Gtdap-1 and the role of autophagy during planarian regeneration and starvation

Planarians have been established as an ideal model organism for stem cell research and regeneration. Planarian regeneration and homeostasis require an exquisite balancing act between cell death and cell proliferation as new tissues are made (epimorphosis) and existing tissues remodeled (morphallaxis). Some of the genes and mechanisms that control cell proliferation and pattern formation are known. However, studies about cell death during remodeling are few and far between. We have studied the gene Gtdap-1, the planarian ortholog of human death-associated protein-1 or DAP-1. DAP-1 together with DAP-kinase has been identified as a positive mediator of programmed cell death induced by gamma-interferon in HeLa cells. We have found that the gene functions at the interface between autophagy and cell death in the remodeling of the organism that occurs during regeneration and starvation in sexual and asexual races of planarians. Our data suggest that autophagy of existing cells may be essential to fuel the continued proliferation and differentiation of stem cells by providing the necessary energy and building blocks to neoblasts.

Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.

AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.

Short-term oleoyl-estrone treatment affects capacity to manage lipids in rat adipose tissue

Background: Short-term OE (oleoyl-estrone) treatment causes significant decreases in rat weight mainly due to adipose tissue loss. The aim of this work was to determine if OE treatment affects the expression of genes that regulate lipid metabolism in white adipose tissue. Results: Gene expression in adipose tissue from female treated rats (48 hours) was analysed by hybridization to cDNA arrays and levels of specific mRNAs were determined by real-time PCR. Treatment with OE decreased the expression of 232 genes and up-regulated 75 other genes in mesenteric white adipose tissue. The use of real-time PCR validate that, in mesenteric white adipose tissue, mRNA levels for Lipoprotein Lipase (LPL) were decreased by 52%, those of Fatty Acid Synthase (FAS) by 95%, those of Hormone Sensible Lipase (HSL) by 32%, those of Acetyl CoA Carboxylase (ACC) by 92%, those of Carnitine Palmitoyltransferase 1b (CPT1b) by 45%, and those of Fatty Acid Transport Protein 1 (FATP1) and Adipocyte Fatty Acid Binding Protein (FABP4) by 52% and 49%, respectively. Conversely, Tumour Necrosis Factor (TNF¿) values showed overexpression (198%). Conclusion: Short-term treatment with OE affects adipose tissue capacity to extract fatty acids from lipoproteins and to deal with fatty acid transport and metabolism.

Role of β-catenin and γ-catenin in haematopoiesis : Generation of floxed Delta like 4 gene targeted mice

Résumé La voie de signalisation de Wnt est extrêmement conservée au cours de l'évolution. Les protéines Wnt sont des molécules sécrétées qui se lient à la famille de récepteurs Frizzled. Cette interaction mène à la stabilisation de la protéine β-caténine, qui va s'accumuler dans le cytoplasme puis migrer dans le noyau où elle peut s'hétérodimériser avec les facteurs de transcription de la famille TCF/LEF. Il a été démontré que cette voie de signalisation joue un rôle important durant la lymphopoïèse et de récents résultats suggèrent un rôle clé de cette voie dans le renouvellement des Cellules Souches Hématopoïétique (CSH). Des études se basant sur un système de surexpression de protéines montrent clairement que la voie Wnt peut influencer l'hématopoïèse. Cependant, le rôle de la protéine β-caténine dans le système hématopoïétique n'a jamais été testé directement. Ce projet de thèse se propose d'étudier la fonction de la protéine β-caténine par sa délétion inductible via le système Cre-loxP. De façon surprenante, nous avons pu démontrer que les progéniteurs de la moelle osseuse, déficients en β-caténine, ne montrent aucune altération dans leur capacité à s'auto-renouveler et/ou à reconstituer toutes les lignées hématopoïétiques (myéloïde, érythroïde et lymphoïde) dans les souris-chimères. De plus, le développement, la survie des thymocytes ainsi que la prolifération des cellules T périphériques induite par un antigène, sont indépendants de β-caténine. Ces résultats suggèrent soit que la protéine β-caténine ne joue pas un rôle primordial dans le système hématopoiétique, soit que son absence pourrait être compensée par une autre protéine. Un candidat privilégié susceptible de se substituer à β-caténine, serait plakoglobine, aussi connu sous le nom de γ-caténine. En effet, ces deux protéines partagent de multiples caractéristiques structurelles. Afin de démontrer que la protéine γ-caténine peut compenser l'absence de β-caténine, nous avons généré des souris dans lesquelles, le système hématopoïétique est déficient pour ces deux protéines. Cette déficience combinée de β- caténine et γ-caténine ne perturbe pas la capacité des Cellules Souche Hématopoïétique-Long Terme (CSH-LT) de se renouveler, par contre elle agit sur un progéniteur précoce déjà différencié de la moelle osseuse. Ces résultats mettent en évidence que la protéine γ-caténine est capable de compenser l'absence de protéine β-caténine dans le système hématopoïétique. Par conséquent, ce travail contribue à une meilleure connaissance de la cascade Wnt dans l'hématopoïèse. Summary The canonical Wnt signal transduction pathway is a developmentally highly conserved. Wnts are secreted molecules which bind to the family of Frizzled receptors in a complex with the low density lipoprotein receptor related protein (LRP-5/6). This initial activation step leads to the stabilization and accumulation of β-catenin, first in the cytoplasm and subsequently in the nucleus where it forms heterodimers with TCF/LEF transcription factor family members. Wnt signalling has been shown to be important during early lymphopoiesis and has more recently, been suggested to be a key player in self-renewal of haematopoietic stem cells (HSCs). Although mostly gain of function studies indicate that components of the Wnt signalling pathway can influence the haematopoietic system, the role of β-catenin has never been directly investigated. The aim of this thesis project is to investigate the putatively critical role of β-catenin in vivo using the Cre-loxP mediated conditional loss of function approach. Surprisingly, β-catenin deficient bone marrow (BM) progenitors arc not impaired in their ability to self-renew and/or to reconstitute all haematopoietic lineages (myeloid, erythroid and lymphoid) in both mixed and straight bone marrow chimeras. In addition, both thymocyte development and survival, and antigen-induced proliferation of peripheral T cells are β- catenin independent. Our results do not necessarily exclude the possibility of an important function for β-catenin mediated Wnt signalling in the haematopoietic system, it rather raises the question that β-catenin is compensated for by another protein. A prime candidate that may take over the function of β-catenin in its absence, is the close relative plakoglobin, also know as γ-catenin. This protein shares multiple structural features with β-catenin. In order to investigate whether γ-catenin can compensate for the loss of β-catenin we have generated mice in which the haematopoietic compartment is deficient for both proteins. Combined deficiency of β-catenin and γ-catenin does not perturb Long Term-Haematopoietic Stem Cells (LT-HSC) self renewal, but affects an already lineage committed progenitor population within the BM. Our results demonstrate that y-catenin can indeed compensate for the loss of β-catenin within the haematopoietie system.

Transcriptional potentiation of the vitellogenin B1 promoter by a combination of both nucleosome assembly and transcription factors: an in vitro dissection.

The Xenopus laevis vitellogenin B1 promoter was assembled into nucleosomes in an oocyte extract. Subsequent RNA polymerase II-dependent transcription from these DNA templates fully reconstituted in chromatin in a HeLa nuclear extract was increased 50-fold compared with naked DNA. Remarkably, under specific conditions, production of a high level of transcripts occurred at very low DNA (1 ng/microliter) and HeLa nuclear protein (1.6 micrograms/microliters) concentrations. When partially reconstituted templates were used, transcription efficiency was intermediate between that of fully reconstituted and naked DNA. These results implicate chromatin in the process of the transcriptional activation observed. Depletion from the oocyte assembly extract of an NF-I-like factor which binds in the promoter region upstream of the TATA box (-114 to -101) or deletion from the promoter of the region interacting with this factor reduced the transcriptional efficiency of the assembled templates by a factor of 5, but transcription of these templates was still 10 times higher than that of naked DNA. Together, these results indicate that the NF-I-like factor participates in the very efficient transcriptional potentiation of the vitellogenin B1 promoter which occurs during nucleosome assembly.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.

PPARgamma in placental angiogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in diverse biological processes including adipocyte differentiation, glucose homeostasis, and inflammatory responses. Analyses of PPARγ knockout animals have been so far preempted by the early embryonic death of PPARγ-/- embryos as a consequence of the severe alteration of their placental vasculature. Using Sox2Cre/PPARγL2/L2 mice, we obtained fully viable PPARγ-null mice through specific and total epiblastic gene deletion, thereby demonstrating that the placental defect is the unique cause of PPARγ-/- embryonic lethality. The vasculature defects observed in PPARγ-/- placentas at embryonic d 9.5 correlated with an unsettled balance of pro- and antiangiogenic factors as demonstrated by increased levels of proliferin (Prl2c2, PLF) and decreased levels of proliferin-related protein (Prl7d1, PRP), respectively. To analyze the role of PPARγ in the later stage of placental development, when its expression peaks, we treated pregnant wild-type mice with the PPARγ agonist rosiglitazone. This treatment resulted in a disorganization of the placental layers and an altered placental microvasculature, accompanied by the decreased expression of proangiogenic genes such as Prl2c2, vascular endothelial growth factor, and Pecam1. Together our data demonstrate that PPARγ plays a pivotal role in controlling placental vascular proliferation and contributes to its termination in late pregnancy.

PPARβ/δ activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells.

Owing to its high fat content, the classical Western diet has a range of adverse effects on the heart, including enhanced inflammation, hypertrophy, and contractile dysfunction. Proinflammatory factors secreted by cardiac cells, which are under the transcriptional control of nuclear factor-κB (NF-κB), may contribute to heart failure and dilated cardiomyopathy. The underlying mechanisms are complex, since they are linked to systemic metabolic abnormalities and changes in cardiomyocyte phenotype. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolism and are capable of limiting myocardial inflammation and hypertrophy via inhibition of NF-κB. Since PPARβ/δ is the most prevalent PPAR isoform in the heart, we analyzed the effects of the PPARβ/δ agonist GW501516 on inflammatory parameters. A high-fat diet induced the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6, and enhanced the activity of NF-κB in the heart of mice. GW501516 abrogated this enhanced proinflammatory profile. Similar results were obtained when human cardiac AC16 cells exposed to palmitate were coincubated with GW501516. PPARβ/δ activation by GW501516 enhanced the physical interaction between PPARβ/δ and p65, which suggests that this mechanism may also interfere NF-κB transactivation capacity in the heart. GW501516-induced PPARβ/δ activation can attenuate the inflammatory response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed a high-fat diet. This is relevant, especially taking into account that PPARβ/δ has been postulated as a potential target in the treatment of obesity and the insulin resistance state.

Estrogen receptor level determines sex-specific in vitro transcription from the Xenopus vitellogenin promoter.

Female-specific expression of the Xenopus laevis vitellogenin gene was reconstituted in vitro by addition of recombinant vaccinia-virus-produced estrogen receptor to nuclear extracts from male livers, in which this gene is silent. Transcription enhancement was at least 30 times and was selectively restricted to vitellogenin templates containing the estrogen-responsive unit. Thus, in male hepatocytes, estrogen receptor is the limiting regulatory factor that in the female liver controls efficient and accurate sex-specific expression of the vitellogenin gene. Furthermore, the Xenopus liver factor B, which is essential in addition to the estrogen receptor for the activation of this gene, was successfully replaced in the Xenopus extract by purified human nuclear factor I, identifying factor B of Xenopus as a functional homolog of this well-characterized human transcription factor.

Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1.

RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317-326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptide containing the 317-326 sequence of RasGAP (TAT-RasGAP₃̀1;₇́1;₃₂₆), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP₃̀1;₇́1;₃₂₆ did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP₃̀1;₇́1;₃₂₆ was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.

Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. Conclusion: We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease. (Hepatology 2014;59:423-433).

Biochemical identification of three sympatric Apodemus species by protein electrophoresis of blood samples

The allelic pattern of seralbumine and general protein 1 of the three sympatric Apodemus species Apodemus sylvaticus, A. flavicollis, and A. alpicola, were studied using electrophoretic analysis of blood samples, This method appears to be a sensitive tool for distinguishing the three Apodemus species in the Alps, Their identification on the basis of external characteristics in the field is sometimes extremely difficult, even more so for juvenile specimens. Compared to previously described methods the electrophoretic analysis does not require killing animals and can be used on juveniles.

Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes.

OBJECTIVE: Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. However, the role of PPARbeta/delta activators in cardiac hypertrophy is not yet known. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the selective PPARbeta/delta activator L-165041 (10 micromol/L) inhibited phenylephrine (PE)-induced protein synthesis ([(3)H]leucine uptake), induction of the fetal-type gene atrial natriuretic factor (ANF) and cardiac myocyte size. Induction of cardiac hypertrophy by PE stimulation also led to a reduction in the transcript levels of both muscle-type carnitine palmitoyltransferase (50%, P<0.05) and pyruvatedehydrogenase kinase 4 (30%, P<0.05), and these changes were reversed in the presence of the PPARbeta/delta agonist L-165041. Stimulation of neonatal rat cardiomyocytes with PE and embryonic rat heart-derived H9c2 cells with lipopolysaccharide (LPS) enhanced the expression of the nuclear factor (NF)-kappaB-target gene monocyte chemoattractant protein 1 (MCP-1). The induction of MCP-1 was reduced in the presence of L-165041, suggesting that this compound prevented NF-kappaB activation. Electrophoretic mobility shift assay (EMSA) revealed that L-165041 significantly decreased LPS-stimulated NF-kappaB binding activity in H9c2 myotubes. Finally, coimmunoprecipitation studies showed that L-165041 strongly enhanced the physical interaction between PPARbeta/delta and the p65 subunit of NF-kappaB, suggesting that increased association between these two proteins is the mechanism responsible for antagonizing NF-kappaB activation by PPARbeta/delta activators. CONCLUSION: These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.