Four-way regulation of mosquito yolk protein precursor genes by juvenile hormone-, ecdysone-, nutrient-, and insulin-like peptide signaling pathways.

Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the vitellogenin-A gene (vg) of the yellow fever mosquito Aedes aegypti. Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., 2005). This review summarizes the role of these four signaling pathways in the regulation of vg-A and focuses upon new findings regarding the interplay between them on an organismal level

Lentiviral Vectors to Probe and Manipulate the Wnt Signaling Pathway

Background: The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling. Methodology/Principal Findings: We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-Puro R (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing Puro R under the control of the 7xTcf promoter. Lastly, we have expressed dominantnegative Tcf4 (dnTcf4) or constitutively active beta-catenin (b-catenin 4A) from the hEF1a promoter in a SV40-Puro R or SV40mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene. Conclusions: These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enable

Divergent Mechanisms Controlling Hypoxic Sensitivity and Lifespan by the DAF-2/Insulin/IGF-Receptor Pathway

Organisms and their cells vary greatly in their tolerance of low oxygen environments (hypoxia). A delineation of the determinants of hypoxia tolerance is incomplete, despite intense interest for its implications in diseases such as stroke and myocardial infarction. The insulin/IGF-1 receptor (IGFR) signaling pathway controls survival of Caenorhabditis elegans from a variety of stressors including aging, hyperthermia, and hypoxia. daf-2 encodes a C. elegans IGFR homolog whose primary signaling pathway modulates the activity of the FOXO transcription factor DAF-16. DAF-16 regulates the transcription of a large number of genes, some of which have been shown to control aging. To identify genes that selectively regulate hypoxic sensitivity, we compared the whole-organismal transcriptomes of three daf-2 reduction-of-function alleles, all of which are hypoxia resistant, thermotolerant, and long lived, but differ in their rank of severities for these phenotypes. The transcript levels of 172 genes were increased in the most hypoxia resistant daf-2 allele, e1370, relative to the other alleles whereas transcripts from only 10 genes were decreased in abundance. RNAi knockdown of 6 of the 10 genes produced a significant increase in organismal survival after hypoxic exposure as would be expected if down regulation of these genes by the e1370 mutation was responsible for hypoxia resistance. However, RNAi knockdown of these genes did not prolong lifespan. These genes definitively separate the mechanisms of hypoxic sensitivity and lifespan and identify biological strategies to survive hypoxic injury

Characterization and Expression Analysis of SOLD1, a Novel Member of the Retrotransposon-Derived Ly-6 Superfamily, in Bovine Placental Villi

BACKGROUND:Ly-6 superfamily members have a conserved Ly-6 domain that is defined by a distinct disulfide bonding pattern between eight or ten cysteine residues. These members are divided into membrane-type and secretory-type proteins. In the present study, we report the identification of a novel Ly-6 domain protein, secreted protein of Ly-6 domain 1 (SOLD1), from bovine placenta. PRINCIPAL FINDINGS:SOLD1 mRNA was expressed in trophoblast mononucleate cells and the protein was secreted into and localized in the extracellular matrix of the mesenchyme in cotyledonary villi. SOLD1 bound mainly with type I collagen telopeptide. We confirmed secretion of SOLD1 from the basolateral surface of a bovine trophoblast cell line (BT-1). It may be related to the organization of the extra-cellular matrix in the mesenchyme of fetal villi. Since trophoblast mononucleate cells are epithelial cells, their polar organization is expected to have a crucial role in the SOLD1 secretion system. We established that SOLD1 is an intronless bovine gene containing the Alu retrotransposon, which was integrated via cytoplasmic reverse transcription. CONCLUSION:We identified a novel retrotransposon-like Ly-6 domain protein in bovine placenta. SOLD1 is a crucial secreted protein that is involved in the organization of the mesenchyme of the cotyledonary villi. Furthermore, the gene encoding SOLD1 has an interesting genomic structure

The Intestinal Peptide Transporter PEPT1 Is Involved in Food Intake Regulation in Mice Fed a High-Protein Diet

High-protein diets are effective in achieving weight loss which is mainly explained by increased satiety and thermogenic effects. Recent studies suggest that the effects of protein-rich diets on satiety could be mediated by amino acids like leucine or arginine. Although high-protein diets require increased intestinal amino acid absorption, amino acid and peptide absorption has not yet been considered to contribute to satiety effects. We here demonstrate a novel finding that links intestinal peptide transport processes to food intake, but only when a protein-rich diet is provided. When mice lacking the intestinal peptide transporter PEPT1 were fed diets containing 8 or 21 energy% of protein, no differences in food intake and weight gain were observed. However, upon feeding a high-protein (45 energy%) diet, Pept1−/− mice reduced food intake much more pronounced than control animals. Although there was a regain in food consumption after a few days, no weight gain was observed which was associated with a reduced intestinal energy assimilation and increased fecal energy losses. Pept1−/− mice on high-protein diet displayed markedly reduced plasma leptin levels during the period of very low food intake, suggesting a failure of leptin signaling to increase energy intake. This together with an almost two-fold elevated plasma arginine level in Pept1−/− but not wildtype mice, suggests that a cross-talk of arginine with leptin signaling in brain, as described previously, could cause these striking effects on food intake

Wnt Signaling Cross-Talks with JH Signaling by Suppressing Met and gce Expression

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, we designed a novel Drosophila genetic screen to isolate mutations that derepress JH-mediated br suppression at early larval stages. We found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous JHA. A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression was suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, we conclude that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages

Role of STAT3 in In Vitro Transformation Triggered by TRK Oncogenes

TRK oncoproteins are chimeric versions of the NTRK1/NGF receptor and display constitutive tyrosine kinase activity leading to transformation of NIH3T3 cells and neuronal differentiation of PC12 cells. Signal Transducer and Activator of Transcription (STAT) 3 is activated in response to cytokines and growth factors and it has been recently identified as a novel signal transducer for TrkA, mediating the functions of NGF in nervous system. In this paper we have investigated STAT3 involvement in signalling induced by TRK oncogenes. We showed that TRK oncogenes trigger STAT3 phosphorylation both on Y705 and S727 residues and STAT3 transcriptional activity. MAPK pathway was involved in the induction of STAT3 phosphorylation. Interestingly, we have shown reduced STAT3 protein level in NIH3T3 transformed foci expressing TRK oncogenes. Overall, we have unveiled a dual role for STAT3 in TRK oncogenes-induced NIH3T3 transformation: i) decreased STAT3 protein levels, driven by TRK oncoproteins activity, are associated to morphological transformation; ii) residual STAT3 transcriptional activity is required for cell growth

Principles and Determinants of G-Protein Coupling by the Rhodopsin-Like Thyrotropin Receptor

In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts

How the Intestinal Peptide Transporter PEPT-1 Contributes to an Obesity Phenotype in Caenorhabditits elegans

Background: Amino acid absorption in the form of di- and tripeptides is mediated by the intestinal proton-coupled peptide transporter PEPT-1 (formally OPT-2) in Caenorhabditits elegans. Transporter-deficient animals (pept-1(lg601)) show impaired growth, slowed postembryonal development and major changes in amino acid status. Principal Findings: Here we demonstrate that abolished intestinal peptide transport also leads to major metabolic alterations that culminate in a two fold increase in total body fat content. Feeding of C. elegans with [U- 13 C]-labelled E. coli revealed a decreased de novo synthesis of long-chain fatty acids in pept-1(lg601) and reduced levels of polyunsaturated fatty acids. mRNA profiling revealed increased transcript levels of enzymes/transporters needed for peroxisomal b-oxidation and decreased levels for those required for fatty acid synthesis, elongation and desaturation. As a prime and most fundamental process that may account for the increased fat content in pept-1(lg601) we identified a highly accelerated absorption of free fatty acids from the bacterial food in the intestine. Conclusions: The influx of free fatty acids into intestinal epithelial cells is strongly dependent on alterations in intracellular pH which is regulated by the interplay of PEPT-1 and the sodium-proton exchanger NHX-2. We here provide evidence for a central mechanism by which the PEPT-1/NHX-2 system strongly influences the in vivo fat content of C. elegans. Loss of PEPT-1 decreases intestinal proton influx leading to a higher uptake of free fatty acids with fat accumulation whereas loss of NHX