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1.  NOX1 is responsible for cell death through STAT3 activation in hyperoxia and is associated with the pathogenesis of Acute Respiratory Distress Syndrome 
Reactive oxygen species (ROS) contribute to alveolar cell death in Acute Respiratory Distress Syndrome (ARDS) and we previously demonstrated that NOX1-derived ROS contributed to hyperoxia-induced alveolar cell death in mice. The study investigates whether NOX1 expression is modulated in epithelial cells concomitantly to cell death and associated to STAT3 signaling in the exudative phase of ARDS. In addition, the role of STAT3 activation in NOX1-dependent epithelial cell death was confirmed by using a lung epithelial cell line and in mice exposed to hyperoxia. NOX1 expression, cell death and STAT3 staining were evaluated in the lungs of control and ARDS patients by immunohistochemistry. In parallel, a stable NOX1-silenced murine epithelial cell line (MLE12) and NOX1-deficient mice were used to characterize signalling pathways. In the present study, we show that NOX1 is detected in alveolar epithelial cells of ARDS patients in the exudative stage. In addition, increased alveolar epithelial cell death and phosphorylated STAT3 are observed in ARDS patients and associated with NOX1 expression. Phosphorylated STAT3 is also correlated with TUNEL staining. We also confirmed that NOX1-dependent STAT3 activation participates to alveolar epithelial cell death. Silencing and acute inhibition of NOX1 in MLE12 led to decreased cell death and cleaved-caspase 3 induced by hyperoxia. Additionally, hyperoxia-induced STAT3 phosphorylation is dependent on NOX1 expression and associated with cell death in MLE12 and mice. This study demonstrates that NOX1 is involved in human ARDS pathophysiology and is responsible for the damage occurring in alveolar epithelial cells at least in part via STAT3 signalling pathways.
PMCID: PMC3925898  PMID: 24551274
NOX1; ARDS; cell death; hyperoxia; STAT3; ROS
2.  TLR-mediated Up-regulation of Serum Retroviral gp70 is Controlled by the Sgp Loci of Lupus-prone Mice 
Journal of autoimmunity  2010;35(2):153-159.
The endogenous retroviral envelope glycoprotein, gp70, implicated in murine systemic lupus erythematosus (SLE), has been considered to be a product of xenotropic, polytropic (PT) and modified PT (mPT) endogenous retroviruses. It is secreted by hepatocytes like an acute phase protein, but its response is under a genetic control. Given critical roles of TLR7 and TLR9 in the pathogenesis of SLE, we assessed their contribution to the acute phase expression of serum gp70, and defined a pivotal role of the Sgp3 (serum gp70 production 3) and Sgp4 loci in this response. Our results demonstrated that serum levels of gp70 were up-regulated in lupus-prone NZB mice injected with TLR7 or TLR9 agonist at levels comparable to those induced by injection of IL-1, IL-6 or TNF. In addition, studies of C57BL/6 Sgp3 and/or Sgp4 congenic mice defined the major roles of these two loci in up-regulated production of serum gp70 during acute phase responses. Finally, the analysis of Sgp3 congenic mice strongly suggests the presence of at least two distinct genetic factors in the Sgp3 interval, one of which controlled the basal-level expression of xenotropic, PT and mPT gp70 and the other which controlled the up-regulated production of xenotropic and mPT gp70 during acute phase responses. Our results uncovered an additional pathogenic role of TLR7 and TLR9 in murine lupus nephritis by promoting the expression of nephritogenic gp70 autoantigen. Furthermore, they revealed the involvement of multiple regulatory genes for the expression of gp70 autoantigen under steady-state and inflammatory conditions in lupus-prone mice.
doi:10.1016/j.jaut.2010.06.001
PMCID: PMC2926185  PMID: 20619604
Toll-like receptor; Systemic lupus erythematosus; Endogenous retrovirus; Acute phase protein
3.  DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells 
Immunome Research  2010;6:10.
Background
The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).
Results
Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.
Conclusions
The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.
doi:10.1186/1745-7580-6-10
PMCID: PMC3000836  PMID: 21092113
4.  Selective Up-regulation of Intact, but not Defective env RNAs of Endogenous Modified Polytropic Retrovirus by the Sgp3 Locus of Lupus-prone Mice1 
Endogenous retroviruses are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Since four different classes of endogenous retroviruses, i.e. ecotropic, xenotropic, polytropic (PT) or modified polytropic (mPT), are expressed in mice, we investigated the possibility that a particular class of endogenous retroviruses is associated with the development of murine SLE. We observed more than 15-fold increased expression of mPT env (envelope) RNA in livers of all four lupus-prone mice, as compared with those of nine non-autoimmune strains of mice. This was not the case for the three other classes of retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many strains of mice expressed two defective mPT env transcripts which carry a deletion in the env sequence of the 3’ portion of the gp70 surface protein and the 5’ portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to non-autoimmune strains of mice, all four lupus-prone mice expressed abundant levels of intact mPT env transcripts, but only low or non-detectable levels of the mutant env transcripts. The Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice was responsible for the selective up-regulation of the intact mPT env RNA. Finally, we observed that single-stranded RNA-specific TLR7 played a critical role in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor through activating TLR7 for the development of autoimmune responses in mice predisposed to SLE.
doi:10.4049/jimmunol.0900263
PMCID: PMC2792900  PMID: 19494335
Autoimmunity; Systemic Lupus Erythematosus; Retrovirus; Rodent
5.  Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site 
Nucleic Acids Research  2009;37(8):2514-2528.
Nucleosome depletion at transcription start sites (TSS) has been documented genome-wide in multiple eukaryotic organisms. However, the mechanisms that mediate this nucleosome depletion and its functional impact on transcription remain largely unknown. We have studied these issues at human MHC class II (MHCII) genes. Activation-induced nucleosome free regions (NFR) encompassing the TSS were observed at all MHCII genes. Nucleosome depletion was exceptionally strong, attaining over 250-fold, at the promoter of the prototypical HLA-DRA gene. The NFR was induced primarily by the transcription factor complex that assembles on the conserved promoter-proximal enhancer situated upstream of the TSS. Functional analyses performed in the context of native chromatin demonstrated that displacing the NFR without altering the sequence of the core promoter induced a shift in the position of the TSS. The NFR thus appears to play a critical role in transcription initiation because it directs correct TSS positioning in vivo. Our results provide support for a novel mechanism in transcription initiation whereby the position of the TSS is controlled by nucleosome eviction rather than by promoter sequence.
doi:10.1093/nar/gkp116
PMCID: PMC2677874  PMID: 19264803
6.  Transcription-coupled deposition of histone modifications during MHC class II gene activation 
Nucleic Acids Research  2007;35(10):3431-3441.
Posttranslational histone modifications associated with actively expressed genes are generally believed to be introduced primarily by histone-modifying enzymes that are recruited by transcription factors or their associated co-activators. We have performed a comprehensive spatial and temporal analyses of the histone modifications that are deposited upon activation of the MHC class II gene HLA-DRA by the co-activator CIITA. We find that transcription-associated histone modifications are introduced during two sequential phases. The first phase precedes transcription initiation and is characterized exclusively by a rapid increase in histone H4 acetylation over a large upstream domain. All other modifications examined, including the acetylation and methylation of several residues in histone H3, are restricted to short regions situated at or within the 5′ end of the gene and are established during a second phase that is concomitant with ongoing transcription. This second phase is completely abrogated when elongation by RNA polymerase II is blocked. These results provide strong evidence that transcription elongation can play a decisive role in the deposition of histone modification patterns associated with inducible gene activation.
doi:10.1093/nar/gkm214
PMCID: PMC1904273  PMID: 17478518
7.  Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity 
Nucleic Acids Research  2007;35(9):3053-3063.
FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3′ flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity.
doi:10.1093/nar/gkm092
PMCID: PMC1888826  PMID: 17452369
8.  Expression of RAB4B, a protein governing endocytic recycling, is co-regulated with MHC class II genes 
Nucleic Acids Research  2006;35(2):595-605.
The small GTPase RAB4 regulates endocytic recycling, a process that contributes to Major Histocompatibility Complex (MHC)-mediated antigen presentation by specialized antigen presenting cells (APC) of the immune system. The gene encoding the RAB4B isoform of RAB4 was singled out by two complementary genome-wide screens. One of these consisted of a computer scan to identify genes containing characteristic MHC class II-related regulatory sequences. The second was the use of chromatin immunoprecipitation coupled to microarrays (ChIP-on-chip) to identify novel targets of a transcriptional co-activator called the MHC class II transactivator (CIITA). We show that the RAB4B gene is regulated by a typical MHC class II-like enhancer that is controlled directly by both CIITA and the multiprotein transcription factor complex known as the MHC class II enhanceosome. RAB4B expression is thus activated by the same regulatory machinery that is known to be essential for the expression of MHC class II genes. This molecular link between the transcriptional activation of RAB4B and MHC class II genes implies that APC boost their antigen presentation capacity by increasing RAB4-mediated endocytic recycling.
doi:10.1093/nar/gkl980
PMCID: PMC1802633  PMID: 17175541
9.  Stress-Activated Protein Kinase Pathway Functions To Support Protein Synthesis and Translational Adaptation in Response to Environmental Stress in Fission Yeast 
Eukaryotic Cell  2005;4(11):1785-1793.
The stress-activated protein kinase (SAPK) pathway plays a central role in coordinating gene expression in response to diverse environmental stress stimuli. We examined the role of this pathway in the translational response to stress in Schizosaccharomyces pombe. Exposing wild-type cells to osmotic stress (KCl) resulted in a rapid but transient reduction in protein synthesis. Protein synthesis was further reduced in mutants disrupting the SAPK pathway, including the mitogen-activated protein kinase Wis1 or the mitogen-activated protein kinase Spc1/Sty1, suggesting a role for these stress response factors in this translational control. Further polysome analyses revealed a role for Spc1 in supporting translation initiation during osmotic stress, and additionally in facilitating translational adaptation. Exposure to oxidative stress (H2O2) resulted in a striking reduction in translation initiation in wild-type cells, which was further reduced in spc1− cells. Reduced translation initiation correlated with phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in wild-type cells. Disruption of Wis1 or Spc1 kinase or the downstream bZip transcription factors Atf1 and Pap1 resulted in a marked increase in eIF2α phosphorylation which was dependent on the eIF2α kinases Hri2 and Gcn2. These findings suggest a role for the SAPK pathway in supporting translation initiation and facilitating adaptation to environmental stress in part through reducing eIF2α phosphorylation in fission yeast.
doi:10.1128/EC.4.11.1785-1793.2005
PMCID: PMC1287851  PMID: 16278445
10.  Definition of a Short Region of XPG Necessary for TFIIH Interaction and Stable Recruitment to Sites of UV Damage 
Molecular and Cellular Biology  2004;24(24):10670-10680.
XPG is the human endonuclease that cuts 3′ to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.
doi:10.1128/MCB.24.24.10670-10680.2004
PMCID: PMC533987  PMID: 15572672
11.  A Fission Yeast Homolog of Int-6, the Mammalian Oncoprotein and eIF3 Subunit, Induces Drug Resistance when Overexpressed 
Molecular Biology of the Cell  2000;11(11):3993-4003.
Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3. Disruption of the murine Int-6 gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood. Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex. Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization. Fission yeast cells lacking the int6 gene grew slowly. This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles. Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.
PMCID: PMC15052  PMID: 11071922
12.  Sum1, a Component of the Fission Yeast eIF3 Translation Initiation Complex, Is Rapidly Relocalized During Environmental Stress and Interacts with Components of the 26S Proteasome 
Molecular Biology of the Cell  2002;13(5):1626-1640.
Eukaryotic translation initiation factor 3 (eIF3) is a multisubunit complex that plays a central role in translation initiation. We show that fission yeast Sum1, which is structurally related to known eIF3 subunits in other species, is essential for translation initiation, whereas its overexpression results in reduced global translation. Sum1 is associated with the 40S ribosome and interacts stably with Int6, an eIF3 component, in vivo, suggesting that Sum1 is a component of the eIF3 complex. Sum1 is cytoplasmic under normal growth conditions. Surprisingly, Sum1 is rapidly relocalized to cytoplasmic foci after osmotic and thermal stress. Int6 and p116, another putative eIF3 subunit, behave similarly, suggesting that eIF3 is a dynamic complex. These cytoplasmic foci, which additionally comprise eIF4E and RNA components, may function as translation centers during environmental stress. After heat shock, Sum1 additionally colocalizes stably with the 26S proteasome at the nuclear periphery. The relationship between Sum1 and the 26S proteasome was further investigated, and we find cytoplasmic Sum1 localization to be dependent on the 26S proteasome. Furthermore, Sum1 interacts with the Mts2 and Mts4 components of the 26S proteasome. These data indicate a functional link between components of the structurally related eIF3 translation initiation and 26S proteasome complexes.
doi:10.1091/mbc.01-06-0301
PMCID: PMC111132  PMID: 12006658

Results 1-12 (12)