. TLR1 is expressed by monocytes, as demonstrated using a novel mAb. Monocytes also express TLR2. TLR1 transfection of HeLa cells, which express neither TLR1 nor TLR2, was not sufficient to confer responsiveness to several microbial extracts. However, cotransfection of TLR1 and TLR2 resulted in enhanced signaling by HeLa cells to soluble factors released from Neisseria meningitidis relative to the response with either TLR alone. This phenomenon was also seen with high concentrations of some preparations of LPS. The N. meningitidis factors recognized by TLR1/TLR2 were not released by N. meningitidis mutant in the LpxA gene. Although LpxA is required for LPS biosynthesis, because cooperation between TLR1 and TLR2 was not seen with all LPS preparations, the microbial component(s) TLR1/2 recognizes is likely to be a complex of LPS and other molecules or a compound metabolically and chemically related to LPS. The functional IL-1R consists of a heterodimer; this report suggests a similar mechanism for TLR1 and TLR2, for certain agonists. These data further suggest that mammalian responsiveness to some bacterial products may be mediated by combinations of TLRs, suggesting a mechanism for diversifying the repertoire of Toll-mediated responses.

A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling.

Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll/IL-1 receptor (TIR) domain, a motif that defines the IL-1/Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NFkappaB, and A52R potently blocked both IL-1- and TLR4-mediated NFkappaB activation. MyD88 is a TIR domain-containing adapter molecule known to have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R are likely to represent a mechanism used by vaccinia virus of suppressing TIR domain-dependent intracellular signaling.

A novel mammalian expression screen exploiting green fluorescent protein-based transcription detection in single cells.

The accumulation of DNA sequence information from large-scale genomic and random library sequencing projects is leading to the rapid identification of many putative genes, virtual transcripts and ESTs of unknown function. There is therefore an increasing need for high throughput, sensitive and robust methods for identification and characterisation of genes, and/or their products, based on function. We describe a high throughput functional expression screen based on semi-quantitative analysis of enhanced green fluorescent protein expression in single cells by confocal microscopy. The assay was implemented in a micro-scale format, requiring around 10(4) cells/test. The system was validated by co-transfection of a series of cDNAs encoding pro-inflammatory cytokine intracellular signal mediators with a d2EGFP reporter containing a cytokine responsive promoter. The majority of the test plasmids gave a detectable signal above background at a pool size of 250-500. Replicate tests indicate that the assay is reproducible at this pool size. At this level we demonstrate that large (>10(6) transformants) libraries can be feasibly screened.

Characterization of the CD55 (DAF)-binding site on the seven-span transmembrane receptor CD97.

CD97 is an activation-induced antigen on leukocytes which belongs to a new group of seven-span transmembrane (7-TM) molecules, designated EGF-TM7 family. Family members, including EMR1 and F4/80, are characterized by an extended extracellular region with several N-terminal epidermal growth factor-like (EGF) domains. Alternative splicing of CD97 results in isoforms possessing either three (EGF1, 2, 5), four (EGF1, 2, 3, 5) or five EGF domains (EGF1, 2, 3, 4, 5). We recently identified decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade, as a cellular ligand of the smallest isoform. Employing mutants of CD97(EGF1, 2, 5) in which the EGF domains have been systematically deleted, we here demonstrate the necessity of at least three tandemly linked EGF domains for the interaction with CD55. Consistent with the involvement of different EGF domains, monoclonal antibodies directed against the first EGF domain as well as the removal of Ca2+, for which binding sites exist in the second and fifth EGF domain, blocked binding to CD55. Compared to CD97(EGF1, 2 ,5) the larger isoforms CD97(EGF1, 2, 3, 5) and CD97(EGF1, 2, 3, 4, 5) have a significantly lower affinity for CD55. Thus, alternative splicing may regulate the ligand specificity of CD97 and probably other members of the EGF-TM7 family.

Transcription factor AP-4 participates in activation of bovine leukemia virus long terminal repeat by p34 Tax.

Three 21bp repeats can be found in the bovine leukemia virus long terminal repeat, which are crucial for the LTR directed gene expression by the trans activator protein Tax. Previous studies demonstrated that the major target of the Tax directed activation are the CRE-like elements in the center of these repeats. In this work we report that another motif of the 21bp repeats is also required for the Tax activation. Gel retardation--with the wild type or mutant 21bp repeats--revealed that cellular factors from HeLa cells were specifically bound to the center (CRE-like element) and the 3' region of the repeats, which contains a CAGCTG consensus AP-4 binding site. In vivo analysis using the synthetic 21bp repeats indicated that beyond the consensus CRE-like motif, the AP-4 site is also essential for Tax activation. To determine the role of AP-4 in BLV Tax trans activation, we used the AP-4 cDNA in antisense transient assays. In the in vivo experiments the antisense AP-4 RNA resulted in strongly decreased Tax activation. On the basis of these results we conclude that AP-4 is a good candidate of cellular factors involved in BLV Tax trans activation.

A downstream regulatory element activates the bovine leukemia virus promoter.

Recent studies demonstrated, that the R/U5 region of the Bovine Leukemia Virus (BLV) long terminal repeat (LTR) up-regulates the virus promoter. It is also known that this effect is independent from the BLV trans activator protein, p34tax, encoded by the virus genome. Deletions were constructed in the R/U5 region to localize the sequences responsible for this effect. The activity of the different constructs was determined in a transient expression system. Our results show that a 64 bp long sequence (called DAS), present at the 3 end of the R region, is involved in the activation. The in vivo results indicate that DAS could be divided into two independent but overlapping elements (DAS1,2). Sequence comparison allows the identification of three conservative boxes in these elements. Our results suggest, that these boxes are functional only together. The gel-shift assay with DAS2, in good agreement with the in vivo data, demonstrates that only the full length element forms a low mobility DNA-protein complex.

Member of the CREB/ATF protein family, but not CREB alpha plays an active role in BLV tax trans activation in vivo.

The trans activator protein of Bovine Leukaemia Virus (tax) increases the rate of transcription from the virus promoter through 21 bp sequences located in three tandem copies in the virus LTR. Based on data obtained by three different experimental approaches we concluded that the central CRE-like motif found in each of the BLV 21 bp repeats plays an important and indispensable role in tax mediated trans activation. These include (i) in vivo analysis of the function of mutant 21 bp sequences in transient transfection, (ii) gel mobility shift assay to show that CREB binds to BLV 21 bp repeats in vitro and (iii) the demonstration that the production of antisense CREB mRNA inhibits tax trans activation. Further studies with different deletion mutant CREB proteins suggest that although CREB alpha can interact with factors involved in BLV trans activation, it does not promote transcription initiation; consequently some other member/s of the CREB/ATF family must be involved.