UCAGenomiX related publications

Du to our strong expertise in "omics" experiments and in microRNAs topics we decided to separate into 3 categories the related publications into which the Functional genomics Platform of Nice-Sophia-Antipolis is involved :
  1. Expression studies (DNA microarrays and high-throughput sequencing experiments)
  2. MicroRNA studies
  3. Miscellaneous

34 publications found

1. Optimizing drug development in oncology by clinical trial simulation: Why and how?
Brief Bioinform. 2017 May 29. doi: 10.1093/bib/bbx055
Gal J, Milano G, Ferrero JM, Saâda-Bouzid E, Viotti J, Chabaud S, Gougis P, Le Tourneau C, Schiappa R, Paquet A, Chamorey E
Epidemiology and Biostatistics Unit, Centre Antoine Lacassagne, 33 avenue de Valombrose, 06189 Nice, France IPMC

In therapeutic research, the safety and efficacy of pharmaceutical products are necessarily tested on humans via clinical trials after an extensive and expensive preclinical development period. Methodologies such as computer modeling and clinical trial simulation (CTS) might represent a valuable option to reduce animal and human assays. The relevance of these methods is well recognized in pharmacokinetics and pharmacodynamics from the preclinical phase to postmarketing. However, they are barely used and are poorly regarded for drug approval, despite Food and Drug Administration and European Medicines Agency recommendations. The generalization of CTS could be greatly facilitated by the availability of software for modeling biological systems, by clinical trial studies and hospital databases. Data sharing and data merging raise legal, policy and technical issues that will need to be addressed. Development of future molecules will have to use CTS for faster development and thus enable better patient management. Drug activity modeling coupled with disease modeling, optimal use of medical data and increased computing speed should allow this leap forward. The realization of CTS requires not only bioinformatics tools to allow interconnection and global integration of all clinical data but also a universal legal framework to protect the privacy of every patient. While recognizing that CTS can never replace 'real-life' trials, they should be implemented in future drug development schemes to provide quantitative support for decision-making. This in silico medicine opens the way to the P4 medicine: predictive, preventive, personalized and participatory.
Pubmed link : 28575140

2. Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region.
Nat Commun. 2016 Feb 17;7:10730. doi: 10.1038/ncomms10730.
Saintamand A, Vincent-Fabert C, Garot A, Rouaud P, Oruc Z, Magnone V, Cogné M, Denizot Y
Université de Limoges, CRIBL, UMR CNRS 7276, Limoges 87025, France. CNRS et Université de Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097, Sophia Antipolis 06560, France. Institut Universitaire de France, Paris 75231, France.

The IgH 3' regulatory region (3'RR) controls class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The mouse 3'RR contains four enhancer elements with hs1,2 flanked by inverted repeated sequences and the centre of a 25-kb palindrome bounded by two hs3 enhancer inverted copies (hs3a and hs3b). hs4 lies downstream of the palindrome. In mammals, evolution maintained this unique palindromic arrangement, suggesting that it is functionally significant. Here we report that deconstructing the palindromic IgH 3'RR strongly affects its function even when enhancers are preserved. CSR and IgH transcription appear to be poorly dependent on the 3'RR architecture and it is more or less preserved, provided 3'RR enhancers are present. By contrast, a 'palindromic effect' significantly lowers VH germline transcription, AID recruitment and SHM. In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance.
Pubmed link : 26883548

3. miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways.
Nat Commun. 2015 Sep 18;6:8386. doi: 10.1038/ncomms9386.
Chevalier B, Adamiok A, Mercey O, Revinski DR, Zaragosi LE, Pasini A, Kodjabachian L, Barbry P, Marcet B
1CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), UMR-7275, 660 route des Lucioles, 06560 Sophia-Antipolis, France. 2University of Nice-Sophia-Antipolis (UNS), Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Valbonne, 06560 Sophia-Antipolis, France. 3Aix-Marseille Université, CNRS, UMR7288, Institut de Biologie du Développement de Marseille (IBDM), 13288 Marseille, France.

Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways.
Pubmed link : 26381333

4. BMP signalling controls the construction of vertebrate mucociliary epithelia.
Development. 2015 Jul 1;142(13):2352-63. doi: 10.1242/dev.118679. Epub 2015 Jun 19.
Cibois M, Luxardi G, Chevalier B, Thomé V, Mercey O, Zaragosi LE, Barbry P, Pasini A, Marcet B, Kodjabachian L
1Aix-Marseille Université, CNRS, IBDM, Marseille 13288, France. 2CNRS, IPMC, Sophia-Antipolis 06560, France. 3CNRS, IPMC, Sophia-Antipolis 06560, France University of Nice Sophia Antipolis (UNS), IPMC, Sophia-Antipolis 06560, France. 4Aix-Marseille Université, CNRS, IBDM, Marseille 13288, France laurent.kodjabachian@univ-amu.fr.

Despite the importance of mucociliary epithelia in animal physiology, the mechanisms controlling their establishment are poorly understood. Using the developing Xenopus epidermis and regenerating human upper airways, we reveal the importance of BMP signalling for the construction of vertebrate mucociliary epithelia. In Xenopus, attenuation of BMP activity is necessary for the specification of multiciliated cells (MCCs), ionocytes and small secretory cells (SSCs). Conversely, BMP activity is required for the proper differentiation of goblet cells. Our data suggest that the BMP and Notch pathways interact to control fate choices in the developing epidermis. Unexpectedly, BMP activity is also necessary for the insertion of MCCs, ionocytes and SSCs into the surface epithelium. In human, BMP inhibition also strongly stimulates the formation of MCCs in normal and pathological (cystic fibrosis) airway samples, whereas BMP overactivation has the opposite effect. This work identifies the BMP pathway as a key regulator of vertebrate mucociliary epithelium differentiation and morphogenesis.
Pubmed link : 26092849

5. Tetraspanin CD63 acts as a pro-metastatic factor via β-catenin stabilization.
Int J Cancer. 2015 May 15;136(10):2304-15. doi: 10.1002/ijc.29296. Epub 2014 Nov 11.
Seubert B, Cui H, Simonavicius N, Honert K, Schäfer S, Reuning U, Heikenwalder M, Mari B, Krüger A
Institute for Experimental Oncology and Therapy Research and Institute of Molecular Immunology, Klinikum rechts der Isar der Technische Universität München, München, Germany.

The tetraspanin CD63 is implicated in pro-metastatic signaling pathways but, so far, it is unclear, how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, β-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. β-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of β-catenin upstream regulators PI3K/AKT or GSK3β could rescue the mesenchymal phenotype underlining the importance of the β-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis whereas CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity.
Pubmed link : 25354204

6. Forkhead Box F1 represses cell growth and inhibits COL1 and ARPC2 expression in lung fibroblasts in vitro.
Am J Physiol Lung Cell Mol Physiol. 2014 Dec 1;307(11):L838-47. doi: 10.1152/ajplung.00012.2014. Epub 2014 Sep 26.
Melboucy-Belkhir S, Pradère P, Tadbiri S, Habib S, Bacrot A, Brayer S, Mari B, Besnard V, Mailleux A, Guenther A, Castier Y, Mal H, Crestani B, Plantier L
1INSERM UMR1152, Labex Inflamex, Paris, France; 2INSERM UMR1152, Labex Inflamex, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, DHU FIRE, Service de Pneumologie A, Paris, France; Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; 3Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR7275, Valbonne, France; 4University of Giessen Lung Centre, Department of Internal Medicine, Giessen, Germany; Lung Clinic Waldhof-Elgershausen, Greifenstein, Germany; 5Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Service de Chirurgie Thoracique et Transplantation Pulmonaire, Paris, France; 6Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Service de Pneumologie B et Transplantation Pulmonaire, Paris, France; and. 7INSERM UMR1152, Labex Inflamex, Paris, France; Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Service de Physiologie-Explorations Fonctionnelles, Paris, France. laurent.plantier@inserm.fr.

Aberrant expression of master phenotype regulators or alterations in their downstream pathways in lung fibroblasts may play a central role in idiopathic pulmonary fibrosis (IPF). Interrogating IPF fibroblast transcriptome datasets, we identified Forkhead Box F1 (FOXF1), a DNA-binding protein required for lung development, as a candidate actor in IPF. Thus we determined FOXF1 expression levels in fibroblasts cultured from normal or IPF lungs in vitro, and explored FOXF1 functions in these cells using transient and stable loss-of-function and gain-of-function models. FOXF1 mRNA and protein were expressed at higher levels in IPF fibroblasts compared with normal fibroblasts (mRNA: +44%, protein: +77%). Immunohistochemistry showed FOXF1 expression in nuclei of bronchial smooth muscle cells, endothelial cells, and lung fibroblasts including fibroblastic foci of IPF lungs. In normal lung fibroblasts, FOXF1 repressed cell growth and expression of collagen-1 (COL1) and actin-related protein 2/3 complex, subunit 2 (ARPC2). ARPC2 knockdown inhibited cell growth and COL1 expression, consistent with FOXF1 acting in part through ARPC2 repression. In IPF fibroblasts, COL1 and ARPC2 repression by FOXF1 was blunted, and FOXF1 did not repress growth. FOXF1 expression was induced by the antifibrotic mediator prostaglandin E2 and repressed by the profibrotic cytokine transforming growth factor-β1 in both normal and IPF lung fibroblasts. Ex vivo, FOXF1 knockdown conferred CCL-210 lung fibroblasts the ability to implant in uninjured mouse lungs. In conclusion, FOXF1 functions and regulation were consistent with participation in antifibrotic pathways. Alterations of pathways downstream of FOXF1 may participate to fibrogenesis in IPF fibroblasts.
Pubmed link : 25260753

7. An ABC of ciliogenesis.
Nat Cell Biol. 2014 Sep;16(9):826-7. doi: 10.1038/ncb3034.
Barbry P, Zaragosi LE
CNRS and University Nice Sophia Antipolis, Institute of Molecular and Cellular Pharmacology, 660 Route des Lucioles, F6560 Sophia Antipolis, France.

ABCC4 is a member of the ATP-binding cassette transporter family known to transport prostaglandin E2 and other molecules across cellular membranes. A mutation in ABCC4 is now shown to cause defects in ciliogenesis, revealing a role for prostaglandin signalling in regulating cilia dynamics.
Pubmed link : 25174819

8. Blocking lipid synthesis overcomes tumor regrowth and metastasis after antiangiogenic therapy withdrawal.
Cell Metab. 2014 Aug 5;20(2):280-94. doi: 10.1016/j.cmet.2014.05.022. Epub 2014 Jul 10.
Sounni NE, Cimino J, Blacher S, Primac I, Truong A, Mazzucchelli G, Paye A, Calligaris D, Debois D, De Tullio P, Mari B, De Pauw E, Noel A
1Laboratory of Tumor and Developmental Biology, GIGA-CANCER, University of Liege, 4000 Liege, Belgium. Electronic address: nesounni@ulg.ac.be. 2Laboratory of Tumor and Developmental Biology, GIGA-CANCER, University of Liege, 4000 Liege, Belgium; Mass Spectrometry Laboratory, GIGA-R, Department of Chemistry, University of Liege, 4000 Liege, Belgium. 3Laboratory of Tumor and Developmental Biology, GIGA-CANCER, University of Liege, 4000 Liege, Belgium. 4Mass Spectrometry Laboratory, GIGA-R, Department of Chemistry, University of Liege, 4000 Liege, Belgium. 5Laboratory of Drug Research Center, University of Liege, 4000 Liege, Belgium. 6UMR-7275 CNRS, University of Nice Sophia-Antipolis, Institute of Molecular and Cellular Pharmacology, 06560 Valbonne, France.

The molecular mechanisms responsible for the failure of antiangiogenic therapies and how tumors adapt to these therapies are unclear. Here, we applied transcriptomic, proteomic, and metabolomic approaches to preclinical models and provide evidence for tumor adaptation to vascular endothelial growth factor blockade through a metabolic shift toward carbohydrate and lipid metabolism in tumors. During sunitinib or sorafenib treatment, tumor growth was inhibited and tumors were hypoxic and glycolytic. In sharp contrast, treatment withdrawal led to tumor regrowth, angiogenesis restoration, moderate lactate production, and enhanced lipid synthesis. This metabolic shift was associated with a drastic increase in metastatic dissemination. Interestingly, pharmacological lipogenesis inhibition with orlistat or fatty acid synthase downregulation with shRNA inhibited tumor regrowth and metastases after sunitinib treatment withdrawal. Our data shed light on metabolic alterations that result in cancer adaptation to antiangiogenic treatments and identify key molecules involved in lipid metabolism as putative therapeutic targets.
Pubmed link : 25017943

9. The small heat shock protein B8 (HSPB8) confers resistance to bortezomib by promoting autophagic removal of misfolded proteins in multiple myeloma cells.
Oncotarget. 2014 Aug 15;5(15):6252-66.
Hamouda MA, Belhacene N, Puissant A, Colosetti P, Robert G, Jacquel A, Mari B, Auberger P, Luciano F
1INSERM U1065, C3M, Team 2, Nice, France; Université de Nice Sophia-Antipolis; Equipe labellisée par la Ligue Nationale Contre le Cancer. 2Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, USA. 3UMR7275 CNRS-UNS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France. 4INSERM U1065, C3M, Team 2, Nice, France; Université de Nice Sophia-Antipolis; Equipe labellisée par la Ligue Nationale Contre le Cancer; These authors contributed equally to this work.

Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. U266-resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells. Elimination of velcade-induced poly-ubiquitinated proteins and protein aggregates was drastically stimulated in the resistant cells and correlated with increased cell survival. Inhibition of the lysosomal activity in velcade-resistant cells resulted in an increase of cell aggregates and decrease survival, indicating that aggregates are eliminated through lysosomal degradation. In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival.
Pubmed link : 25051369

10. Phenotypic and genotypic characterization of azacitidine-sensitive and resistant SKM1 myeloid cell lines.
Oncotarget. 2014 Jun 30;5(12):4384-91.
Cluzeau T, Dubois A, Jacquel A, Luciano F, Renneville A, Preudhomme C, Karsenti JM, Mounier N, Rohrlich P, Raynaud S, Mari B, Robert G, Auberger P
1INSERM U1065, Mediterranean Center for Molecular Medicine (C3M), Nice, France; University of Nice, Nice, France. 2INSERM U1065, Mediterranean Center for Molecular Medicine (C3M), Nice, France; University of Nice, Nice, France; Department of Clinical hematology and Transplantation, CHU of Nice, Nice, France.

In the present study, we provide a comparative phenotypic and genotypic analysis of azacitidine-sensitive and resistant SKM-1 cell lines. Morphologically, SKM1-R exhibited increase in cell size that accounts for by enhanced ploidy in a majority of cells as shown by cell cycle and karyotype analysis. No specific Single Nucleotide Polymorphism (SNP) alteration was found in SKM1-R cells compared to their SKM1-S counterpart. Comparative pangenomic profiling revealed the up-regulation of a panel of genes involved in cellular movement, cell death and survival and down-regulation of genes required for cell to cell signaling and free radical scavenging in SKM1-R cells. We also searched for mutations frequently associated with myelodysplastic syndromes (MDS) and found that both cell lines harbored mutations in TET2, ASLX1 and TP53. Collectively, our data show that despite their different morphological and phenotypic features, SKM1-S and SKM1-R cells exhibited similar genotypic characteristics. Finally, pangenomic profiling identifies new potential pathways to be targeted to circumvent AZA-resistance. In conclusion, SKM1-R cells represent a valuable tool for the validation of new therapeutic intervention in MDS.
Pubmed link : 24962689

11. ⁹⁹mTcO₄--, auger-mediated thyroid stunning: dosimetric requirements and associated molecular events.
PLoS One. 2014 Mar 24;9(3):e92729. doi: 10.1371/journal.pone.0092729. eCollection 2014.
Cambien B, Franken PR, Lamit A, Mauxion T, Richard-Fiardo P, Guglielmi J, Crescence L, Mari B, Pourcher T, Darcourt J, Bardiès M, Vassaux G
Laboratoire TIRO, UMRE 4320, iBEB, DSV, CEA, Nice, France; Université de Nice-Sophia Antipolis, Nice, France; Centre Antoine Lacassagne, Department of nuclear medicine, Nice, France. 2UMR 1037 INSERM/UPS, Centre de Recherche en Cancérologie, Toulouse, France. 3Université de Nice-Sophia Antipolis, Nice, France; Institut de Pharmacologie Moléculaire et Cellulaire-IPMC, CNRS UMR 7275, Sophia Antipolis, France.

Low-energy Auger and conversion electrons deposit their energy in a very small volume (a few nm3) around the site of emission. From a radiotoxicological point of view the effects of low-energy electrons on normal tissues are largely unknown, understudied, and generally assumed to be negligible. In this context, the discovery that the low-energy electron emitter, 99mTc, can induce stunning on primary thyrocytes in vitro, at low absorbed doses, is intriguing. Extrapolated in vivo, this observation suggests that a radioisotope as commonly used in nuclear medicine as 99mTc may significantly influence thyroid physiology. The aims of this study were to determine whether 99mTc pertechnetate (99mTcO4-) is capable of inducing thyroid stunning in vivo, to evaluate the absorbed dose of 99mTcO4- required to induce this stunning, and to analyze the biological events associated/concomitant with this effect. Our results show that 99mTcO4--mediated thyroid stunning can be observed in vivo in mouse thyroid. The threshold of the absorbed dose in the thyroid required to obtain a significant stunning effect is in the range of 20 Gy. This effect is associated with a reduced level of functional Na/I symporter (NIS) protein, with no significant cell death. It is reversible within a few days. At the cellular and molecular levels, a decrease in NIS mRNA, the generation of double-strand DNA breaks, and the activation of the p53 pathway are observed. Low-energy electrons emitted by 99mTc can, therefore, induce thyroid stunning in vivo in mice, if it is exposed to an absorbed dose of at least 20 Gy, a level unlikely to be encountered in clinical practice. Nevertheless this report presents an unexpected effect of low-energy electrons on a normal tissue in vivo, and provides a unique experimental setup to understand the fine molecular mechanisms involved in their biological effects.
Pubmed link : 24663284

12. Predicting HIV-1 broadly neutralizing antibody epitope networks using neutralization titers and a novel computational method.
BMC Bioinformatics. 2014 Mar 19;15:77. doi: 10.1186/1471-2105-15-77.
Evans MC, Phung P, Paquet AC, Parikh A, Petropoulos CJ, Wrin T, Haddad M
1Monogram Biosciences Inc,, 345 Oyster Point Blvd,, South San Francisco, CA 94080, USA. mojganhd@yahoo.com.

BACKGROUND: Recent efforts in HIV-1 vaccine design have focused on immunogens that evoke potent neutralizing antibody responses to a broad spectrum of viruses circulating worldwide. However, the development of effective vaccines will depend on the identification and characterization of the neutralizing antibodies and their epitopes. We developed bioinformatics methods to predict epitope networks and antigenic determinants using structural information, as well as corresponding genotypes and phenotypes generated by a highly sensitive and reproducible neutralization assay.282 clonal envelope sequences from a multiclade panel of HIV-1 viruses were tested in viral neutralization assays with an array of broadly neutralizing monoclonal antibodies (mAbs: b12, PG9,16, PGT121 - 128, PGT130 - 131, PGT135 - 137, PGT141 - 145, and PGV04). We correlated IC50 titers with the envelope sequences, and used this information to predict antibody epitope networks. Structural patches were defined as amino acid groups based on solvent-accessibility, radius, atomic depth, and interaction networks within 3D envelope models. We applied a boosted algorithm consisting of multiple machine-learning and statistical models to evaluate these patches as possible antibody epitope regions, evidenced by strong correlations with the neutralization response for each antibody. RESULTS: We identified patch clusters with significant correlation to IC50 titers as sites that impact neutralization sensitivity and therefore are potentially part of the antibody binding sites. Predicted epitope networks were mostly located within the variable loops of the envelope glycoprotein (gp120), particularly in V1/V2. Site-directed mutagenesis experiments involving residues identified as epitope networks across multiple mAbs confirmed association of these residues with loss or gain of neutralization sensitivity. CONCLUSIONS: Computational methods were implemented to rapidly survey protein structures and predict epitope networks associated with response to individual monoclonal antibodies, which resulted in the identification and deeper understanding of immunological hotspots targeted by broadly neutralizing HIV-1 antibodies.
Pubmed link : 24646213

13. A decade of HIV-1 drug resistance in the United States: trends and characteristics in a large protease/reverse transcriptase and co-receptor tropism database from 2003 to 2012.
Antivir Ther. 2014;19(4):435-41. doi: 10.3851/IMP2748. Epub 2014 Feb 12.
Paquet AC, Solberg OD, Napolitano LA, Volpe JM, Walworth C, Whitcomb JM, Petropoulos CJ, Haddad M
Monogram Biosciences, South San Francisco, CA, USA.

BACKGROUND: Drug resistance testing and co-receptor tropism determination are key components of the management of antiretroviral therapy for HIV-1-infected individuals. The purpose of this study was to examine trends of HIV-1 resistance and viral evolution in the past decade by surveying a large commercial patient testing database. METHODS: Temporal trends of drug resistance, viral fitness and co-receptor usage among samples submitted for routine phenotypic and genotypic resistance testing to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), as well as for tropism determination were investigated. RESULTS: Within 62,397 resistant viruses reported from 2003 to 2012, we observed a decreasing trend in the prevalence of three-class resistance (from 25% to 9%) driven by decreased resistance to PIs (43% to 21%) and NRTIs (79% to 57%), while observing a slight increase in NNRTI resistance (68% to 75%). The prevalence of CXCR4-mediated entry among tropism testing samples (n=52,945) declined over time from 47% in 2007 to 40% in 2012. A higher proportion of CXCR4-tropic viruses was observed within samples with three-class resistance (50%) compared with the group with no resistance (36%). CONCLUSIONS: Decreased prevalence of three-class resistance and increased prevalence of one-class resistance was observed within samples reported between 2003 and 2012. The fraction of CXCR4-tropic viruses has decreased over time; however, CXCR4 usage was more prevalent among multi-class-resistant samples, which may be due to the more advanced disease stage of treatment-experienced patients. These trends have important implications for clinical practice and future drug discovery and development.
Pubmed link : 24518099

14. Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance.
Cancer Res. 2012 Apr 15;72(8):2140-50. doi: 10.1158/0008-5472.CAN-11-3940. Epub 2012 Mar 2.
Brahimi-Horn MC, Ben-Hail D, Ilie M, Gounon P, Rouleau M, Hofman V, Doyen J, Mari B, Shoshan-Barmatz V, Hofman P, Pouysségur J, Mazure NM
Institute of Developmental Biology and Cancer Research, University of Nice, CNRS-UMR 6543, Centre Antoine Lacassagne, Nice, France.

Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae. In these cells, we found that a major mitochondrial protein regulating metabolism and apoptosis, the voltage-dependent anion channel 1 (VDAC1), was linked to chemoresistance when in a truncated (VDAC1-ΔC) but active form. The formation of truncated VDAC1, which had a similar channel activity and voltage dependency as full-length, was hypoxia-inducible factor-1 (HIF-1)-dependent and could be inhibited in the presence of the tetracycline antibiotics doxycycline and minocycline, known inhibitors of metalloproteases. Its formation was also reversible upon cell reoxygenation and associated with cell survival through binding to the antiapoptotic protein hexokinase. Hypoxic cells containing VDAC1-ΔC were less sensitive to staurosporine- and etoposide-induced cell death, and silencing of VDAC1-ΔC or treatment with the tetracycline antibiotics restored sensitivity. Clinically, VDAC1-ΔC was detected in tumor tissues of patients with lung adenocarcinomas and was found more frequently in large and late-stage tumors. Together, our findings show that via induction of VDAC1-ΔC, HIF-1 confers selective protection from apoptosis that allows maintenance of ATP and cell survival in hypoxia. VDAC1-ΔC may also hold promise as a biomarker for tumor progression in chemotherapy-resistant patients.
Pubmed link : 22389449

15. Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR.
Retrovirology. 2012 Feb 8;9:13. doi: 10.1186/1742-4690-9-13.
Nakamura M, Basavarajaiah P, Rousset E, Beraud C, Latreille D, Henaoui IS, Lassot I, Mari B, Kiernan R
Laboratoire de Régulation des Gènes, Institut de Génétique Humaine, CNRS UPR1142, Montpellier, France.

BACKGROUND: Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. RESULTS: Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. CONCLUSION: These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.
Pubmed link : 22316138

16. The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats.
Cell Res. 2011 Jul;21(7):1028-38. doi: 10.1038/cr.2011.40. Epub 2011 Mar 22.
Simonet T, Zaragosi LE, Philippe C, Lebrigand K, Schouteden C, Augereau A, Bauwens S, Ye J, Santagostino M, Giulotto E, Magdinier F, Horard B, Barbry P, Waldmann R, Gilson E
Laboratoire de Biologie Moléculaire de la Cellule-UMR 5239 CNRS/ENS Lyon/ Université Lyon, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, Lyon 69364, France.

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.
Pubmed link : 21423270

17. In utero exposure to maternal diabetes impairs vascular expression of prostacyclin receptor in rat offspring.
Diabetes. 2010 Oct;59(10):2597-602.
Duong Van Huyen JP, Vessieres E, Perret C, Troise A, Prince S, Guihot AL, Barbry P, Henrion D, Bruneval P, Laurent S, Lelievre-Pegorier M, Fassot C
INSERM U872, Centre de Recherche des Cordeliers, Universite's Pierre et Marie Curie et Paris-Descartes, Paris, France.

OBJECTIVE: To evaluate modifications of arterial structure, gene expression, and function in our model of rats exposed to maternal diabetes. RESEARCH DESIGN AND METHODS: Morphometric analyses of elastic vessels structure and determination of thoracic aortic gene expression profile with oligonucleotide chips (Agilent, G4130, 22k) were performed before the onset of established hypertension (3 months). RESULTS: Arterial parameters of in situ fixed thoracic aorta were not significantly different between control mother offspring and diabetic mother offspring (DMO). The aortic gene expression profile of DMO is characterized by modifications of several members of the arachidonic acid metabolism including a twofold underexpression of prostacyclin receptor, which could contribute to decreased vasodilatation. This was confirmed by ex vivo experiments on isolated aortic rings. Pharmacological studies on conscious rats showed that systolic blood pressure decline in response to a PGI(2) analog was impaired in DMO rats. CONCLUSIONS: These results suggest an abnormal vascular fetal programming of prostacyclin receptor in rats exposed in utero to maternal hyperglycemia that is associated with impaired vasodilatation and may be involved in the pathophysiology of hypertension in this model.
Pubmed link : 20622163

18. Activin a plays a critical role in proliferation and differentiation of human adipose progenitors.
Diabetes. 2010 Oct;59(10):2513-21. Epub 2010 Jun 8.
Zaragosi LE, Wdziekonski B, Villageois P, Keophiphath M, Maumus M, Tchkonia T, Bourlier V, Mohsen-Kanson T, Ladoux A, Elabd C, Scheideler M, Trajanoski Z, Takashima Y, Amri EZ, Lacasa D, Sengenes C, Ailhaud G, Clément K, Bouloumie A, Kirkland JL, Dani C
UMR6543 Centre National de la Recherche Scientifique, Institute of Developmental Biology and Cancer, University of Nice Sophia-Antipolis, Nice, France.

OBJECTIVE: Growth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process. RESEARCH DESIGN AND METHODS: Expression of INHBA/activin A was investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression. RESULTS: INHBA/activin A is expressed by adipose progenitors from various fat depots, and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes, respectively, adipocyte differentiation via the C/EBPβ-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared with the expression levels in lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue. CONCLUSIONS: Altogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages that are located in adipose tissue regulate adipose progenitor self-renewal through activin A.
Pubmed link : 20530742

19. A Western-like fat diet is sufficient to induce a gradual enhancement in fat mass over generations.
J Lipid Res. 2010 Aug;51(8):2352-61
Massiera F, Barbry P, Guesnet P, Joly A, Luquet S, Moreilhon-Brest C, Mohsen-Kanson T, Amri EZ, Ailhaud G
Universite de Nice Sophia-Antipolis, CNRS, IBDC, UMR 6543, 06107 Nice, France.

The prevalence of obesity has steadily increased over the last few decades. During this time, populations of industrialized countries have been exposed to diets rich in fat with a high content of linoleic acid and a low content of alpha-linolenic acid compared with recommended intake. To assess the contribution of dietary fatty acids, male and female mice fed a high-fat diet (35% energy as fat, linoleic acid:alpha-linolenic acid ratio of 28) were mated randomly and maintained after breeding on the same diet for successive generations. Offspring showed, over four generations, a gradual enhancement in fat mass due to combined hyperplasia and hypertrophy with no change in food intake. Transgenerational alterations in adipokine levels were accompanied by hyperinsulinemia. Gene expression analyses of the stromal vascular fraction of adipose tissue, over generations, revealed discrete and steady changes in certain important players, such as CSF3 and Nocturnin. Thus, under conditions of genome stability and with no change in the regimen over four generations, we show that a Western-like fat diet induces a gradual fat mass enhancement, in accordance with the increasing prevalence of obesity observed in humans.
Pubmed link : 20410018

20. A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates.
J Clin Invest. 2010 Apr 1;120(4):1125-39
Blin G, Nury D, Stefanovic S, Neri T, Guillevic O, Brinon B, Bellamy V, Rucker-Martin C, Barbry P, Bel A, Bruneval P, Cowan C, Pouly J, Mitalipov S, Gouadon E, Binder P, Hagege A, Desnos M, Renaud JF, Menasche P, Puceat M
INSERM U633, Avenir Program, Embryonic Stem Cells and Cardiogenesis, Evry, France.

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.
Pubmed link : 20335662

21. Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium.
BMC Mol Biol. 2009 Dec 24;10:111.
Emboule L, Daigle F, Meyer DF, Mari B, Pinarello V, Sheikboudou C, Magnone V, Frutos R, Viari A, Barbry P, Martinez D, Lefrançois T, Vachiery N
UMR 15 CIRAD-INRA, Controle des maladies animales exotiques et emergentes, Site de Duclos, Prise d'Eau 97170, Petit Bourg, Guadeloupe. loic.emboule@cirad.fr

BACKGROUND: Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. RESULTS: We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. CONCLUSIONS: We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.
Pubmed link : 20034374

22. The caspase-cleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice.
EMBO J. 2009 Aug 19;28(16):2449-60. Epub 2009 Jul 9.
Marchetti S, Gamas P, Belhacene N, Grosso S, Pradelli LA, Colosetti P, Johansen C, Iversen L, Deckert M, Luciano F, Hofman P, Ortonne N, Khemis A, Mari B, Ortonne JP, Ricci JE, Auberger P
INSERM, U895, Centre Mediterraneen de Medecine Moleculaire (C3M), Team 2, Nice, France.

We showed previously that Lyn is a substrate for caspases, a family of cysteine proteases, involved in the regulation of apoptosis and inflammation. Here, we report that expression of the caspase-cleaved form of Lyn (LynDeltaN), in mice, mediates a chronic inflammatory syndrome resembling human psoriasis. Genetic ablation of TNF receptor 1 in a LynDeltaN background rescues a normal phenotype, indicating that LynDeltaN mice phenotype is TNF-alpha-dependent. The predominant role of T cells in the disease occurring in LynDeltaN mice was highlighted by the distinct improvement of LynDeltaN mice phenotype in a Rag1-deficient background. Using pan-genomic profiling, we also established that LynDeltaN mice show an increased expression of STAT-3 and inhibitory members of the NFkappaB pathway. Accordingly, LynDeltaN alters NFkappaB activity underlying a link between inhibition of NFkappaB and LynDeltaN mice phenotype. Finally, analysis of Lyn expression in human skin biopsies of psoriatic patients led to the detection of Lyn cleavage product whose expression correlates with the activation of caspase 1. Our data identify a new role for Lyn as a regulator of psoriasis through its cleavage by caspases.
Pubmed link : 19590497

23. Dax-1 knockdown in mouse embryonic stem cells induces loss of pluripotency and multilineage differentiation.
Stem Cells. 2009 Jul;27(7):1529-37.
Khalfallah O, Rouleau M, Barbry P, Bardoni B, Lalli E
Institut de Pharmacologie Moleculaire et Cellulaire CNRS UMR 6097, Valbonne, France.

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here, we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of beta-catenin-dependent transcription in mouse ES cells. Furthermore, Dax-1 knockdown induced upregulation of multilineage differentiation markers, and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis, we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly, the great majority of these genes are upregulated, showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells, as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1-regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis, respectively.
Pubmed link : 19530134

24. Isoform-specific contribution of protein kinase C to prion processing.
Mol Cell Neurosci. 2008 Nov;39(3):400-10. Epub 2008 Jul 29.
Alfa Cisse M, Louis K, Braun U, Mari B, Leitges M, Slack BE, Fisher A, Auberger P, Checler F, Vincent B
Institut de Pharmacologie Moleculaire et Cellulaire du CNRS, UMR6097, UNSA, Equipe labellisee Fondation pour la Recherche Medicale, Sophia-Antipolis, Valbonne, France.

The cellular prion protein (PrP(c)) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrP(c). First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCalpha lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCalpha-, PKCdelta- and PKCepsilon-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCepsilon-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCalpha, delta and epsilon, but not zeta, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production.
Pubmed link : 18722532

25. Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia-warm reperfusion injury.
Hepatology. 2008 Jan;47(1):177-85.
Defamie V, Laurens M, Patrono D, Devel L, Brault A, Saint-Paul MC, Yiotakis A, Barbry P, Gugenheim J, Crenesse D, Dive V, Huet, PM, Mari B
Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, UMR6097, Universite de Nice-Sophia Antipolis, France.

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after two conditions of CI (4oC for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, while gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10µM) was added to the UW solution and livers were preserved for 42 hours (4oC), then reperfused for 1 hour in Krebs solution (37oC), containing 20% erythrocytes. Liver viability parameters were recorded and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2 to 60% of liver biopsies, mean: 26 ± 9%) to isolated necrotic hepatocytes as found in treated livers (0.2 to 12%, mean 3 ± 2%) (p<0.05). In conclusion, these data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.
Pubmed link : 18008367

26. Effect of caspase inhibition on thymic apoptosis in hemorrhagic shock.
J Invest Surg. 2007 Mar-Apr;20(2):97-103
Bini R, Cursio R, Belhacene N, Giudicelli J, Ferrua B, Olivero G, Auberger P, Mari B, Gugenheim J, Cotogni P
Chirurgia d'Urgenza, Dipartimento di Discipline Medico-Chirurgiche, Universit a di Torino, Torino, Italy.

In hemorrhagic shock (HS) an increased thymic apoptosis (TA) was described. The aim of this study was to evaluate the effect of administration of the caspase inhibitor N-benzyloxy-carbonil-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) during the resuscitation phase on TA, organ dysfunctions, and tumor necrosis factor (TNF)-alpha release in HS. Forty rats were randomly assigned to four groups: no HS/resuscitation (sham); HS/resuscitation with shed blood and normal saline (control); HS/resuscitation with shed blood and phosphate-buffered solution (PBS) (vehicle); and HS/resuscitation with shed blood and Z-VAD-FMK (inhibitor). Rats were subjected to HS by blood removal to a MAP of 35-40 mmHg. After a 1-h shock period, the animals were resuscitated according to the protocol. At 1 and 3 h after resuscitation, transaminases, creatinine, urea, lipase, TNF-alpha, and TA were evaluated. Our study showed that a nonlethal HS is early able to induce organ dysfunctions and increased TA. Administration of Z-VAD-FMK did not significantly decrease organ dysfunctions, while it induced a significant TNF-alpha release. TA was significantly reduced by Z-VAD-FMK after 1 h, but not after 3 h. Our results suggest that postinjury caspase inhibition does not attenuate organ dysfunctions, and also does not permanently reduce TA induced by HS and resuscitation in rats.
Pubmed link : 17454394

27. Delta-subunit confers novel biophysical features to alpha beta gamma-human epithelial sodium channel (ENaC) via a physical interaction.
J Biol Chem. 2006 Mar 24;281(12):8233-41. Epub 2006 Jan 19.
Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ
Department of Anesthesiology, University of Alabama at Birmingham, Alabama 35205, USA.

Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of delta-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the delta-subunit may form multimeric channels with alpha beta gamma-ENaC. Reverse transcription-PCR revealed that delta-ENaC is co-expressed with alpha beta gamma-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that delta-ENaC is co-expressed with alpha-, beta-, and gamma-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for delta alpha beta gamma-ENaC differed from those of both alpha beta gamma- and delta beta gamma-ENaC (n = 6). The contribution of the delta subunit to P(Li)/P(Na) ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of delta alpha beta gamma-ENaC differed from those of alpha beta gamma- and delta beta gamma-ENaC. Co-immunoprecipitation of delta-ENaC with alpha- and gamma-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of delta-ENaC with other subunits could account for heterogeneity of native epithelial channels.
Pubmed link : 16423824

28. Hypoxia-inducible factor 1{alpha} is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells.
J Cell Biol. 2005 Jul 4;170(1):49-59. Epub 2005 Jun 27.
Busca R, Berra E, Gaggioli C, Khaled M, Bille K, Marchetti B, Thyss R, Fitsialos G, Larribere L, Bertolotto C, Virolle T, Barbry P, Pouyssegur J, Ponzio G, Ballotti R
INSERM U597, Biologie et physiopathologie des cellules melanocytaires, Faculty of Medicine, 06107 Nice cedex 2, France.

In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell-specific manner and increases the expression of a functional hypoxia-inducible factor 1alpha (HIF1alpha) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF "silencing" abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.
Pubmed link : 15983061

29. Cooperation of amphiregulin and insulin-like growth factor-1 inhibits Bax- and Bad-mediated apoptosis via a protein kinase C-dependent pathway in non-small cell lung cancer cells.
J Biol Chem. 2005 May 20;280(20):19757-67. Epub 2005 Mar 14.
Hurbin A, Coll JL, Dubrez-Daloz L, Mari B, Auberger P, Brambilla C, Favrot MC
Groupe de Recherche sur le Cancer du Poumon, INSERM U578, Institut Albert Bonniot, La Tronche, France.

Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.
Pubmed link : 15767261

30. CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes.
J Physiol. 2005 May 1;564(Pt 3):671-82. Epub 2005 Mar 3.
Nagel G, Barbry P, Chabot H, Brochiero E, Hartung K, Grygorczyk R
Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.
Pubmed link : 15746174

31. Tumor cell-mediated induction of the stromal factor stromelysin-3 requires heterotypic cell contact-dependent activation of specific protein kinase C isoforms.
J Biol Chem. 2005 Jan 14;280(2):1272-83. Epub 2004 Oct 27.
Louis K, Guerineau N, Fromigue O, Defamie V, Collazos A, Anglard P, Shipp MA, Auberger P, Joubert D, Mari B
INSERM U526, IFR50, Faculte de Medecine Pasteur, 06107 Nice, France.

Stromelysin-3 (ST3, MMP-11) has been shown to be strongly overexpressed in stromal fibroblasts of most invasive human carcinomas. However, the molecular mechanisms leading to ST3 expression in nonmalignant fibroblasts remain unknown. The aim of the present study was to analyze the signaling pathways activated in normal pulmonary fibroblasts after their interaction with non-small cell lung cancer (NSCLC) cells and leading to ST3 expression. The use of selective signaling pathway inhibitors showed that conventional and novel protein kinase Cs (PKC) were required for ST3 induction, whereas Src kinases exerted a negative control. We observed by both conventional and real time confocal microscopy that green fluorescent protein-tagged PKCalpha and PKCepsilon, but not PKCdelta, transfected in fibroblasts, accumulate selectively at the cell-cell contacts between fibroblasts and tumor cells. In agreement, RNAi-mediated depletion of PKCalpha and PKCepsilon, but not PKCdelta significantly decreased co-culture-dependent ST3 production. Finally, a tetracycline-inducible expression model allowed us to confirm the central role of these PKC isoforms and the negative regulatory function of c-Src in the control of ST3 expression. Altogether, our data emphasize signaling changes occurring in the tumor microenvironment that may define new stromal targets for therapeutic intervention.
Pubmed link : 15509588

32. Escherichia coli cytotoxic necrotizing factor 1 inhibits intestinal epithelial wound healing in vitro after mechanical injury.
Infect Immun. 2004 Oct;72(10):5733-40.
Brest P, Turchi L, Le'Negrate G, Berto F, Moreilhon C, Mari B, Ponzio G, Hofman P
Equipe INSERM 0215, Faculte de Medecine, Nice, France.

Cytotoxic necrotizing factor type 1 (CNF1) from Escherichia coli activates the small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42) by catalyzing their deamidation at a specific glutamine residue. Since RhoA, Rac, and Cdc42 play a pivotal role in cell migration during the early phase of wound repair, we investigated whether CNF1 was able to interfere with wound healing in intestinal epithelial monolayers (T84 cells). After mechanical injury, we found that CNF1 blocks epithelial wound repair within 48 h. This effect was characterized by cell elongation and filopodium formation on the leading edge, in association with permanent phosphorylation of the focal adhesion kinase (FAK) via Rho activation. Moreover, inhibition of Rho kinase with Y-27632 decreased CNF1-mediated permanent FAK phosphorylation, leading to complete restitution of wound repair within 24 h. In addition, we found that CNF1 induced upregulation of mitogen-activated protein kinases (MAPK) activation. Moreover, activation of Rac and MAPK by CNF1 increased matrix metalloproteinase 9 expression in wounded T84 monolayers. Taken together, these results provide evidence that CNF1 strongly impairs intestinal epithelial wound healing.
Pubmed link : 15385472

33. Active stromelysin-3 (MMP-11) increases MCF-7 survival in three-dimensional Matrigel culture via activation of p42/p44 MAP-kinase.
Int J Cancer. 2003 Sep 1;106(3):355-63.
Fromigue O, Louis K, Wu E, Belhacene N, Loubat A, Shipp M, Auberger P, Mari B
INSERM U526, Equipe labellisee LNC, Faculte de Medecine Pasteur, Avenue de Valombrose, 06107 Nice Cedex 2, France.

Stromelysin-3 (ST3) has the characteristic structure of matrix metalloproteinases (MMP), but its substrate specificity and pattern of expression differ markedly from that of other MMP family members. ST3 was originally isolated on the basis of its expression in primary breast cancers and has been shown to be overexpressed in virtually all primary carcinomas, suggesting that ST3 participates in the initial development of epithelial malignancies. Recent data using murine models reported that ST3 expression was able to increase tumor take by suppressing cell apoptosis. Our present goal was to set up an in vitro model in which we could study this new function. For this purpose, we analyzed survival of MCF-7 transfectants expressing either wild-type or catalytically inactive ST3 (ST3wt or ST3cat-) in three-dimensional (3-D) culture conditions by inclusion in Matrigel. In such conditions, that mimic the in vivo microenvironment, we found a marked decrease in the percentage of cell death when active ST3 was expressed (ST3wt transfectants vs. ST3cat- or vector only transfectants) as assessed by FACS and TUNEL analysis. The addition of batimastat, a broad spectrum MMP inhibitor, reversed the increased cell survival in ST3wt transfectants, confirming that ST3 enzymatic activity was required for this effect. Finally, we analyzed the expression of anti- and pro-apoptotic proteins as well as activation of cell survival pathways and we found that ST3-mediated cell survival was accompanied by activation of both p42/p44 MAPK and AKT. Our data confirm and extend the anti-apoptotic function of ST3 and provide a useful model to dissect this new role and identify new physiological substrates.
Pubmed link : 12845673

34. TWIK-2, an inactivating 2P domain K+ channel
J Biol Chem. 2000 Sep 15;275(37):28722-30.
Patel AJ, Maingret F, Magnone V, Fosset M, Lazdunski M, Honore E
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France.

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.
Pubmed link : 10887187