Alternative transcription factor sigma(B) is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate.

Rachid S, Ohlsen K, Wallner U, Hacker J, Hecker M, Ziebuhr W. Institut für Molekulare Infektionsbiologie, D-97070 Würzburg, Germany. Osmotic stress was found to induce biofilm formation in a Staphylococcus aureus mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor sigma(B), resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding sigma(B) completely restored the wild-type phenotype. The combined data suggest a critical role of sigma(B) in S. aureus biofilm regulation under environmental stress conditions.

The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections.

The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.

STAT3, p38 MAP Kinase and NF-{kappa}B Drive Unopposed Monocyte-dependent Fibroblast MMP-1 Secretion in Tuberculosis.

Tissue destruction characterizes infection with Mycobacterium tuberculosis (Mtb). Type I collagen provides the lung's tensile strength, is extremely resistant to degradation, but is cleaved by matrix metalloproteinase (MMP)-1. Fibroblasts potentially secrete quantitatively more MMP-1 than other lung cells. We investigated mechanisms regulating Mtb-induced collagenolytic activity in fibroblasts in vitro and in patients. Lung fibroblasts were stimulated with conditioned media from Mtb-infected monocytes (CoMTb). CoMTb induced sustained increased MMP-1 (74 versus 16 ng/ml) and decreased tissue inhibitor of metalloproteinase (TIMP)-1 (8.6 versus 22.3 ng/ml) protein secretion. CoMTb induced a 2.7-fold increase in MMP-1 promoter activation and a 2.5-fold reduction in TIMP-1 promoter activation at 24 hours (P = 0.01). Consistent with this, TIMP-1 did not co-localize with fibroblasts in patient granulomas. MMP-1 up-regulation and TIMP-1 down-regulation were p38 (but not extracellular signal–regulated kinase or c-Jun N-terminal kinase) mitogen-activated protein kinase–dependent. STAT3 phosphorylation was detected in fibroblasts in vitro and in tuberculous granulomas.STAT3 inhibition reduced fibroblast MMP-1 secretion by 60% (P = 0.046). Deletion of the MMP-1 promoter NF-B–binding site abrogated promoter induction in response to CoMTb. TNF-, IL-1ß, or Oncostatin M inhibition in CoMTb decreased MMP-1 secretion by 65, 63, and 25%, respectively. This cytokine cocktail activated the same signaling pathways in fibroblasts and induced MMP-1 secretion similar to that induced by CoMTb. This study demonstrates in a cellular model and in patients with tuberculosis that in addition to p38 and NF-B, STAT3 has a key role in driving fibroblast-dependent unopposed MMP-1 production that may be key in tissue destruction in patients.

A20 Regulation of NF-{kappa}B: Perspectives for Inflammatory Lung Disease.

Persistent activation of NF-B is central to the pathogenesis of many inflammatory lung disorders including Cystic Fibrosis, Asthma and Chronic Obstructive Pulmonary Disease. A20 is an endogenous negative regulator of NF-B signalling which has been widely described in autoimmune and inflammatory disorders including Diabetes and Crohn’s disease, but which has received little attention in terms of chronic lung disorders. This review examines the existing body of research on A20 regulation of NF-B signalling and details the mechanism and regulation of A20 action focusing, where possible, on pulmonary inflammation. A20 and its associated signalling molecules are highlighted as being of potential therapeutic interest for the treatment of inflammatory disorders and a proposed model of A20 activity in inflammatory lung disease is provided.

Akt-Mediated Proinflammatory Response of Mononuclear Phagocytes Infected with Burkholderia cenocepacia Occurs by a Novel GSK3 beta-Dependent, I kappa B Kinase-Independent Mechanism

The environmental bacterium Burkholderia cenocepacia causes opportunistic lung infections in immunocompromised individuals, particularly in patients with cystic fibrosis. Infections in these patients are associated with exacerbated inflammation leading to rapid decay of lung function, and in some cases resulting in cepacia syndrome, which is characterized by a fatal acute necrotizing pneumonia and sepsis. B. cenocepacia can survive intracellularly in macrophages by altering the maturation of the phagosome, but very little is known on macrophage responses to the intracellular infection. In this study, we have examined the role of the PI3K/Akt signaling pathway in B. cenocepacia-infected monocytes and macrophages. We show that PI3K/Akt activity was required for NF-kappa B activity and the secretion of proinflammatory cytokines during infection with B. cenocepacia. In contrast to previous observations in epithelial cells infected with other Gram-negative bacteria, Akt did not enhance I kappa B kinase or NF-kappa B p65 phosphorylation, but rather inhibited GSK3 beta, a negative regulator of NF-kappa B transcriptional activity. This novel mechanism of modulation of NF-kappa B activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection. The Journal of Immunology, 2011, 187: 635-643.

Nuclear factor-kappaB as a potential therapeutic target for the novel cytotoxic agent LC-1 in acute myeloid leukaemia

Nuclear factor-kappaB (NF-kappaB) has been implicated in a number of malignancies and has been suggested to be a potential molecular target in the treatment of leukaemia. This study demonstrated the constitutive activation of NF-kappaB in human myeloid blasts and a clear correlation between NF-kappaB expression and in vitro cytoprotection. High NF-kappaB expression was found in many of the poor prognostic acute myeloid leukaemia (AML) subtypes, such as French-American-British classification M0 and M7, and the poor cytogenetic risk group. The in vitro effects of LC-1, a novel dimethylamino-parthenolide analogue, were assessed in 62 primary untreated AML samples. LC-1 was found to be cytotoxic to AML cells in a dose-dependent manner, mediated through the induction of apoptosis. The median drug concentration necessary to kill 50% of the cells was 4.5 micromol/l for AML cells, compared with 12.8 micromol/l for normal marrow cells. LC-1 was shown to reduce the five individual human NF-kappaB Rel proteins in a dose-dependent manner. The subsequent inhibition of many NF-kappaB-regulated cytokines was also demonstrated. Importantly, sensitivity to LC-1 was correlated with the basal NF-kappaB activity. Consequently, LC-1 treatment provides a proof of principle for the use of NF-kappaB inhibitors in the treatment of AML.

Potentiation of Inflammatory CXCL8 Signalling Sustains Cell Survival in PTEN-deficient Prostate Carcinoma

Background: Inflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised. <br/><br/>Objective: To determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa. <br/><br/>Design, setting and participants: Experiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten(+/-)) mice harbouring inactivation of one PTEN allele. <br/><br/>Interventions: Small interfering RNA (siRNA)- or small hairpin RNA (shRNA)-directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions. <br/><br/>Outcome measurements and statistical analysis: Changes in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays. <br/><br/>Results and limitations: Transient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten(+/-) murine prostate tissue relative to normal epithelium in wild-type PTEN (Pten(WT)) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study. <br/><br/>Conclusions: PTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Les azasulfurylpeptides : synthèse, analyse conformationnelle et applications biologiques

Les azasulfurylpeptides sont des mimes peptidiques auxquels le carbone en position alpha et le carbonyle d’un acide aminé sont respectivement remplacés par un atome d’azote et un groupement sulfonyle (SO2). Le but premier de ce projet a été de développer une nouvelle méthode de synthèse de ces motifs, également appelés N-aminosulfamides. À cette fin, l’utilisation de sulfamidates de 4-nitrophénol s’est avérée importante dans la synthèse des azasulfuryltripeptides, permettant le couplage d’hydrazides avec l’aide d’irradiation aux micro-ondes (Chapitre 2). Par la suite, en quantité stoechiométrique d’une base et d’un halogénure d’alkyle, les azasulfurylglycines (AsG) formés peuvent être chimiosélectivement alkylés afin d’y insérer diverses chaînes latérales. Les propriétés conformationnelles des N-aminosulfamides à l’état solide ont été élucidées grâce à des études cristallographiques par rayons X : elles possèdent une structure tétraédrique autour de l’atome de soufre, des traits caractéristiques des azapeptides et des sulfonamides, ainsi que du potentiel à favoriser la formation de tours gamma (Chapitre 3). Après le développement d’une méthode de synthèse des N-aminosulfamides en solution, une approche combinatoire sur support solide a également été élaborée sur la résine amide de Rink afin de faciliter la génération d’une librairie d’azasulfurylpeptides. Cette étude a été réalisée en employant le growth hormone releasing peptide 6 (GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2). Ce dernier est un hexapeptide possédant une affinité pour deux récepteurs, le growth hormone secretagogue receptor 1a (GHS-R1a) et le récepteur cluster of differenciation 36 (CD36). Une affinité sélective envers le récepteur CD36 confère des propriétés thérapeutiques dans le traitement de la dégénérescence maculaire liée à l’âge (DMLA). Six analogues d’azasulfurylpeptides de GHRP-6 utilisés comme ligands du CD36 ont été synthétisés sur support solide, mettant en évidence le remplacement du tryptophane à la position 4 de GHRP-6 (Chapitre 4). Les analogues de GHRP-6 ont été ensuite analysés pour leur capacité à moduler les effets de la fonction et de la cascade de signalisation des ligands spécifiques au Toll-like receptor 2 (TLR2), en collaboration avec le Professeur Huy Ong du département de Pharmacologie à la Faculté de Pharmacie de l’Université de Montréal. Le complexe TLR2-TLR6 est reconnu pour être co-exprimé et modulé par CD36. En se liant au CD36, certains ligands de GHRP-6 ont eu un effet sur la signalisation du TLR2. Par exemple, les azasulfurylpeptides [AsF(4-F)4]- et [AsF(4-MeO)4]-GHRP-6 ont démontré une capacité à empêcher la surproduction du monoxyde d’azote (NO), un sous-produit réactif formé suite à l’induction d’un signal dans les macrophages par des ligands spécifiques liés au TLR2, tel le fibroblast-stimulating lipopeptide 1 (R-FSL-1) et l’acide lipotéichoïque (LTA). En addition, la sécrétion du tumor necrosis factor alpha (TNFa) et du monocyte chemoattractant protein 1 (MCP-1), ainsi que l’activation du nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), ont été réduites. Ces résultats démontrent le potentiel de ces azasulfurylpeptides à pouvoir réguler le rôle du TLR2 qui déclenche des réponses inflammatoires et immunitaires innées (Perspectives). Finalement, le potentiel des azasulfurylpeptides d’inhiber des métallo-bêta-lactamases, tels le New-Delhi Metallo-bêta-lactamase 1 (NDM-1), IMP-1 et le Verona Integron-encoded Metallo-bêta-lactamase 2 (VIM-2), a été étudié en collaboration avec le Professeur James Spencer de l’Université de Bristol (Royaumes-Unis). Certains analogues ont été des inhibiteurs micromolaires du IMP-1 (Perspectives). Ces nouvelles voies de synthèse des azasulfurylpeptides en solution et sur support solide devraient donc permettre leur utilisation dans des études de relations structure-activité avec différents peptides biologiquement actifs. En plus d'expandre l'application des azasulfurylpeptides comme inhibiteurs d'enzymes, cette thèse a révélé le potentiel de ces N-aminosulfamides à mimer les structures secondaires peptidiques, tels que les tours gamma. À cet égard, l’application des azasulfurylpeptides a été démontrée par la synthèse de ligands du CD36 présentant des effets modulateurs sur le TLR2. Compte tenu de leur synthèse efficace et de leur potentiel en tant qu’inhibiteurs, les azasulfurylpeptides devraient trouver une large utilisation dans les sciences de peptides pour des applications dans la médecine et de la chimie biologique.

Na+-glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: Involvement of hepatocyte nuclear factor-1 alpha expression and activity

Mutations in Na+-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1 alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1 alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1 alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1 alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1 alpha mRNA expression (similar to 50%) and binding of nuclear proteins to a HNF-1 consensus motif (similar to 100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1 alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1 alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1 alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.