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Complete proteomic datasets are available in the PRIDE partner repository (Perez-Riverol et al., 2019) under identification numbers: PXD030822 and 10.6019/PXD030822 for HepaRG cells, PXD031045 and 10.6019/PXD031045 for HepaRG culture media, PXD030887 and 10.6019/PXD030887 for PHHs, and PXD030892 and 10.6019/PXD030892 for PRHs. Metabolomic data are available in the MetaboLights repository (Haug et al., 2020) under the identification number: www.ebi.ac.uk/metabolights/MTBLS4063.; International audience; The biological effects of the pesticide and mitochondrial complex I inhibitor tebufenpyrad (TEBU) on liver cells were investigated by combining proteomics and metabolomics. Both cell culture media and cellular lysates were analyzed in dose-response and kinetic experiments on the HepaRG cell line. Responses were compared with those obtained on primary human and rat hepatocytes. A multitude of phase I and II metabolites (>80) mainly common to HepaRG cells and primary hepatocytes and an increase in metabolization enzymes were observed. Synthesis of mitochondrion and oxidative phosphorylation complex constituents, fatty acid oxidation, and cellular uptake of lipids were induced to compensate for complex I inhibition and the decrease in ATP intracellular contents caused by TEBU. Secretion of the 20 S circulating proteasome and overall inhibition of acute inflammation followed by IL-6 secretion in later stages were observed in HepaRG cells. These effects were associated with a decrease in STAT1 and STAT3 transcription factor abundances, but with different kinetics. Based on identified TEBU targets, docking experiments, and nuclear receptor reporter assays, we concluded that liver cell response to TEBU is mediated by its interaction with the PPARγ transcription factor.

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we recently developed viral vectors targeting astrocytes that either activate or inhibit the JAK2-STAT3 pathway, a central cascade controlling astrocyte reaction.We aimed to evaluate whether reactive astrocytes contribute to Tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops Tau hyperphosphorylation and aggregation in addition to amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in the hippocampus of 3xTg-AD mice, did not significantly influence Tau phosphorylation or amyloid processing and deposition, at early, advanced and terminal stage of the disease. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve short-term spatial memory in the Y maze but it reduced anxiety in the elevated plus maze. Our unique approach to specifically manipulate reactive astrocytes in situ show these cells may impact behavioral outcomes without influencing Tau or amyloid pathology.

Introduction. Signal transducer and activator of transcription 3 (STAT3) deficiency is a rare primary immunodeficiency associated with increased susceptibility to bacterial and fungal infections, notably pulmonary aspergillosis. Aim. We describe the emergence of azole-resistant Aspergillus fumigatus infections in STAT3-deficient patients. Methodology. During a retrospective study of 13 pulmonary aspergillosis cases in STAT3-deficient patients conducted in France, we identified patients infected with azole-resistant A. fumigatus isolates. Results. Two out of the 13 STAT3-deficient patients with aspergillosis had azole-resistant A. fumigatus infection, indicating an unexpectedly high prevalence of resistance. The first patient with STAT3 deficiency presented several flares of allergic bronchopulmonary aspergillosis-like episodes. He was chronically infected with two azole-resistant A. fumigatus isolates (TR34/L98). Despite prolonged antifungal treatment, including caspofungin and amphotericin B, the patient was not able to clear the azole-resistant A. fumigatus. The second patient had chronic cavitary pulmonary aspergillosis (CCPA). The A. fumigatus isolate was initially azole susceptible but harboured three F46Y, M172V and E427K point mutations. Despite prolonged antifungal therapies, lesions worsened and the isolate became resistant to all azoles. Surgery and caspofungin treatments were then required to cure CCPA. Resistance was probably acquired from the environment (TR34/L98) in the first case whereas resistance developed under antifungal treatments in the second case. These infections required long-term antifungal treatments and surgery. Conclusions. The emergence of azole-resistant A. fumigatus infections in STAT3-deficiency dramatically impacts both curative and prophylactic antifungal strategies. Physicians following patients with primary immune-deficiencies should be aware of this emerging problem as it complicates management of the patient.

Type I IFN can exert pro- and anti-inflammatory activities in the immune system. Here, we have investigated the mechanism by which IFN-α enhances early expression of the anti-inflammatory cytokine IL-10 in human CD45RA+CD4+ T cells. With the use of transcriptomic and biochemical approaches, we found distinct and combined contributions of the IFN and the TCR signaling pathways to the induction of STAT1/2/3 and the basic leucine zipper activating transcription factor-like (BATF) family members. Moreover, IFN-induced STAT3 phosphorylation was prolonged by the TCR response, whereas IFN-induced STAT2 phosphorylation was of long duration. With the use of RNA interference (RNAi), we identified STAT3 as the major actor and STAT2 as a contributor of the IFN action on IL-10. Upon TCR/IFN costimulation, STAT3 directly bound at the IL-10 conserved noncoding sequence (CNS)- 9, an enhancer element known to recruit BATF in CD4 T cells. The cosilencing of the 3 BATFs resulted in an overall reduction of IL-10 expression, but the promoting activity of IFN-α was retained. These results support the notion that the IFN action is indexed on BATF function and provide evidence for a cooperation between BATFs and STAT3, the latter being activated via early IFN and delayed TCR effects.

International audience; Aberrant activation of the SRC family kinase hematopoietic cell kinase (HCK) triggers hematological malignancies as a tumor cell-intrinsic oncogene. Here we find that high HCK levels correlate with reduced survival of colorectal cancer patients. Likewise, increased Hck activity in mice promotes the growth of endogenous colonic malignancies and of human colorectal cancer cell xenografts. Furthermore, tumor-associated macrophages of the corresponding tumors show a pronounced alternatively activated endotype, which occurs independently of mature lymphocytes or of Stat6-dependent Th2 cytokine signaling. Accordingly, pharmacological inhibition or genetic reduction of Hck activity suppresses alternative activation of tumor-associated macrophages and the growth of colon cancer xenografts. Thus, Hck may serve as a promising therapeutic target for solid malignancies.

International audience; Germline mutations in the gene encoding tumor suppressor kinase LKB1 lead to gastrointestinal tumorigenesis in Peutz-Jeghers syndrome (PJS) patients and mouse models; however, the cell types and signaling pathways underlying tumor formation are unknown. Here, we demonstrated that mesenchymal progenitor- or stromal fibroblast-specific deletion of Lkb1 results in fully penetrant polyposis in mice. Lineage tracing and immunohistochemical analyses revealed clonal expansion of Lkb1-deficient myofibroblast-like cell foci in the tumor stroma. Loss of Lkb1 in stromal cells was associated with induction of an inflammatory program including IL-11 production and activation of the JAK/STAT3 pathway in tumor epithelia concomitant with proliferation. Importantly, treatment of LKB1-defcient mice with the JAK1/2 inhibitor ruxolitinib dramatically decreased polyposis. These data indicate that IL-11-mediated induction of JAK/STAT3 is critical in gastrointestinal tumorigenesis following Lkb1 mutations and suggest that targeting this pathway has therapeutic potential in Peutz-Jeghers syndrome.

B lymphocytes from chronic lymphocytic leukemia (CLL) display some CD5 transcripts for CD5 containing the known exon 1 (E1A) and other CD5 transcripts containing the new exon 1 (E1B). These malignant B cells, as well as B cell lines transfected with cDNA for E1A-cd5 or with cDNA for E1B-cd5 produce IL-10, raising the possibility that CD5 participates in the secretion of IL-10. We identified transcription factors involved in this production in CD5+ B lymphocytes from CLL patients and in E1A-cd5–transfected or E1B-cd5–transfected Jok cells. STAT3 is activated via phosphorylation of serine 727 but also NFAT2 through its translocation into the nucleus. Chromatin immunoprecipitation experiments confirmed the role of STAT3 and allowed the discovery of a role for NFAT2 in IL-10 production. Both transcription factors bind not only to the enhancer of the Il-10 gene but also to the promoter of the Il-5 and Il-13 genes. Furthermore, transfection of B cell lines with E1A-cd5 or E1B-cd5 established that activation of STAT3 and NFAT2 is regulated by CD5. The same holds true for the production of IL-10, IL-5, and IL-13 and the expression of the receptors for these cytokines. This interpretation was confirmed by two experiments. In the first, downregulation of CD5 by small interfering RNAs lowered the production of IL-10. In the second experiment, transfection of the GFP-NFAT2 gene into B lymphocytes induced nuclear translocation of NFAT2 in CD5+ but not in CD5− B cells. Thus, CD5 expression is associated with NFAT2 activity (and mildly STAT3 activity), indicating that CD5 controls IL-10 secretion.

International audience; BACKGROUND & AIMS: Liver resection includes temporal vascular inflow occlusion resulting in ischemia/reperfusion injury in the remnant liver. Here, we developed a rat model of selective lobe occlusion to isolate reperfusion stress from ischemia and to analyze its effect on liver regeneration. METHODS: Left lateral and median lobes of liver were either mobilized or subjected twice for 10min to ischemia followed by 5min reperfusion prior to resection while the regenerative lobes were only subjected to reperfusion. RESULTS: Although intermittent reperfusion stress induced higher levels of serum transaminases, analysis of cell cycle regulators revealed accelerated regenerative response compared to standard partial hepatectomy. The G0/G1 transition occurred before tissue resection, as evidenced by c-fos, junB, and IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition, and cell division occurred earlier than normal. Unexpectedly, liver mobilization, a component of the clamping procedure, also resulted in earlier G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal Kinase pathway and was associated with an oxidative stress response as evidenced by the expression of inducible nitric oxide synthase. CONCLUSION: Intermittent selective clamping of lobes to be resected induced reperfusion stress on remnant liver that was beneficial for liver regeneration, suggesting this procedure could be applied in clinical practice.

International audience; Aging is associated with changes in the immune response which are collectively called immunosenescence. The changes mainly affect the adaptive immune response and especially the T cell-mediated cellular immune response. There are a few data indicating that the cytokine signalling in T cells is altered with aging. Zinc has been specifically shown to have potent immunomodulatory effects. The aim of the present work was to study the IL-2 and IL-6 cytokine signalling and activation induced cell death (AICD) in T cells of elderly subjects of various ages and from various European countries. These experiments were performed in the frame of European Community financed project called ZINCAGE "Nutritional zinc, oxidative stress and immunosenescence: biochemical, genetic and lifestyle implications for healthy ageing", assembling 17 laboratories from 8 countries through Europe. The study was carried out in a total of 312 French and a group of 201 (26 from Italy, 63 from France, 57 from Greece, 24 from Poland and 30 from Germany) healthy non-institutionalized men and women older than 60 years of age, with available dietary data. Human peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood and were stimulated in vitro by IL-2 or IL-6 for various periods and the phosphorylation of STAT3 and STAT5 was measured by FACScan. The activation induced cell death (AICD) was measured after anti-CD3 and CD28 restimulation for 48h by using the Annexin:FITC Apoptosis Kit. We found that there is an IL-2 signalling defect with aging up to 90 years of age which cannot be modulated by zinc. In contrast at 90 years and over the zinc could reverse the negative signalling effect of IL-2. There is also a signalling defect for STAT3 and STAT5 activation in T cells under IL-6 stimulation with aging and the zinc supplementation could potentiate only the STAT5 activation in the age-group 90 years and over. Studying signalling in PBL from different countries we detected less activation in T cells of subjects from France and the most changes occurred in T cells of subjects from Poland, suggesting no correlation with the plasma zinc status observed in these countries. In vivo zinc supplementation had no effect on IL-2 and IL-6-modulated STAT3 and STAT5 activation. Zinc added in vitro to these T cells even inhibited the stimulation either by IL-2 or by IL-6. Zinc supplementation improved the susceptibility of T cells to AICD in both age-groups, with more efficiency in later ages. Our results suggest that zinc can have a potent immunomodulatory effect via the modulation of cytokine signalling and AICD, however this effect depends on the function and the activation status of the T cells.

International audience; To date, the mutational status of EGFR and PTEN has been shown as relevant for favoring pro- or anti-tumor functions of STAT3 in human glioblastoma multiforme (GBM). We have screened genomic data from 154 patients and have identified a strong positive correlation between STAT3 and HDAC7 expression. In the current work we show the existence of a subpopulation of patients overexpressing HDAC7 and STAT3 that has particularly poor clinical outcome. Surprisingly, the somatic mutation rate of both STAT3 and HDAC7 was insignificant in GBM comparing with EGFR, PTEN or TP53. Depletion of HDAC7 in a range of GBM cells induced the expression of tyrosine kinase JAK1 and the tumor suppressor AKAP12. Both proteins synergistically sustained the activity of STAT3 by inducing its phosphorylation (JAK1) and protein expression (AKAP12). In absence of HDAC7, activated STAT3 was responsible for significant imbalance of secreted pro-/anti-angiogenic factors. This inhibited the migration and sprouting of endothelial cells in paracrine fashion in vitro as well as angiogenesis in vivo. In a murine model of GBM, induced HDAC7-silencing decreased the tumor burden by threefold. The current data show for the first time that silencing HDAC7 can reset the tumor suppressor activity of STAT3, independently of the EGFR/PTEN/TP53 background of the GBM. This effect could be exploited to overcome tumor heterogeneity and provide a new rationale behind the development of specific HDAC7 inhibitors for clinical use.

International audience; Interferon gamma (IFN-γ) and leukemia inhibitory factor (LIF) are key gestational factors that may differentially affect leukocyte function during gestation. Because IFN-γ induces a pro-inflammatory phenotype in macrophages and because trophoblast cells are principal targets of LIF in the placenta, we investigated whether and how soluble factors from trophoblast cells regulate the effects of IFN-γ on macrophage activation. IFN-γ reduces macrophage motility, but enhances Stat1 activation, pro-inflammatory gene expression and cytotoxic functions. Soluble factors from villous cytotrophoblasts (vCT+LIF cells) and BeWo cells (BW/ST+LIF cells) that were differentiated in the presence of LIF inhibit macrophage Stat1 activation but inversely sustain Stat3 activation in response to IFN-γ. vCT+LIF cells produce soluble factors that induce Stat3 activation; this effect is partially abrogated in the presence of neutralizing anti-interleukin 10 (IL-10) antibodies. Moreover, soluble factors from BW/ST+LIF cells reduce cell proliferation but enhance the migratory responses of monocytes. In addition, these factors reverse the inhibitory effect of IFN-γ on monocyte/macrophage motility. BW/ST+LIF cells also generate IFN-γ-activated macrophages with enhanced IL-10 expression, but reduced tumor-necrosis factor alpha (TNF-α), CD14 and CD40 expression as well as impaired cytotoxic function. Additional assays performed in the presence of neutralizing anti-IL-10 antibodies and exogenous IL-10 demonstrate that reduced macrophage cytotoxicity and proliferation, but increased cell motility result from the ability of trophoblast IL-10 to sustain Stat3 activation and suppress IFN-γ-induced Stat1 activation. These in vitro studies are the first to describe the regulatory role of the LIF-trophoblast-IL-10 axis in the process of macrophage activation in response to pro-inflammatory cytokines.

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

International audience; Skin inflammation is a complex process that involves interactions between various cell types residing in different skin compartments. Using mice with conditionally targeted I kappa B kinase 2 (IKK2) alleles, we have previously shown that epidermal keratinocytes can play a dominant role in the initiation of an inflammatory reaction. In order to investigate long-term consequences of IKK2 deletion in adult skin, we have generated mice with floxed IKK2 alleles in which expression of a Tamoxifen-inducible Cre recombinase construct is targeted to epidermal keratinocytes (K14-Cre-ER(T2)IKK2(fl/fl) mice). K14-Cre-ER(T2)IKK2(fl/fl) mice are born normally and do not show signs of a skin disease until the age of 6 months. Deletion of IKK2 can be observed after Tamoxifen application to the back skin or spontaneously, without Tamoxifen application, in mice older than 6 months. This deletion is accompanied by dramatic, localized skin changes that are characterized by invasion of inflammatory cells, hair follicle disruption, and pseudoepitheliomatous hyperplasia of the epidermis, but not by tumor formation. The hyperplastic epithelium shows increased phosphorylation of signal transducer and activator of transcription 3 and extracellular signal-regulated protein kinase 1/2, typical features of psoriatic epidermis. Our results identify a primary role for IKK2 in the development of skin inflammation and confirm its requirement for the maintenance of skin homeostasis.

International audience; As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCR-stimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67(phox). We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression.

PubMedID: 25724123 Background Mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency (CID) also classified as autosomal recessive (AR) hyper-IgE syndrome (HIES). Recognizing patients with CID/HIES is of clinical importance because of the difference in prognosis and management. Objectives We sought to define the clinical features that distinguish DOCK8 deficiency from other forms of HIES and CIDs, study the mutational spectrum of DOCK8 deficiency, and report on the frequency of specific clinical findings. Methods Eighty-two patients from 60 families with CID and the phenotype of AR-HIES with (64 patients) and without (18 patients) DOCK8 mutations were studied. Support vector machines were used to compare clinical data from 35 patients with DOCK8 deficiency with those from 10 patients with AR-HIES without a DOCK8 mutation and 64 patients with signal transducer and activator of transcription 3 (STAT3) mutations. Results DOCK8-deficient patients had median IgE levels of 5201 IU, high eosinophil levels of usually at least 800/µL (92% of patients), and low IgM levels (62%). About 20% of patients were lymphopenic, mainly because of low CD4+ and CD8+ T-cell counts. Fewer than half of the patients tested produced normal specific antibody responses to recall antigens. Bacterial (84%), viral (78%), and fungal (70%) infections were frequently observed. Skin abscesses (60%) and allergies (73%) were common clinical problems. In contrast to STAT3 deficiency, there were few pneumatoceles, bone fractures, and teething problems. Mortality was high (34%). A combination of 5 clinical features was helpful in distinguishing patients with DOCK8 mutations from those with STAT3 mutations. Conclusions DOCK8 deficiency is likely in patients with severe viral infections, allergies, and/or low IgM levels who have a diagnosis of HIES plus hypereosinophilia and upper respiratory tract infections in the absence of parenchymal lung abnormalities, retained primary teeth, and minimal trauma fractures. © 2015 American Academy of Allergy, Asthma & Immunology. National Institutes of Health: R01AI065617 National Institutes of Health: R21AI087627

IL-6 is a multifaceted pleiotropic cytokine, which is produced by a variety of cell types and targets different cells and tissues. In physiological conditions, IL-6 can be locally and transiently produced by skeletal muscle and plays an important role in muscle homeostasis. Circulating IL-6 levels are normally very low or undetectable but are dramatically increased in several pathologic conditions. In this study, we aimed to define the potential molecular mechanisms underlying the effects of IL-6 on myogenic program. We explored the molecular mechanisms through which exogenous IL-6, or the conditioned medium from the murine C-26 adenocarcinoma cells (a cellular model that secretes high levels of IL-6 and induces cancer cachexia in mice), interferes with the myogenic program. Our study revealed that IL-6 induces the activation of the Stat3 signaling and promotes the downmodulation of the p90RSK/eEF2 and mTOR/p70S6K axes, while it does not affect the activation of AKT. We thus identified potential molecular mediators of the inhibitory effects of IL-6 on myogenic program.

Engagement of NKG2D and DNAX accessory molecule-1 (DNAM-1) receptors on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the glycogen synthase kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in multiple myeloma (MM) cells. GSK3 is a pleiotropic serine–threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 upregulates both MICA protein surface and mRNA expression in MM cells, with little or no effects on the basal expression of the MICB and DNAM-1 ligand poliovirus receptor/CD155. Moreover, exposure to GSK3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and to enhance the ability of myeloma cells to trigger NK cell–mediated cytotoxicity. We could exclude that increased expression of β-catenin or activation of the heat shock factor-1 (transcription factors inhibited by active GSK3) is involved in the upregulation of MICA expression, by using RNA interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr705 phosphorylation and binding of STAT3 on MICA promoter are reduced by GSK3 inhibitors; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional immune-mediated mechanism supporting the antimyeloma activity of GSK3 inhibitors.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

International audience; The capacity of a virus to cross species barriers is determined by the development of bona fide interactions with cellular components of new hosts, and in particular its ability to block IFN-α/β antiviral signaling. Tioman virus (TioV), a close relative of mumps virus (MuV), has been isolated in giant fruit bats in Southeast Asia. Nipah and Hendra viruses, which are present in the same bat colonies, are highly pathogenic in human. Despite serological evidences of close contacts between TioV and human populations, whether TioV is associated to some human pathology remains undetermined. Here we show that in contrast to the V protein of MuV, the V protein of TioV (TioV-V) hardly interacts with human STAT2, does not degrade STAT1, and cannot block IFN-α/β signaling in human cells. In contrast, TioV-V properly binds to human STAT3 and MDA5, and thus interferes with IL-6 signaling and IFN-β promoter induction in human cells. Because STAT2 binding was previously identified as a host restriction factor for some Paramyxoviridae, we established STAT2 sequence from giant fruit bats, and binding to TioV-V was tested. Surprisingly, TioV-V interaction with STAT2 from giant fruit bats is also extremely weak and barely detectable. Altogether, our observations question the capacity of TioV to appropriately control IFN-α/β signaling in both human and giant fruit bats that are considered as its natural host.

Background & Aims Mosaic G-protein alpha-subunit ( GNAS )-activating mutations are responsible for the McCune–Albright (MCA) syndrome. This oncogene that activates the adenylate cyclase is also mutated in various tumor types leading to the accumulation of cyclic-AMP. Identification of a hepatocellular adenoma (HCA) in two MCA patients led us to search for GNAS activation in benign and malignant hepatocellular carcinogenesis. Methods GNAS mutations were screened by sequencing 164 HCA, 245 hepatocellular carcinoma (HCC), and 17 fibrolamellar carcinomas. Tumors were characterized by quantitative RT-PCR, gene mutation screening and pathological reviewing. The consequences of wild type and mutant GNAS expression were analyzed in hepatocellular cell lines. Results A somatic GNAS -activating mutation was identified in 5 benign tumors and in 2 HCC. In benign tumors, GNAS mutations were exclusive from HNF1A , CTNNB1 , and IL6ST mutations whereas one HCC demonstrated both CTNNB1 and GNAS mutations. Quantitative RT-PCR showed an activation of the IL-6 and interferon pathways in GNAS -mutated tumor tissues. Accordingly, pathological reviewing identified in GNAS -mutated tumors an inflammatory phenotype characterized by fibrosis and STAT3 activation. We further demonstrated in HCC cell lines that GNAS mutant expression induced inflammatory response and STAT3 activation. Conclusions We identified for the first time the association between two rare diseases, MCA syndrome and HCA occurrence, but also that somatic GNAS -activating mutations in sporadic benign and malignant liver tumors are characterized by an inflammatory phenotype. These results showed a cross-talk between cyclic-AMP and JAK/STAT pathways in liver tumors and they reinforce the role of STAT3 activation in liver tumorigenesis.

International audience; Erythropoietin-producing hepatocellular kinases (Eph kinases) constitute the largest family of cell membrane receptor tyrosine kinases, and their ligand ephrins are also cell surface molecules. Because of promiscuous interaction between Ephs and ephrins, there is considerable redundancy in this system, reflecting the essential roles of these molecules in the biological system through evolution. In this study, both Efnb1 and Efnb2 were null-mutated in the T cell compartment of mice through loxP-mediated gene deletion. Mice with this double conditional mutation (double KO mice) showed reduced thymus and spleen size and cellularity. There was a significant decrease in the DN4, double positive, and single positive thymocyte subpopulations and mature CD4 and CD8 cells in the periphery. dKO thymocytes and peripheral T cells failed to compete with their WT counterparts in irradiated recipients, and the T cells showed compromised ability of homeostatic expansion. dKO naive T cells were inferior in differentiating into Th1 and Th17 effectors in vitro. The dKO mice showed diminished immune response against LCMV infection. Mechanistic studies revealed that IL-6 signaling in dKO T cells was compromised, in terms of abated induction of STAT3 phosphorylation upon IL-6 stimulation. This defect likely contributed to the observed in vitro and in vivo phenotype in dKO mice. This study revealed novel roles of Efnb1 and Efnb2 in T cell development and function.

The protein ERp57/GRP58 is a stress-responsive protein and a component of the protein disulfide isomerase family. Its functions in the endoplasmic reticulum are well known, concerning mainly the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class 1. However, ERp57 is present in many other subcellular locations, where it is involved in a variety of functions, primarily suggested by its participation in complexes with other proteins and even with DNA. While in some instances these roles need to be confirmed by further studies, a great number of observations support the participation of ERp57 in signal transduction from the cell surface, in regulatory processes taking place in the nucleus, and in multimeric protein complexes involved in DNA repair.

Summary The signal transducer and activator of transcription 3 (STAT3) transcription factor pathway plays an important role in many biological phenomena. STAT3 transcription is triggered by cytokine-associated signals. Here, we use isolated human B cells to analyse the role of STAT3 in interleukin (IL)-10 induced terminal B cell differentiation and in immunoglobulin (Ig)A production as a characteristic readout of IL-10 signalling. We identified optimal conditions for inducing in-vitro IgA production by purified blood naive B cells using IL-10 and soluble CD40L. We show that soluble CD40L consistently induces the phosphorylation of nuclear factor (NF)-κB p65 but not of STAT3, while IL-10 induces the phosphorylation of STAT3 but not of NF-κB p65. Interestingly, while soluble CD40L and IL-10 were synergistic in driving the terminal maturation of B cells into IgA-producing plasma cells, they did not co-operate earlier in the pathway with regard to the transcription factors NF-κB p65 or STAT3. Blocking either NF-κB p65 or STAT3 profoundly altered the production of IgA and mRNA for activation-induced cytidine deaminase (AID), an enzyme strictly necessary for Ig heavy chain recombination. Finally, the STAT3 pathway was directly activated by IL-10, while IL-6, the main cytokine otherwise known for activating the STAT3 pathway, did not appear to be involved in IL-10-induced-STAT3 activation. Our results suggest that STAT3 and NF-κB pathways co-operate in IgA production, with soluble CD40L rapidly activating the NF-κB pathway, probably rendering STAT3 probably more reactive to IL-10 signalling. This novel role for STAT3 in B cell development reveals a potential therapeutic or vaccine target for eliciting IgA humoral responses at mucosal interfaces.

International audience; Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumors. 60% of these tumors have IL-6 signal transducer (IL6ST; gp130) mutations that activate interleukin 6 (IL-6) signaling. Here, we report that 12% of IHCA subsets lacking IL6ST mutations harbor somatic signal transducer and activator of transcription 3 (STAT3) mutations (6/49). Most of these mutations are amino acid substitutions in the SH2 domain that directs STAT3 dimerization. In contrast to wild-type STAT3, IHCA STAT3 mutants constitutively activated the IL-6 signaling pathway independent of ligand in hepatocellular cells. Indeed, the IHCA STAT3 Y640 mutant homodimerized independent of IL-6 and was hypersensitive to IL-6 stimulation. This was associated with phosphorylation of tyrosine 705, a residue required for IL-6-induced STAT3 activation. Silencing or inhibiting the tyrosine kinases JAK1 or Src, which phosphorylate STAT3, impaired constitutive activity of IHCA STAT3 mutants in hepatocellular cells. Thus, we identified for the first time somatic STAT3 mutations in human tumors, revealing a new mechanism of recurrent STAT3 activation and underscoring the role of the IL-6-STAT3 pathway in benign hepatocellular tumorigenesis.

The role of natural killer group 2, member D receptor (NKG2D)–expressing natural killer (NK) cells in tumor immunosurveillance is now well established. Nevertheless, tumor progression occurs despite tumor immunosurveillance, leading to cancer persistence in immunocompetent hosts. STAT3 plays a pivotal role both in oncogenic functions and in immunosuppression. In this study, we investigated the role of STAT3 in suppressing NK cell–mediated immunosurveillance. Using a colorectal cancer cell line (HT29) that can poorly activate NK, we neutralized STAT3 with pharmacologic inhibitors or siRNA and found that this led to an increase in NK degranulation and IFN-γ production in a TGF-β1–independent manner. Exposure to NKG2D-neutralizing antibodies partially restored STAT3 activity, suggesting that it prevented NKG2D-mediated NK cell activation. On this basis, we investigated the expression of NKG2D ligands after STAT3 activation in HT29, mesenchymal stem cells, and activated lymphocytes. The NK cell recognition receptor MHC class I chain–related protein A (MICA) was upregulated following STAT3 neutralization, and a direct interaction between STAT3 and the MICA promoter was identified. Because cross-talk between DNA damage repair and NKG2D ligand expression has been shown, we assessed the influence of STAT3 on MICA expression under conditions of genotoxic stress. We found that STAT3 negatively regulated MICA expression after irradiation or heat shock, including in lymphocytes activated by CD3/CD28 ligation. Together, our findings reveal a novel role for STAT3 in NK cell immunosurveillance by modulating the MICA expression in cancer cells. Cancer Res; 71(5); 1615–26. ©2011 AACR.

International audience; Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumors. 60% of these tumors have IL-6 signal transducer (IL6ST; gp130) mutations that activate interleukin 6 (IL-6) signaling. Here, we report that 12% of IHCA subsets lacking IL6ST mutations harbor somatic signal transducer and activator of transcription 3 (STAT3) mutations (6/49). Most of these mutations are amino acid substitutions in the SH2 domain that directs STAT3 dimerization. In contrast to wild-type STAT3, IHCA STAT3 mutants constitutively activated the IL-6 signaling pathway independent of ligand in hepatocellular cells. Indeed, the IHCA STAT3 Y640 mutant homodimerized independent of IL-6 and was hypersensitive to IL-6 stimulation. This was associated with phosphorylation of tyrosine 705, a residue required for IL-6-induced STAT3 activation. Silencing or inhibiting the tyrosine kinases JAK1 or Src, which phosphorylate STAT3, impaired constitutive activity of IHCA STAT3 mutants in hepatocellular cells. Thus, we identified for the first time somatic STAT3 mutations in human tumors, revealing a new mechanism of recurrent STAT3 activation and underscoring the role of the IL-6-STAT3 pathway in benign hepatocellular tumorigenesis.

International audience; Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.

International audience; BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) has been implicated as an oncogene in several neoplastic diseases. However, the biological effects of STAT3 have not been extensively studied in rectal carcinogenesis. AIMS: To evaluate STAT3 activation in advanced rectal cancers and its association with clinicopathological variables and prognosis. METHODS: Nuclear immunohistochemical expression of phosphorylated STAT3 (p-STAT3) was studied in 104 advanced rectal cancers (T3-T4). All patients were participating in the EORTC 22921 trial to assess whether preoperative chemoradiotherapy followed by postoperative chemotherapy improved overall and progression-free survival. RESULTS: Nuclear p-STAT3 expression was detected in 37.5% of rectal cancer patients. No correlation was observed between p-STAT3 and any clinicopathological variables tested. However, patients with tumours positive for p-STAT3 had significantly improved overall survival. CONCLUSION: These results highlight an unexpected role for nuclear p-STAT3 expression in advanced rectal cancers and need further investigation to clarify this finding.

International audience; During the inflammatory process, hepcidin overexpression favours the development of anaemia of chronic diseases which represents the second most common form of anaemia worldwide. The identification of therapeutic agents decreasing hepcidin expression is therefore an important goal. The aim of this study was to target the STAT3 signalling involved in the development of increased hepcidin expression related to chronic inflammation. In a co-culture model associating mouse hepatocytes and rat liver epithelial cells, the mRNA levels of hepcidin1, albumin, aldolase B, Cyp3a4, Stat3, Smad4 and iron regulatory genes were measured by real-time PCR. STAT3 and phosphorylated SMAD1/5/8 proteins were analysed by Western blot. At variance of hepatocyte pure culture, co-culture provided high levels of hepcidin1 mRNA, reaching 400% of the freshly isolated hepatocyte values after 6 days of culture. Hepcidin expression was associated with the maintenance of hepatocyte phenotype, STAT3 phosphorylation and functional BMP/SMAD pathway. Stat3 siRNAs inhibited the hepcidin1 mRNA expression. STAT3 inhibitors, including curcumin, AG490 and a peptide (PpYLKTK), reduced hepcidin1 mRNA expression even when cells were additionally exposed to IL-6. Hepcidin1 mRNA was expressed at high levels by hepatocytes in the co-culture model, and STAT3 pathway activation was controlled through STAT3 inhibitors. Such inhibitors could be useful to prevent anaemia related to hepcidin overexpression during chronic inflammation.

International audience; Acute leptin increase as well as endogenous hyperleptinemia evoked by high-fat diets (HF) activate fatty acid metabolism in nonadipose tissues. This supports the notion that hyperleptinemia is pivotal to prevent/delay steatosis during periods of positive energy balance. We have previously shown that long-term HF spares ectopic accumulation of lipids specifically in the miocardium. Because carnitine palmitoyltransferase I (CPT-I) allows mitochondrial uptake/oxidation of fatty acids, we have hypothesized that leptin drives cardiac CPT-I activity. In the current study, hyperleptinemia was induced in C57BL/6J mice either by exogenous leptin administration or by means of HF, and the ability of malonyl-coenzyme A (malonyl-CoA) (the main endogenous inhibitor of CPT-I) to inhibit cardiac CPT was analyzed. IC(50) values of malonyl-CoA were 8.1 +/- 1.5 micromol/liter in controls vs. 69.3 +/- 5.2 micromol/liter (P < 0.01) in leptin-treated mice. This effect was also observed in cardiac explants incubated with leptin and was blocked by triciribine, a compound shown to inhibit protein kinase B (Akt) phosphorylation (pAkt). In accordance, acute leptin evoked an increase of cardiac pAkt levels, which correlated with CPT sensitivity to malonyl-CoA. Otherwise, the inhibitory effect of malonyl-CoA was hindered in HF hyperleptinemic mice, and in this case, pAkt levels also correlated with CPT sensitivity to malonyl-CoA. Our data show that leptin reduces the sensitivity of cardiac CPT-I to malonyl-CoA and suggest the involvement of an Akt-related signaling pathway in this effect. This mechanism appears to be sensitive to both acute and chronic hyperleptinemia. We conclude that this action of leptin is pivotal to drive cardiac metabolism under situations associated to hyperleptinemia.

International audience; Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular bacterium typically found in myeloid cells. The infection is a source of severe obstetrical complications in humans and cattle and can undergo chronic evolution in a minority of pregnant women. Because C. burnetii is found in the placentas of aborted fetuses, we investigated the possibility that it could infect trophoblasts. Here, we show that C. burnetii infected and replicated in BeWo trophoblasts within phagolysosomes. Using pangenomic microarrays, we found that C. burnetii induced a specific transcriptomic program. This program was associated with the modulation of inflammatory responses that were shared with inflammatory agonists, such as TNF, and more specific responses involving genes related to pregnancy development, including EGR-1 and NDGR1. In addition, C. burnetii stimulated gene networks organized around the IL-6 and IL-13 pathways, which both modulate STAT3. Taken together, these results revealed that trophoblasts represent a protective niche for C. burnetii. The activation program induced by C. burnetii in trophoblasts may allow bacterial replication but seems unable to interfere with the development of normal pregnancy. Such pathophysiologocal processes should require the activation of immune placental cells associated with trophoblasts.

International audience; Inflammatory hepatocellular adenomas are benign liver tumours defined by the presence of inflammatory infiltrates and by the increased expression of inflammatory proteins in tumour hepatocytes. Here we show a marked activation of the interleukin (IL)-6 signalling pathway in this tumour type; sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the IL6ST gene, which encodes the signalling co-receptor gp130. Indeed, 60% of inflammatory hepatocellular adenomas harbour small in-frame deletions that target the binding site of gp130 for IL-6, and expression of four different gp130 mutants in hepatocellular cells activates signal transducer and activator of transcription 3 (STAT3) in the absence of ligand. Furthermore, analysis of hepatocellular carcinomas revealed that rare gp130 alterations are always accompanied by beta-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant conversion of hepatocytes. The recurrent gain-of-function gp130 mutations in these human hepatocellular adenomas fully explains activation of the acute inflammatory phase observed in tumourous hepatocytes, and suggests that similar alterations may occur in other inflammatory epithelial tumours with STAT3 activation.

International audience; The maintenance of bone mass requires a strict balance between bone formation by osteoblasts and bone resorption by osteoclasts. In tumoral bone environment, tumor cells frequently disturb this balance by interaction with bone cells to create a favorable site for tumor growth, and promote pathological bone changes. Thus, elucidation of the mechanisms underlying interaction between tumor cells and bone cells might eventually lead to a more rational strategy for therapeutic intervention for bone tumors and better understanding of bone biology. In the present study, the effects of mouse osteosarcoma cells on mouse preosteoblastic cells were determined by assessment of cell viability, osteoblastic differentiation and signal transduction pathways. MOS-J/POS-1 conditioned media (CM) significantly induced MC3T3-E1 cell proliferation in a dose-dependent manner and reduced both alkaline phosphatase activity and mineralized nodule formation. Piceatannol, AG490, LY294002 and rapamycin significantly abrogated this up-regulated cell proliferation; however, UO126 and STAT3 inhibitor peptide did not affect this up-regulated cell proliferation. MOS-J/POS-1 CM activated ERK 1/2, STAT3 and Akt signal transduction pathways; however, pro-proliferating signal induced by MOS-J/POS-1 CM was transmitted via Akt not ERK 1/2 and STAT3 pathways. Furthermore, Western blot analyses clearly revealed novel signal crosstalk between JAKs and PI3-K/Akt in osteoblastic cells. The specific factor(s) involved in MOS-J/POS-1 CM-induced MC3T3-E1 cell proliferation that use JAKs/PI3-K/Akt/mTOR pathway remain(s) to be determined. Determination of the specific factor(s) responsible for JAKs and PI3-K/Akt signal crosstalk that results in up-regulated preosteoblast proliferation will offer new insight into the pathology of osteosarcoma as well as other bone-related diseases.

International audience; Tyk2 is a Jak family member involved in cytokine signaling through heterodimeric-type receptors. Here, we analyzed the impact of the Val(678)-to-Phe substitution on Tyk2 functioning. This mutation is homologous to the Jak2 Val(617)-to-Phe mutation, implicated in myeloproliferative disorders. We studied ligand-independent and ligand-dependent Jak/Stat signaling in cells expressing Tyk2 V678F. Moreover, the effect of Tyk2 V678F was monitored in the context of the native heterodimeric interferon alpha receptor and in the context of a homodimeric receptor chimera, EpoR/R1, containing the ectodomain of the erythropoietin receptor. We show that Tyk2 V678F has increased catalytic potential in vivo and in vitro and more so when it is anchored to the homodimeric receptor. Tyk2 V678F leads to constitutive Stat3 phosphorylation but has no notable effect on the canonical interferon alpha-induced signaling. However, if anchored to the homodimeric EpoR/R1, the mutant confers to the cell increased sensitivity to erythropoietin. Thus, despite the catalytic gain of function of Tyk2 V678F, the effect on ligand-induced signaling is manifest only when two mutant enzymes are juxtaposed via the homodimeric receptor.

International audience; Osteoclasts are bone-resorptive cells that differentiate from hematopoietic precursors upon receptor activator of nuclear factor kappaB ligand (RANKL) activation. Previous studies demonstrated that IL-6 indirectly stimulates osteoclastogenesis through the production of RANKL by osteoblasts. However, few data described the direct effect of IL-6 on osteoclasts. To investigate this effect, we used several models: murine RAW264.7 cells, mouse bone marrow, and human blood monocytes. In the three models used, the addition of IL-6 inhibited RANKL-induced osteoclastogenesis. Furthermore, IL-6 decreased the expression of osteoclast markers and up-modulated macrophage markers. To elucidate this inhibition, signal transducer and activator of transcription (STAT) 3, the main signaling molecule activated by IL-6, was analyzed. Addition of two STAT3 inhibitors completely abolished RANKL-induced osteoclastogenesis, revealing a key role of STAT3. We demonstrated that a basal level of phosphorylated-STAT3 on Serine(727) associated with an absence of phosphorylation on Tyrosine(705) is essential for osteoclastogenesis. Furthermore, a decrease of Serine(727) phosphorylation led to an inhibition of osteoclast differentiation, whereas an increase of Tyrosine(705) phosphorylation upon IL-6 stimulation led to the formation of macrophages instead of osteoclasts. In conclusion, we showed for the first time that IL-6 inhibits RANKL-induced osteoclastogenesis by diverting cells into the macrophage lineage, and demonstrated the functional role of activated-STAT3 and its form of phosphorylation in the control of osteoclastogenesis.

Immunological tolerance is maintained by specialized subsets of T cells including CD4+CD25+FOXP3+ regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. Moreover, foxp3 gene transfer in murine CD4+CD25− T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4+CD25− T lymphocytes leads to STAT3 Tyr705 phosphorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4+CD25− T cell conversion into Treg through costimulation with TCR/CD28 and TGF-β1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-γ. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differentiated CD4+CD25+ T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4+CD25+ T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4+ lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance.

The basic helix-loop-helix protein Neurogenin3 specifies precursor cells of the endocrine pancreas during embryonic development, and is thought to be absent postnatally. We have studied Ngn3 expression during in vitro generation of beta-cells from adult rat exocrine pancreas tissue treated with epidermal growth factor and leukaemia inhibitory factor. This treatment induced a transient expression of both Ngn3 and its upstream activator hepatocyte nuclear factor 6. Inhibition of EGF and LIF signalling by pharmacological antagonists of the JAK2/STAT3 pathway, or knockdown of Ngn3 by RNA interference prevented the generation of new insulin-positive cells. This study demonstrates that in vitro growth factor stimulation can induce recapitulation of an embryonic endocrine differentiation pathway in adult dedifferentiated exocrine cells. This could prove to be important for understanding the mechanism of beta-cell regeneration and for therapeutic ex vivo neogenesis of beta cells.

International audience; Prostate cancer metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer, frequently resulting in significant morbidity and mortality. As the underlying mechanisms are not fully characterized, understanding the biological mechanisms that govern prostate cancer metastases to bone at the molecular level should lead to the determination of new potential therapeutic targets. Receptor activator of NFkappaB ligand (RANKL)/RANK/Osteoprotegerin (OPG) are the key regulators of bone metabolism both in normal and pathological condition, including prostate cancer bone metastases. In the present study, we demonstrated that human prostate cancer cell lines, DU145 and PC3 express biologically functional RANK. Indeed, soluble human RANKL (shRANKL, 100 ng/ml) treatment induced ERK 1/2, p38 and IkappaB phosphorylations in these cells. shRANKL administration also promoted DU145 and PC3 prostate cancer cell invasion in vitro. Whereas human OPG (hOPG) administration alone (100 ng/ml) had no marked effect, combined association of both agents abolished the RANKL-induced DU145 cell invasion. As RANKL had no direct effect on DU145 cell proliferation, the observed effects were indeed related to RANKL-induced cell migration. DU145 human prostate cancer cells promoted osteoclastogenesis of osteoclast precursors generated from mouse bone marrow. Moreover, DU145 cells produced soluble factor(s) that up-regulate the proliferation of MC3T3-E1 pre-osteoblasts through the activation of the ERK 1/2 and STAT3 signal transduction pathways. This stimulation of pre-osteoblast proliferation resulted in an increased local RANKL expression that can activate both osteoclasts/osteoclast precursors and prostate cancer cells, thus facilitating prostate cancer metastasis development in bone. We confirm that RANKL is a factor that facilitates metastasis to bone by acting as an activator of both osteoclasts and RANK-positive prostate cancer cells in our model. Furthermore, the present study provides the evidence that blocking RANKL-RANK interaction offer new therapeutic approach not only at the level of bone resorbing cells, but also by interfering with RANK-positive prostate cancer cells in the prostate cancer bone metastasis development.

Leptin resistance contributes to the pathogenesis of common obesity related metabolic diseases, including insulin resistance. However, the relationship between leptin and insulin resistance is not clearly established. Here, we show that induced hyperleptinemia by leptin infusion alters insulin signalling in rat liver. Leptin infusion clearly reduced the insulin or leptin dependent IRS-1/IRS-2 association to p85 regulatory subunit of PI 3-kinase. Leptin infusion also abolished STAT-3 phosphorylation in response to insulin or leptin and similar results were obtained for MAP-kinase phosphorylation. Hypothalamic leptin resistance was also induced by leptin infusion since leptin was unable to induce STAT-3 phosphorylation. These results provide evidence that induced hyperleptinemia can contribute to the onset of insulin resistance at least at the hepatic level.

International audience; Although Th17 cells are known to promote tissue inflammation and autoimmunity, their role during cancer progression remains elusive. Here, we showed that in vitro Th17 cells generated with the cytokines IL-6 and TGF-β expressed CD39 and CD73 ectonucleotidases, leading to adenosine release and the subsequent suppression of CD4(+) and CD8(+) T cell effector functions. The IL-6-mediated activation of the transcription factor Stat3 and the TGF-β-driven downregulation of Gfi-1 transcription factor were both essential for the expression of ectonucleotidases during Th17 cell differentiation. Stat3 supported whereas Gfi-1 repressed CD39 and CD73 expression by binding to their promoters. Accordingly, Th17 cells differentiated with IL-1β, IL-6, and IL-23 but without TGF-β did not express ectonucleotidases and were not immunosuppressive. Finally, adoptive transfer of Th17 cells induced by TGF-β and IL-6 promoted tumor growth in a CD39-dependent manner. Thus, ectonucleotidase expression supports the immunosuppressive fate of Th17 cells in cancer.