Negative feedback loops in inflammation

Entzündung ist im Prinzip eine schützende Reaktion des Organismus auf verschiedenste schädigende Reize. Kommt es aber zu einer übertriebenen oder ständigen Entzündungsreaktion, so werden durch den ursprünglich nutzbringenden Abwehrmechanismus Schädigungen verursacht. Chronische Entzündungen sind in die Pathogenese von vielen unterschiedlichen Erkrankungen involviert. Die Aufklärung von Mechanismen, die die adäquate Beendigung dieser Reaktion ermöglichen ist daher ein wichtiges Ziel der Entzündungsforschung. Neben verschiedenen Immunzellen tragen auch endotheliale Zellen aktiv zu einer Entzündung bei. Das Endothelium kleidet die Vaskulatur aus und bildet unter normalen Bedingungen eine anti-koagulante Oberfläche und eine Barriere zwischen dem Blutstrom und dem umliegenden Gewebe. Als Reaktion auf pro-inflammatorische Stimulation kommt es zu phenotypischen Veränderungen der endothelialen Zellen, die es den Immunzellen erlauben diese Schranke zu passieren und ins Gewebe einzuwandern. Auf molekularer Ebene sind diese Prozesse hauptsächlich durch verschiedene induzierbare Transkriptionsfaktoren (e.g. NF-κB, AP-1, NFAT) reguliert. Ihre Aktivierung führt nicht nur zur Expression einer Vielzahl von pro-inflammatorischen Genen, sondern auch von solchen, die in einem späteren Stadium einer Entzündungsreaktion deren Ende induzieren. Die Proteine, die von solchen Genen codiert werden, formen so genannte „negative feedback loops“. Um neue „negative feedback loops“ zu identifizieren wurden Gene selektiert, die in Endothelzellen aus der humane Nabelschnur (HUVECs) durch das pro-inflammatorische Cytokin IL-1 induziert werden und inhibitorisches Potential aufweisen. Im Rahmen dieser Diplomarbeit wurden einige diese Kandidatengene in passende Expressionsvektoren kloniert und ihr Einfluss auf Transkriptionsfaktoren und Signaltransduktionswege die bei der Entzündung eine wichtige Rolle spielen, mittels Reportergen Assays analysiert. Eines der untersuchten Gene, GG2-1, kodiert ein FLICE–inhibierendes, anti-apoptotisches Protein mit onkogenem Potential. Reportergen Assays in HEK293 zeigten, dass Überexpression von GG2-1 einen signifikanten inhibitorischen Effekt auf die Aktivität von NF-κB und AP-1 hat, wenn deren Aktivierung durch Transfektion der MAP3 Kinase MEKK1 erreicht wurde. Damit übereinstimmend wurde beobachtet, dass die MEKK1-induzierte Aktivität des IL-8-Promoters ebenfalls proportional zu steigenden Mengen von GG2-1 sinkt. Detektierung des Proteins mittels „Western Blotting“ zeigte, dass die Inhibierung der Reportergen Expression mit abnehmenden Proteinmengen von MEKK1 assoziiert ist. Die Konzentrationen von anderen MAP3 Kinasen wurde durch die Anwesenheit von GG2-1 nicht beeinflusst, was die Vermutung erlaubt, dass der beobachtete Effekt spezifisch für MEKK1 ist. Durch Co-Immunopräzipitaton wurde eine direkte Interaktion zwischen den beiden Proteinen ausgeschlossen. JAG1 kodiert Jagged-1, einen der vier Liganden von Notch 1. Aktives Notch setzt Signalkaskaden in Gang, die essentiell für die Zell-Zell Kommunikation sowie diverse Entwicklungsprozesse sind. Mutationen in JAG1 verursachen das Alagille Syndrom (AGS), eine autosomal dominate Erbkrankheit, die Auswirkungen auf verschiedenste Bereiche des Organismus hat. In endothelialen Zellen ist Jagged-1 in die Kontaktinhibierung involviert, wahrscheinlich durch Regulierung des Retinoblastoma Proteins (Rb). In anderen Zelltypen zeigt Jagged-1 onkogene Eigenschaften. Die durchgeführten Reportergen Assays zeigten keinen signifikanten inhibitorischen Effekt von Jagged-1 auf pro-inflammatorische wirkende Transkriptionsfaktoren. Im Gegensatz dazu wurde die Aktivität des Tumorsuppressors p53 stark unterdrückt, was vermuten lässt, dass Jagged-1 auch in endothelialen Zellen einen Einfluss auf onkogene Prozesse haben könnte.Inflammation is a beneficial response of the organism to harmful stimuli, but it can have detrimental effects if not resolved in time or occurs exaggerated. Chronic inflammatory conditions play a role in the pathogenesis of various severe diseases. Thus, mechanisms that cause shutting down of inflammation are important targets of studies. Endothelial cells lining the vasculature are actively contributing to inflammatory reactions. Upon pro-inflammatory stimulation they undergo phenotypic changes that enable the passage of immune cells through the endothelium to the side of injury. On the molecular level these processes are mainly regulated by inducible transcription factors like NF-κB, AP-1 and NFAT. Amongst their target genes are not only pro-inflammatory genes, but also genes encoding proteins that cause the resolution of the reaction at later stages. These gene products represent “negative feedback loops”. For the identification of novel negative feedback loops, some genes were selected that were found previously to be inducible in human vascular endothelial cells (HUVEC) by the pro-inflammatory cytokine IL-1, and are supposed to have repressive potential. These genes of interest were cloned into mammalian expression vectors and used to perform different reporter gene assays. One of the examined genes, GG2-1, encodes a member of the FLICE-inhibitory family of anti-apoptotic proteins that possesses oncogenic potential. In AP-1 and NF-κB reporter gene assays, the protein exhibited significant inhibitory effects on the transcription factors when their activation was achieved by co-transfection of MEKK1. Accordingly, the activity of the IL-8 promoter was suppressed by GG2-1. Western blotting revealed that the decrease of MEKK1 on the protein level correlated with increasing concentrations of GG2-1. This effect seemed to be specific for MEKK1, since protein levels of other MAP3K were not influenced by GG2-1 overexpression. Co-immunoprecipitation excluded direct protein interaction between the two proteins. Another target of studies was JAG1 that encodes one of the ligands of Notch1, Jagged-1. Notch signalling is involved in cell-cell communication and developmental processes. Mutations of JAG1 cause the Alagille syndrom (AGS), an autosomal dominant disorder affecting various systems of the body. In endothelial cells Jagged-1 plays a role in contact-inhibition by regulating the Retinoblastoma (Rb) protein, in other cell types Jagged-1 exhibits oncogenic properties. In reporter gene assays examining pro-inflammatory transcription factors, Jagged-1 did not show considerable inhibitory effects, whereas the activity of the tumor-suppressor p53 was significantly suppressed

Relationship of aerobic fitness and motor skills with memory and attention in preschoolers (Ballabeina): A cross-sectional and longitudinal study

BACKGROUND: The debate about a possible relationship between aerobic fitness and motor skills with cognitive development in children has recently re-emerged, because of the decrease in children's aerobic fitness and the concomitant pressure of schools to enhance cognitive performance. As the literature in young children is scarce, we examined the cross-sectional and longitudinal relationship of aerobic fitness and motor skills with spatial working memory and attention in preschool children. METHODS: Data from 245 ethnically diverse preschool children (mean age: 5.2 (0.6) years, girls: 49.4%) analyzed at baseline and 9 months later. Assessments included aerobic fitness (20 m shuttle run) and motor skills with agility (obstacle course) and dynamic balance (balance beam). Cognitive parameters included spatial working memory (IDS) and attention (KHV-VK). All analyses were adjusted for age, sex, BMI, migration status, parental education, native language and linguistic region. Longitudinal analyses were additionally adjusted for the respective baseline value. RESULTS: In the cross-sectional analysis, aerobic fitness was associated with better attention (r=0.16, p=0.03). A shorter time in the agility test was independently associated with a better performance both in working memory (r=-0.17, p=0.01) and in attention (r=-0.20, p=0.01). In the longitudinal analyses, baseline aerobic fitness was independently related to improvements in attention (r=0.16, p=0.03), while baseline dynamic balance was associated with improvements in working memory (r=0.15, p=0.04). CONCLUSIONS: In young children, higher baseline aerobic fitness and motor skills were related to a better spatial working memory and/or attention at baseline, and to some extent also to their future improvements over the following 9 months. TRIAL REGISTRATION: clinicaltrials.gov NCT0067454

Functional interplay of two SWI/SNF chromatin-remodeling accessory subunits during C. elegans development

El complexos SWI/SNF són remodeladors de cromatina dependents d’ATP que es troben altament conservats al llarg de l’evolució. Aquests complexos poden regular l’accessibilitat a una zona genòmica alterant l’estat de la cromatina i actuant com a reguladors transcripcionals. A més d’una subunitat enzimàtica central i diverses proteïnes que conformen el nucli, els complexos SWI/SNF incorporen subunitats accessòries que confereixen especificitat i varien segons el tipus cellular i el context del desenvolupament. Les poteïnes accessòries humanes BAF60a, BAF60b i BAF60c representen proteïnes paràlogues amb funcions especialitzades, de manera que mutacions en elles s’han relacionat amb diverses malalties (p. ex. càncer). Per tal de tenir una millor visió de les funcions de les unitats accessòries del complex SWI/SNF hem estudiat els paràlegs de les proteïnes BAF, codificades pels gens ham-3 i swsn-2.2. Hem investigat les funcions de ham-3 i swsn-2.2 en diversos teixits i processos del desenvolupament i hem observat que els dos gens són redundants en diversos contextos. Tot i així, i com ocorre amb els gens humans, HAM-3 i SWSN-2.2 també han adquirit funcions específiques.SWI/SNF complexes are ATP-dependent chromatin remodelers highly conserved through evolution. By altering the chromatin state, these complexes can regulate the accessibility of a given genomic region and thereby perform transcriptional regulation. Besides a central enzymatic subunit and various core proteins, SWI/SNF complexes incorporate accessory subunits that confer specificity to a given complex and vary depending on the cell type and developmental context. The human accessory proteins BAF60a, BAF60b and BAF60c represent paralog proteins with specialized functions, and mutations in the BAF60 genes are involved in human disease (e.g. cancer). To get closer insight in the functions of SWI/SNF accessory subunits, we studied C. elegans homologs of the BAF60 proteins, encoded by the paralog gene pair ham-3 and swsn-2.2. We investigated ham-3 and swsn-2.2 functions in various tissues and developmental processes and observed that the two genes act redundantly in many contexts. However, as their human counterparts, HAM-3 and SWSN-2.2 have also acquired specialized functions

Functional interplay of two paralogs encoding SWI/SNF chromatin-remodeling accessory subunits during Caenorhabditis elegans development

SWI/SNF ATP-dependent chromatin-remodeling complexes have been related to several cellular processes such as transcription, regulation of chromosomal stability, and DNA repair. The Caenorhabditis elegans gene ham-3 (also known as swsn-2.1) and its paralog swsn-2.2 encode accessory subunits of SWI/SNF complexes. Using RNA interference (RNAi) assays and diverse alleles we investigated whether ham-3 and swsn-2.2 have different functions during C. elegans development since they encode proteins that are probably mutually exclusive in a given SWI/SNF complex. We found that ham-3 and swsn-2.2 display similar functions in vulva specification, germline development, and intestinal cell proliferation, but have distinct roles in embryonic development. Accordingly, we detected functional redundancy in some developmental processes and demonstrated by RNA sequencing of RNAi-treated L4 animals that ham-3 and swsn-2.2 regulate the expression of a common subset of genes but also have specific targets. Cell lineage analyses in the embryo revealed hyper-proliferation of intestinal cells in ham-3 null mutants whereas swsn-2.2 is required for proper cell divisions. Using a proteomic approach, we identified SWSN-2.2-interacting proteins needed for early cell divisions, such as SAO-1 and ATX-2, and also nuclear envelope proteins such as MEL-28. swsn-2.2 mutants phenocopy mel-28 loss-of-function, and we observed that SWSN-2.2 and MEL-28 colocalize in mitotic and meiotic chromosomes. Moreover, we demonstrated that SWSN-2.2 is required for correct chromosome segregation and nuclear reassembly after mitosis including recruitment of MEL-28 to the nuclear periphery.Some strains were provided by the Caenorhabditis Genetics Center, which is funded by National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). I.E. was supported by an IDIBELL (Bellvitge Biomedical Research Institute) Ph.D. fellowship. J.C. is a Miguel Servet Researcher (ISCIII). This study was supported by a grant from the Instituto de Salud Carlos III (ISCIII) (PI12/01554), which is cofunded by FEDER funds/European Regional Development Fund—a way to build Europe. The P.A. laboratory was supported by funding from the Spanish Ministry of Economy and Competitiveness (BFU2013-42709-P).Peer Reviewe

The risk of posttraumatic stress disorder after trauma depends on traumatic load and the catechol-o-methyltransferase Val(158)Met polymorphism

Kolassa I-T, Kolassa S, Ertl V, Papassotiropoulos A, De Quervain DJ. The risk of posttraumatic stress disorder after trauma depends on traumatic load and the catechol-o-methyltransferase Val(158)Met polymorphism. Biological Psychiatry. 2010;67(4):304-308.BACKGROUND: The risk for posttraumatic stress disorder (PTSD) depends on the number of traumatic event types experienced in a dose-response relationship, but genetic factors are known to also influence the risk of PTSD. The catechol-O-methyltransferase (COMT) Val158Met polymorphism has been found to affect fear extinction and might play a role in the etiology of anxiety disorders. METHODS: Traumatic load and lifetime and current diagnosis of PTSD and COMT genotype were assessed in a sample of 424 survivors of the Rwandan Genocide living in the Nakivale refugee camp in southwestern Uganda. RESULTS: Higher numbers of different lifetime traumatic event types led to a higher prevalence of lifetime PTSD in a dose-response relationship. However, this effect was modulated by the COMT genotype: whereas Val allele carriers showed the typical dose-response relationship, Met/Met homozygotes exhibited a high risk for PTSD independently of the severity of traumatic load. CONCLUSIONS: The present findings indicate a gene-environment interaction between the human COMT Val158Met polymorphism and the number of traumatic event types experienced in the risk of developing PTSD

Genes influence emotional memory: A deletion variant of the alpha(2B)-adrenoceptor is related to enhanced intrusion symptoms in posttraumatic stress disorder

Kolassa I-T, Ertl V, Onyut LP, et al. Genes influence emotional memory: A deletion variant of the alpha(2B)-adrenoceptor is related to enhanced intrusion symptoms in posttraumatic stress disorder. Presented at the XXIX International Congress of Psychology

Association study of trauma load and SLC6A4 promoter polymorphism in posttraumatic stress disorder: evidence from survivors of the Rwandan genocide

Kolassa I-T, Ertl V, Eckart C, et al. Association study of trauma load and SLC6A4 promoter polymorphism in posttraumatic stress disorder: evidence from survivors of the Rwandan genocide. Journal of Clinical Psychiatry. 2010;71(05):543-547.OBJECTIVE: As exposure to different types of traumatic stressors increases, the occurrence of posttraumatic stress disorder (PTSD) increases. However, because some people exhibit either surprising resilience or high vulnerability, further influencing factors have been conjectured, such as gene-environment interactions. The SLC6A4 gene, which encodes serotonin transporter, has been identified as predisposing toward differential emotional processing between genotypes of its promoter polymorphism. METHOD: We investigated 408 refugees from the Rwandan genocide and assessed lifetime exposure to traumatic events, PTSD (according to DSM-IV) status, and genotype of the SLC6A4 promoter polymorphism. The study was conducted from March 2006 to February 2007. RESULTS: The prevalence of PTSD approached 100% when traumatic exposure reached extreme levels. However, persons homozygous for the short allele of the SLC6A4 promoter polymorphism showed no dose-response relationship but were at high risk for developing PTSD after very few traumatic events. This genotype influence vanished with increasing exposure to traumatic stressors. CONCLUSION: We find evidence for a gene-environment interplay for PTSD and show that genetic influences lose importance when environmental factors cause an extremely high trauma burden to an individual. In the future, it may be important to determine whether the effectiveness of therapeutic interventions in PTSD is also modulated by the SLC6A4 genotype

The SMARCD Family of SWI/SNF Accessory Proteins Is Involved in the Transcriptional Regulation of Androgen Receptor-Driven Genes and Plays a Role in Various Essential Processes of Prostate Cancer

Previous studies have demonstrated an involvement of chromatin-remodelling SWI/SNF complexes in the development of prostate cancer, suggesting both tumor suppressor and oncogenic activities. SMARCD1/BAF60A, SMARCD2/BAF60B, and SMARCD3/BAF60C are mutually exclusive accessory subunits that confer functional specificity and are components of all known SWI/SNF subtypes. To assess the role of SWI/SNF in prostate tumorigenesis, we studied the functions and functional relations of the SMARCD family members. Performing RNA-seq in LnCAP cells grown in the presence or absence of dihydrotestosterone, we found that the SMARCD proteins are involved in the regulation of numerous hormone-dependent AR-driven genes. Moreover, we demonstrated that all SMARCD proteins can regulate AR-downstream targets in androgen-depleted cells, suggesting an involvement in the progression to castration-resistance. However, our approach also revealed a regulatory role for SMARCD proteins through antagonization of AR-signalling. We further demonstrated that the SMARCD proteins are involved in several important cellular processes such as the maintenance of cellular morphology and cytokinesis. Taken together, our findings suggest that the SMARCD proteins play an important, yet paradoxical, role in prostate carcinogenesis. Our approach also unmasked the complex interplay of paralogue SWI/SNF proteins that must be considered for the development of safe and efficient therapies targeting SWI/SNF

The SMARCD Family of SWI/SNF Accessory Proteins Is Involved in the Transcriptional Regulation of Androgen Receptor-Driven Genes and Plays a Role in Various Essential Processes of Prostate Cancer

Previous studies have demonstrated an involvement of chromatin-remodelling SWI/SNF complexes in the development of prostate cancer, suggesting both tumor suppressor and oncogenic activities. SMARCD1/BAF60A, SMARCD2/BAF60B, and SMARCD3/BAF60C are mutually exclusive accessory subunits that confer functional specificity and are components of all known SWI/SNF subtypes. To assess the role of SWI/SNF in prostate tumorigenesis, we studied the functions and functional relations of the SMARCD family members. Performing RNA-seq in LnCAP cells grown in the presence or absence of dihydrotestosterone, we found that the SMARCD proteins are involved in the regulation of numerous hormone-dependent AR-driven genes. Moreover, we demonstrated that all SMARCD proteins can regulate AR-downstream targets in androgen-depleted cells, suggesting an involvement in the progression to castration-resistance. However, our approach also revealed a regulatory role for SMARCD proteins through antagonization of AR-signalling. We further demonstrated that the SMARCD proteins are involved in several important cellular processes such as the maintenance of cellular morphology and cytokinesis. Taken together, our findings suggest that the SMARCD proteins play an important, yet paradoxical, role in prostate carcinogenesis. Our approach also unmasked the complex interplay of paralogue SWI/SNF proteins that must be considered for the development of safe and efficient therapies targeting SWI/SNF

Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans

Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo- or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16::GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance