Modeling time delay in the NFκB signaling pathway following low dose IL-1 stimulation

Stimulation of human epithelial cells with IL-1 (10 ng/ml) + UVB radiation results in sustained NFκB activation caused by continuous IKKβ phosphorylation. We have recently published a strictly reduced ordinary differential equation model elucidating the involved mechanisms. Here, we compare model extensions for low IL-1 doses (0.5 ng/ml), where delayed IKKβ phosphorylation is observed. The extended model including a positive regulatory element, most likely auto-ubiquitination of TRAF6, reproduces the observed experimental data most convincingly. The extension is shown to be consistent with the original model and contains very sensitive processes which may serve as potential intervention targets

Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore–microtubule attachments

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)–microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT–MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT–MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 “phospho-switch” that temporally regulates KT–MT attachment stability.National Institutes of Health (U.S.) (NIH/National Institute of General Medical Sciences grant GM088313)National Institutes of Health (U.S.) (NIH grant 5R01-GM078373)American Heart Association (grant-in-aid 10GRNT4230026)National Institutes of Health (U.S.) (NIH grant GM51542)Fundação para a Ciência e a Tecnologia (FCT grant REEQ/564/BIO/2005 (EU-FEDER), POCI 2010

Mechanism of PP2A-mediated IKK beta dephosphorylation: a systems biological approach.

BACKGROUND: Biological effects of nuclear factor-kappaB (NF kappaB) can differ tremendously depending on the cellular context. For example, NF kappaB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NF kappaB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NF kappaB, involving a lack of inhibitor of kappaB (I kappaB alpha) protein reappearance. Permanent activation of the upstream kinase IKK beta results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized I kappaB alpha. RESULTS: To investigate the mechanism underlying the general PP2A-mediated tuning of IKK beta phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKK beta and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKK beta phosphorylation thus allowing for I kappaB alpha resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKK beta phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of I kappaB alpha reappearance, is not a direct UVB action but requires instrumentality. CONCLUSION: The model developed here can be used as a reliable building block of larger NF kappa B models and offers comprehensive simplification potential for future modeling of NF kappa B signaling. It gives more insight into the newly discovered mechanisms for IKK deactivation and allows for substantiated predictions and investigation of different hypotheses. The evidence of constitutive activity of PP2Ac at the IKK complex provides new insights into the feedback regulation of NF kappa B, which is crucial for the development of new anti-cancer strategies

Severe Congenital Heart Defects and Cerebral Palsy

Objective: To report the prevalence of cerebral palsy (CP) in children with severe congenital heart defects (sCHD) and the outcome/severity of the CP. Methods: Population-based, data linkage study between CP and congenital anomaly registers in Europe and Australia. The EUROCAT definition of severe CHD (sCHD) was used. Linked data from 4 regions in Europe and 2 in Australia were included. All children born in the regions from 1991 through 2009 diagnosed with CP and/or sCHD were included. Linkage was completed locally. Deidentified linked data were pooled for analyses. Results:The study sample included 4989 children with CP and 3684 children with sCHD. The total number of livebirths in the population was 1 734 612. The prevalence of CP was 2.9 per 1000 births (95% CI, 2.8-3.0) and the prevalence of sCHD was 2.1 per 1000 births (95% CI, 2.1-2.2). Of children with sCHD, 1.5% (n = 57) had a diagnosis of CP, of which 35 (61%) children had prenatally or perinatally acquired CP (resulting from a brain injury at £28 days of life) and 22 (39%) children had a postneonatal cause (a brain injury between 28 days and 2 years). Children with CP and sCHD more often had unilateral spastic CP and more intellectual impairments than children with CP without congenital anomalies.Conclusions: In high-income countries, the proportion of children with CP is much higher in children with sCHD than in the background population. The severity of disease in children with CP and sCHD is milder compared with children with CP without congenital anomaliesFunding support received for the overarching Comprehensive CA-CP Study: the Cerebral Palsy Alliance Research Foundation (The Comprehensive CA-CP Study PG1215 and PG2816 and salary support from Cerebral Palsy Alliance Research Foundation (S.G., S.M., H.S.S., N.B.).info:eu-repo/semantics/publishedVersio

Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach

BACKGROUND: Biological effects of nuclear factor-kappaB (NF kappaB) can differ tremendously depending on the cellular context. For example, NF kappaB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NF kappaB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NF kappaB, involving a lack of inhibitor of kappaB (I kappaB alpha) protein reappearance. Permanent activation of the upstream kinase IKK beta results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized I kappaB alpha. RESULTS: To investigate the mechanism underlying the general PP2A-mediated tuning of IKK beta phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKK beta and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKK beta phosphorylation thus allowing for I kappaB alpha resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKK beta phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of I kappaB alpha reappearance, is not a direct UVB action but requires instrumentality. CONCLUSION: The model developed here can be used as a reliable building block of larger NF kappa B models and offers comprehensive simplification potential for future modeling of NF kappa B signaling. It gives more insight into the newly discovered mechanisms for IKK deactivation and allows for substantiated predictions and investigation of different hypotheses. The evidence of constitutive activity of PP2Ac at the IKK complex provides new insights into the feedback regulation of NF kappa B, which is crucial for the development of new anti-cancer strategies

Genetic determinants of response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer identified by whole exome sequencing

The cornerstone in the treatment of locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision. Reliable predictors of response to nCRT in LARC remain an unmet need in colorectal cancer research. This study used high throughput DNA analysis to investigate genetic differences between highly responsive tumors and tumors resistant to nCRT.European Human Genetics Conference Hybrid Conference Glasgow, Scotland, UK JUNE 10–13, 202

The role of Ser/Thr phosphatase PP2A in the regulation of transcription factor NFkB

Der Transkriptionsfaktor NFkB kann durch eine Vielzahl von Stimuli, wie durch das pro-inflammatorische Zytokin IL-1, aktiviert werden. NFkB ist generell dafür bekannt, dass es die Transkription von Genen verstärkt, deren Produkte das Überleben der Zelle fördern und die Apoptose inhibieren. In diesem Zusammenhang konnte beobachtet werden, dass die TRAIL-induzierte Apoptose durch eine IL-1-vermittelte NFkB-Aktivierung stark vermindert werden kann. Paradoxerweise, wird die durch UVB-Strahlung ausgelöste Apoptose durch Ko-Stimulation mit IL-1 nicht reduziert, sondern sogar noch verstärkt. Dieser Effekt konnte auf eine NFkB-abhängige Repression anti-apoptotischer Proteine und auf eine erhöhte Freisetzung des Zytokins TNF zurückgeführt werden. Sezerniertes TNF induziert durch autokrine Bindung an den TNF-R1 den apoptotischen Signalweg, wodurch die UVB-induzierte Apoptose additiv verstärkt wird. Ein Ziel dieser Arbeit war es, die molekularen Mechanismen, die für die beobachtete NFkB-abhängige Freisetzung von TNF verantwortlich sind, aufzuklären. In unstimulierten Zellen liegt NFkB, durch Bindung an IkBa, inaktiv im Zytoplasma vor. Nach der IL-1-vermittelten Phosphorylierung der Ser/Thr Kinase IKKbeta an Ser177/181 wird IkBalpha phosphoryliert, polyubiquitiniert und umgehend proteasomal degradiert. Daraufhin wird NFkB aktiviert und induziert neben seinen Zielgenen, auch die Transkription von IkBalpha. Nach kurzer Zeit beenden resynthetisierte IkBalpha-Moleküle, durch eine erneute Bindung an NFkB, dessen Aktivität. Überraschenderweise findet nach Stimulation von Zellen mit IL-1 und UVB keine Re-Synthese von IkBalpha mehr statt. Es konnte gezeigt werden, dass die daraus resultierende konstitutive Aktivierung von NFkB für eine massiv erhöhte Transkription von TNF und somit auch indirekt für dessen Sezernierung verantwortlich ist. Unsere Untersuchungen ergaben, dass die Re-Synthese von IkBalpha dadurch inhibiert wird, dass IkBalpha zwar NFkB-abhängig transkribiert, das resynthetisierte Protein aber anschließend sofort wieder phosphoryliert und proteasomal degradiert wird. Als Ursache hierfür wurde eine kontinuierliche Phosphorylierung und damit Aktivität von IKKbeta festgestellt. Verantwortlich für diese chronische IKKbeta Phosphorylierung ist die UVB-induzierte Inhibierung der Ser/Thr Phosphatase PP2A, die so als ein neuer und kritischer Modulator der IKKbeta-Aktivität identifiziert werden konnte. Die Tyrosinkinase c-Src ist an der Regulation vieler zellulärer Prozesse, die das Wachstum und das Überleben der Zelle fördern, beteiligt. Eine unkontrollierte Aktivität dieses Proto-Onkogens spielt, vorwiegend durch die Aktivierung des PI3K/Akt-, Ras/MAPK-, aber auch des NFkB-Signalweges, eine bedeutende Rolle bei der Entstehung vieler Arten von Tumoren. In der vorliegenden Arbeit konnte ein bisher unbekannter Mechanismus der Src-vermittelten konstitutiven NFkB-Aktivierung nachgewiesen werden. Unter Verwendung des Tyr-Phosphatase Inhibitors Orthovanadat kommt es in Kombination mit IL-1 Stimulation zunächst zu einer stabilen aktivierenden Tyr416 Phosphorylierung von Src. Diese wiederum inaktiviert PP2A, durch deren Phosphorylierung an Tyr307, die gleichzeitig IL-1-vermittelte IKKbeta-Aktivität verlängert wird. Folglich wird auch in diesem Fall resynthetisiertes IkBalpha phosphoryliert und proteasomal abgebaut, wodurch die NFkB-abhängige Transkription anti-apoptotischer Gene massiv verstärkt wird.The transcription factor can be activated by a variety of stimuli, such as the pro-inflammatory cytokine IL-1. NFkB is generally known to control the expression of many genes that promote cell survival and protect cells from apoptosis. Accordingly, IL-1-mediated NFkB activation significantly reduces TRAIL-induced apoptosis. Paradoxically, UVB-induced apoptosis is not inhibited but even enhanced upon co-stimulation with IL-1. This effect was associated with NFkB-dependent repression of anti-apoptotic genes and sustained secretion of TNF which additively induces apoptosis upon autocrine activation of TNF-R1. The aim of this study was to elucidate the molecular mechanisms underlying NFkB-dependent enhancement of TNF release. In resting cells, inactive NFkB is kept in the cytoplasm by binding to its inhibitor IkBalpha. Upon stimulation with IL-1, the Ser/Thr Kinase IKKbeta becomes phosphorylated at Ser 177/181, which in turn causes phosphorylation of IkBalpha, leading to its subsequent polyubiquitination and proteasomal degradation. As a result, NFkB becomes activated and induces the transcription of target genes, including the one encoding its inhibitor IkBalpha. Newly synthesized IkBalpha consequently binds to NFkB again, thereby terminating its transcriptional activity. Surprisingly, recurrence of IkBalpha is completely blocked following co-stimulation with IL-1 and UVB, consequently leading to constitutive NFkB activation. We could show permanent NFkB activity to be a prerequisite for enhanced TNF transcription and release. Furthermore our studies revealed lack of IkBalpha recurrence not to be a result of inhibition of NFkB-dependent transcription, but to be mediated by immediate phosphorylation and proteasomal degradation of newly-synthesized IkBalpha due to persistent IKKbeta phosphorylation and activation. In this context, we revealed the Ser/Thr phosphatase PP2A to play a crucial role in the control of IKKbeta activity. Furthermore, we could show that UVB-induced inhibition of PP2A is responsible for constitutive IKKbeta phosphorylation and activation. The Tyrosine-Kinase c-Src is involved in the regulation of many cellular processes, such as growth and survival. Uncontrolled activity of the proto-oncogene Src has been linked to the development of many different types of tumors, mainly by inducing activation of the PI3/Akt, Ras/MAPK, but also the NFkB activating pathways. Utilizing the Tyr-phosphatase inhibitor orthovanadate combined with IL-1 stimulation we could demonstrate stable and activating Tyr416 phosphorylation of Src to occur. Consequently, stably activated c-Src induces the inhibitory Tyr307 phosphorylation of PP2A, thereby maintaining the IL-1-mediated activity of IKKbeta. Accordingly, also under these circumstances, resynthesized IkBalpha undergoes phosphorylation and proteasomal degradation, finally leading to a strong increase of NFkB-dependent transcription of anti-apoptotic genes

Modeling time delay in the NFκB signaling pathway following low dose IL-1 stimulation

Stimulation of human epithelial cells with IL-1 (10 ng/ml) + UVB radiation results in sustained NFκB activation caused by continuous IKKbeta phosphorylation. We have recently published a strictly reduced ordinary differential equation model elucidating the involved mechanisms. Here, we compare model extensions for low IL-1 doses (0.5 ng/ml), where delayed IKKbeta phosphorylation is observed. The extended model including a positive regulatory element, most likely auto-ubiquitination of TRAF6, reproduces the observed experimental data most convincingly. The extension is shown to be consistent with the original model and contains very sensitive processes which may serve as potential intervention targets

Modeling and Analysis of UVB Influence on IL-1 induced NF-kappaB Signaling

Enhancement of UVB-induced apoptosis has recently been reported upon co-stimulation of cells with IL-1. The phenomenon has been shown to be NF-kappaB dependent and coincides with a sustained absence of the inhibitor of NF-kappaB, IkappaBalpha. The exact mechanisms are the subject of present research. In order to investigate the behavior of IkappaBalpha, a mathematical model for the IL-1 receptor is developed based on ordinary differential equations. It is coupled with an existing NF-kappaB signaling module and parameterized using experimental and literature data. The model provides a more realistic input for the NF-kappaB module and is suitable for fitting the experimental data of IkappaBalpha following IL-1 stimulation. It is used to test and reject the hypothesis that the sustained absence of IkappaBalpha upon IL-1+UVB stimulation is due to an altered internalization behavior of the IL-1 receptor

The Cofilin Phosphatase Slingshot Homolog 1 (SSH1) Links NOD1 Signaling to Actin Remodeling

NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-kappa B activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-kappa B activation and innate immune responses