The functional effects of ceramide on the monocyte CD36 scavenger receptor and NADPH oxidase

Atherosclerosis is the principal cause of death in the United States, Europe and much of Asia. During the last decade, inflammation has been suggested to play a key role in the development of atherosclerosis. Reactive oxygen species (ROS) released during inflammation additionally oxidize LDL, which is subsequently taken up in an unregulated way through scavenger receptors on macrophages to form foam cells, the hallmark of atherosclerotic lesions. Previous work has shown that the lipid ceramide, which is found in aggregated LDL and in atherosclerotic plaques, decreases intracellular peroxide most likely through reducing NADPH oxidase activity. Ceramide is an important component of membrane microdomains called lipid rafts which are important for membrane protein function. Endogenous ceramide enhances lipid raft f'ormation and alters theirs composition. NADPH oxidase membrane subunits cytochrome b558 (which includes gp91) strongly associates with lipid rafts Therefore present study investigated whether short chain ceramides reduce NADPH oxidase in U937 monocytes by disrurting the membrane component of NADPH oxidase. Results showed that C2 ceramide alters the distribution of raft marker, flottillin and the raft environment. NADPH oxidase membrane component gp9J phox and cytosolic component p47 phox were identified in rafts. C2 ceramide reduces both gp91 and p47 phox in rafts, which leads to the decrease of peroxide production by NADPH oxidase. Ceramide is also an important second messenger involved in many different signaling pathways associated with atherogenesis from the activation of sphingomyelinase (SMase). It has been reported that SMase enhances LDL receptor mediated LDL endocytosis. However, no study has been done to investigate the effect of ceramide on scavenger receptors such as CD36 and oxidized LDL (OxLDL) uptake. CD36 is the major recertor far OxLDL. Reduced CD36 expression results in less foam cell formation and less atherosclerotic lesion without disrupting the clearance of OxLDL from plasma. This thesis shows that ceramides significantly reduce CD36 surface expression on U937 monocytes, macrophages and human primary monocytes. This effect is seen using both synthetic short chain ceramide and SMase catalysed long chain ceramide treatment. To investigate whether the effect of ceramide on CD36 is functional, OxLOL uptake was measured in ceramide treated cells. Ceramide reduces the uptake of OxLOL by both U937 monocytes and PMA-differentiated macrophages. The mechanism of ceramide reduction of CD36 expression was studied by measuring the surface antigen using flow cytometry and fluorescence microscopy, whole cellular CD36 expression and shedding of C036 by Western blotting of cell lysates and cell culture supernatants and mRNA level of CD36 using RT-PCR. Ceramide reduces shedding of CD36, activates mRNA expression of CD36 and induces intracellular CD36 accumulation probably through retaining the receptor inside cells. In summary, ceramides modulate several of the processes involved in LOL oxidation and uptake by CD36 receptors on monocytes/macrophages in a way which may protect against atherosclerosis.

Activity-regulated cytoskeleton-associated protein controls AMPAR endocytosis through a direct interaction with clathrin-adaptor protein 2

The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc-AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2.

The anxiolytic-like effects of cannabidiol injected into the bed nucleus of the stria terminalis are mediated by 5-HT1A receptors

Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic-like effects in rodents and humans after systemic administration. Previous results from our group showed that CBD injection into the bed nucleus of the stria terminalis (BNST) attenuates conditioned aversive responses. The aim of this study was to further investigate the role of this region on the anxiolytic effects of the CBD. Moreover, considering that CBD can activate 5-HT1A receptors, we also verified a possible involvement of these receptors in those effects. Male Wistar rats received injections of CBD (15, 30, or 60 nmol) into the BNST and were exposed to the elevated plus-maze (EPM) or to the Vogel conflict test (VCT), two widely used animal models of anxiety. CBD increased open arms exploration in the EPM as well as the number of punished licks in the VCT, suggesting an anxiolytic-like effect. The drug did not change the number of entries into the enclosed arms of the EPM nor interfered with water consumption or nociceptive threshold, discarding potential confounding factors in the two tests. Moreover, pretreatment with the 5-HT1A receptor antagonist WAY100635 (0.37 nmol) blocked the effects of CBD in both models. These results give further support to the proposal that BNST is involved in the anxiolytic-like effects of CBD observed after systemic administration, probably by facilitating local 5-HT1A receptor-mediated neurotransmission.

Dynamin-dependent endocytosis is necessary for convergent-extension movements in Xenopus animal cap explants

Cadherin cell-cell adhesion molecules are important determinants of morphogenesis and tissue patterning. C-cadherin plays a key role in the cell-upon-cell movements seen during Xenopus gastrulation. In particular, regulated changes in C-cadherin adhesion critically influence convergence-extension movements, thereby determining organization of the body plan. It is also predicted that remodelling of cadherin adhesive contacts is important for such cell-on-cell movements to occur. The recent demonstration that Epithelial (E-) cadherin is capable of undergoing endocytic trafficking to and from the cell surface presents a potential mechanism for rapid remodelling of such adhesive contacts. To test the potential role for C-cadherin endocytosis during convergence-extension, we expressed in early Xenopus embryos a dominantly-inhibitory mutant of the GTPase, dynamin, a key regulator of clathrin-mediated endocytosis. We report that this dynamin mutant significantly blocked the elongation of animal cap explants in response to activin, accompanied by inhibition of C-cadherin endocytosis. We propose that dynamin-dependent endocytosis of C-cadherin plays an important role in remodelling adhesive contacts during convergence-extension movements in the early Xenopus embryo.

The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel

In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.

Facilitation of renal autoregulation by angiotensin II is mediated through modulation of nitric oxide

Aims: This study was designed to investigate the influence of angiotensin II (Ang II) and nitric oxide (NO) on autoregulation of renal perfusion. Methods: Autoregulation was investigated in isolated perfused kidneys (IPRK) from Sprague-Dawley rats during stepped increases in perfusion pressure. Results: Ang II (75-200 pM) produced dose-dependent enhancement of autoregulation whereas phenylephrine produced no enhancement and impaired autoregulation of GFR. Enhancement by Ang II was inhibited by the AT(1) antagonist, Losartan, and the superoxide scavenger, Tempol. Under control conditions nitric oxide synthase (NOS) inhibition by 10 muM N-omega-nitro-L-arginine methyl ester (L-NAME) facilitated autoregulation in the presence of non-specific cyclooxygenase (COX) inhibition by 10 muM indomethacin. Both COX and combined NOS/COX inhibition reduced the autoregulatory threshold concentration of Ang II. Facilitation by 100 pM Ang II was inhibited by 100 muM frusemide. Methacholine (50 nM) antagonised Ang II-facilitated autoregulation in the presence and absence of NOS/COX inhibition. Infusion of the NO donor, 1 muM sodium nitroprusside, inhibited L-NAME enhancement of autoregulation under control conditions and during Ang II infusion. Conclusions: The results suggest than an excess of NO impairs autoregulation under control conditions in the IPRK and that endogenous and exogenous NO, vasodilatory prostaglandins and endothelium-derived hyperpolarizing factor (EDHF) activity antagonise Ang II-facilitated autoregulation. Ang II also produced a counterregulatory vasodilatory response that included prostaglandin and NO release. We suggest that Ang II facilitates autoregulation by a tubuloglomerular feedback-dependent mechanism through AT(1) receptor-mediated depletion of nitric oxide, probably by stimulating generation of superoxide.

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Complete hypogonadotropic hypogonadism associated with a novel inactivating mutation of the gonadotropin-releasing hormone receptor.

In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism who presented primary failure of pulsatile GnRH therapy, but responded to exogenous gonadotropin administration. This patient bore a novel point mutation (T for A) at codon 168 of the gene encoding the GnRH receptor (GnRH-R), resulting in a serine to arginine change in the fourth transmembrane domain of the receptor. This novel mutation was present in the homozygous state in the patient, whereas it was in the heterozygous state in both phenotypically normal parents. When introduced into the complementary DNA coding for the GnRH-R, this mutation resulted in the complete loss of the receptor-mediated signaling response to GnRH. In conclusion, we report the first mutation of the GnRH-R gene that can induce a total loss of function of this receptor and is associated with a phenotype of complete hypogonadotropic hypogonadism.

Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

IL-5 and IL-5 receptor in asthma

Eosinophils, along with mast cells are key cells involved in the innate immune response against parasitic infection whereas the adaptive immune response is largely dependent on lymphocytes. In chronic parasitic disease and in chronic allergic disease, IL-5 is predominantly a T cell derived cytokine which is particularly important for the terminal differentiation, activation and survival of committed eosinophil precursors. The human IL-5 gene is located on chromosome 5 in a gene cluster that contains the evolutionary related IL-4 family of cytokine genes. The human IL-5 receptor complex is a heterodimer consisting of a unique a subunit (predominantly expressed on eosinophils) and a beta subunit which is shared between the receptors for IL-3 & GM-CSF (more widely expressed). The a subunit is required for ligand-specific binding whereas association with the beta subunit results in increased binding affinity. The alternative splicing of the alphaIL-5R gene which contains 14 exons can yield several alphaIL-5R isoforms including a membrane-anchored isoform (alphaIL-5Rm) and a soluble isoform (alphaIL-5Rs). Cytokines such as IL-5 produce specific and non-specific cellular responses through specific cell membrane receptor mediated activation of intracellular signal transduction pathways which, to a large part, regulate gene expression. The major intracellular signal transduction mechanism is activation of non-receptor associated tyrosine kinases including JAK and MAP kinases which can then transduce signals via a novel family of transcriptional factors named signal transducers and activators of transcription (STATS). JAK2, STAT1 and STAT 5 appear to be particularly important in IL-5 mediated eosinophil responses. Asthma is characterized by episodic airways obstruction, increased bronchial responsiveness, and airway inflammation. Several studies have shown an association between the number of activated T cells and eosinophils in the airways and abnormalities in FEV1, airway reactivity and clinical severity in asthma. It has now been well documented that IL-5 is highly expressed in the bronchial mucosa of atopic and intrinsic asthmatics and that the increased IL-5 mRNA present in airway tissues is predominantly T cell derived. Immunocytochemical staining of bronchial biopsy sections has confirmed that IL-5 mRNA transcripts are translated into protein in asthmatic subjects. Furthermore, the number of activated CD 4 + T cells and IL-5 mRNA positive cells are increased in asthmatic airways following antigen challenge and studies that have examined IL-5 expression in asthmatic subjects before and after steroids have shown significantly decreased expression following oral corticosteroid treatment in steroid-sensitive asthma but not in steroid resistant and chronic severe steroid dependent asthma. The link between T cell derived IL-5 and eosinophil activation in asthmatic airways is further strengthened by the demonstration that there is an increased number of alphaIL-5R mRNA positive cells in the bronchial biopsies of atopic and non-atopic asthmatic subjects and that the eosinophil is the predominant site of this increased alphaIL-5R mRNA expression. We have also shown that the subset of activated eosinophils that expressed mRNA for membrane bound alpha IL5r inversely correlated with FEV1, whereas the subset of activated eosinophils that expressed mRNA for soluble alphaIL5r directly correlated with FEV1. Hence, not only does this data suggest that the presence of eosinophils expressing alphaIL-5R mRNA contribute towards the pathogenesis of bronchial asthma, but also that the eosinophil phenotype with respect to alphaIL-5R isoform expression is of central importance. Finally, there are several animal, and more recently in vitro lung explant, models of allergen induced eosinophilia, late airway responses(LARS), and bronchial hyperresponsiveness(BHR) - all of which support a link between IL-5 and airway eosinophila and bronchial hyperresponsiveness. The most direct demonstration of T cell involvement in LARS is the finding that these physiological responses can be transferred by CD4+ but not CD8+ T cells in rats. The importance of IL-5 in animal models of allergen induced bronchial hyperresponsiveness has been further demonstrated by a number of studies which have indicated that IL-5 administration is able to induce late phase responses and BHR and that anti-IL-5 antibody can block allergen induced late phase responses and BHR. In summary, activated T lymphocytes, IL5 production and eosinophil activation are particularly important in the asthmatic response. Human studies in asthma and studies in allergic animal models have clearly emphasised the unique role of IL-5 in linking T lymphocytes and adaptive immunity, the eosinophil effector cell, and the asthma phenotype. The central role of activated lymphocytes and eosinophils in asthma would argue for the likely therapeutic success of strategies to block T cell and eosinophil activation (eg steroids). Importantly, more targeted therapies may avoid the complications associated with steroids. Such therapies could target key T cell activation proteins and cytokines by various means including blocking antibodies (eg anti-CD4, anti-CD40, anti-IL-5 etc), antisense oligonucleotides to their specific mRNAs, and/or selective inhibition of the promoter sites for these genes. Another option would be to target key eosinophil activation mechanisms including the aIL5r. As always, the risk to benefit ratio of such strategies await the results of well conducted clinical trials.

Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the alpha 1B-adrenergic receptor.

The alpha 1B-adrenergic receptor (alpha 1BAR) and its truncated mutant T368 lacking the last 147 amino acids were stably expressed in Rat1 fibroblasts. The wild type alpha 1BAR was rapidly phosphorylated upon exposure to the agonist epinephrine as well as to phorbol ester as assessed by immunoprecipitation of the receptor with antiserum raised against its amino-terminal portion. Exposure of cells expressing the wild type alpha 1BAR to epinephrine resulted also in rapid homologous desensitization of receptor-mediated response on polyphosphoinositide hydrolysis. On the other hand, truncation of the serine- and threonine-rich carboxyl portion of the alpha 1BAR abolished agonist-induced phosphorylation and greatly impaired homologous desensitization of the receptor. The truncated receptor T368 could undergo agonist-induced decrease of cell surface receptors but to a lesser extent, as compared with the wild type alpha 1BAR. These results demonstrate that the carboxyl portion of the alpha 1BAR plays a crucial role in the regulation of receptor function. They also suggest a strong relationship between agonist-induced phosphorylation and desensitization of the alpha 1BAR, which were both insensitive to the inhibitor of protein kinase C RO-318220. Our findings support the emerging hypothesis that the biochemical mechanisms involved in rapid agonist-dependent regulation of G protein-coupled receptors, which activate polyphosphoinositide hydrolysis, do not primarily involve protein kinase C.

Analysis of GPCR properties of Frizzled receptors and establishment of the "in vitro" platform for screening of Frizzled agonists/antagonists as potential therapeutic agents

Morphogens of the Wnt protein family are the secreted lipoglycoprotein ligands which initiate several pathways heavily involved in the coordination of various developmental stages of organisms in the majority of animal species. Deregulation of these pathways in the adult leads to formation and sustaining of multiple types of cancer. The latter notion is reinforced by the fact that the very discovery of the first Wnt ligand was due to its role as the causative factor of carcinogenic transformation (Nusse and Varmus, 1982). Nowadays our knowledge on Wnt signaling has "moved with the times" and these pathways were identified to be often crucial for tumor formation, its interactions with the microenvironment, and promotion of the metastases (Huang and Du, 2008; Zerlin et al., 2008; Jessen, 2009). Thus the relevance of the pathway as the target for drug development has further increased in the light of modern paradigms of the complex cancer treatments which target also spreading and growth- promoting factors of tumors by specific and highly efficient substances (Pavet et al., 2010). Presently the field of the Wnt-targeting drug research is almost solely dominated by assays based on transcriptional activation induced by the signaling. This approach resulted in development of a number of promising substances (Lee et al., 2011). Despite its effectiveness, the method nevertheless suffers from several drawbacks. Among the major ones is the fact that this approach is prone to identify compounds targeting rather downstream effectors of the pathway, which are indiscriminately used by all the subtypes of the Wnt signaling. Additionally, proteins which are involved in several signaling cascades and not just the Wnt pathway turn out as targets of the new compounds. These issues increase risks of side effects due to off-target interactions and blockade of the pathway in healthy cells. In the present work we put forward a novel biochemical approach for drug development on the Wnt pathway. It targets Frizzleds (Fzs) - a family of 7-transmbembrane proteins which serve as receptors for Wnt ligands. They offer unique properties for the development of highly specific and effective drugs as they control all branches of the Wnt signaling. Recent advances in the understanding of the roles of heterotrimeric G proteins downstream from Fzs (Katanaev et al., 2005; Liu et al., 2005; Jernigan et al., 2010) suggest application of enzymatic properties of these effectors to monitor the receptor-mediated events. We have applied this knowledge in practice and established a specific and efficient method based on utilization of a novel high-throughput format of the GTP-binding assay to follow the activation of Fzs. This type of assay is a robust and well-established technology for the research and screenings on the GPCRs (Harrison and Traynor, 2003). The conventional method of detection involves the radioactively labeled non-hydrolysable GTP analog [35S]GTPyS. Its application in the large-scale screenings is however problematic which promoted development of the novel non-radioactive GTP analog GTP-Eu. The new molecule employs phenomenon of the time-resolved fluorescence to provide sensitivity comparable to the conventional radioactive substance. Initially GTP-Eu was tested only in one of many possible types of GTP-binding assays (Frang et al., 2003). In the present work we expand these limits by demonstrating the general comparability of the novel label with the radioactive method in various types of assays. We provide a biochemical characterization of GTP-Eu interactions with heterotrimeric and small GTPases and a comparative analysis of the behavior of the new label in the assays involving heterotrimeric G protein effectors. These developments in the GTP-binding assay were then applied to monitor G protein activation by the Fz receptors. The data obtained in mammalian cultured cell lines provides for the first time an unambiguous biochemical proof for direct coupling of Fzs with G proteins. The specificity of this interaction has been confirmed by the experiments with the antagonists of Fz and by the pertussis toxin-mediated deactivation. Additionally we have identified the specificity of Wnt3a towards several members of the Fz family and analyzed the properties of human Fz-1 which was found to be the receptor coupled to the Gi/o family of G proteins. Another process playing significant role in the functioning of every GPCR is endocytosis. This phenomenon can also be employed for drug screenings on GPCRs (Bickle, 2010). In the present work we have demonstrated that Drosophila Fz receptors are involved in an unusual for many GPCRs manifestation of the receptor-mediated internalization. Through combination of biochemical approaches and studies on Drosophila as the model organism we have shown that direct interactions of the Fzs and the α-subunit of the heterotrimeric G protein Go with the small GTPase Rab5 regulate internalization of the receptor in early endosomes. We provide data uncovering the decisive role of this self-promoted endocytosis in formation of a proper signaling output in the canonical as well as planar cell polarity (PCP) pathways regulated by Fz. The results of this work thus establish a platform for the high-throughput screening to identify substances active in the cancer-related Wnt pathways. This methodology has been adjusted and applied to provide the important insights in Fz functioning and will be instrumental for further investigations on the Wnt-mediated pathways.

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

The challenge of modeling nuclear receptor regulatory networks in mammalian cells.

Nuclear receptors are a major component of signal transduction in animals. They mediate the regulatory activities of many hormones, nutrients and metabolites on the homeostasis and physiology of cells and tissues. It is of high interest to model the corresponding regulatory networks. While molecular and cell biology studies of individual promoters have provided important mechanistic insight, a more complex picture is emerging from genome-wide studies. The regulatory circuitry of nuclear receptor regulated gene expression networks, and their response to cellular signaling, appear highly dynamic, and involve long as well as short range chromatin interactions. We review how progress in understanding the kinetics and regulation of cofactor recruitment, and the development of new genomic methods, provide opportunities but also a major challenge for modeling nuclear receptor mediated regulatory networks.

Formation of the long range Dpp morphogen gradient.

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution.