TY  - THES
A1  - Niederlechner, Stefanie
T1  - Assessment of the basic molecular mechanisms underlying L-glutamine's cytoprotective effects after intestinal injury
T1  - Untersuchung und Auswertung der molekularen Mechanismen, die L-Glutamin's protektivem Zellmechanismus nach Intestinaler Verletzung zugrunde liegen
N2  - Critical illness like sepsis, shock, and intestinal bowel disease are one of the leading causes of morbidity and mortality in the US and around the world. At present, studies to define new therapeutic interventions that can protect tissues and cells against injury and attenuate inflammation are fields of intense investigation. While research over the past decade has clearly identified GLN as a vital stress substrate facilitating cellular survival following injury, the initiation steps in GLN’s cytoprotective molecular mechanism still remain elusive. Previously published work suggested that stabilization of ECM proteins and activation of ECM receptor osmosignaling may play a central role in the orchestration of many cellular pathways following stress. Thus, I hypothesized that preservation of ECM protein and EGFR levels as well as ECM receptor signaling play key roles in the molecular mechanisms underlying GLN’s protection against thermal injury in the intestine. I was able to confirm via Western blotting and by using silencing RNA against FN, Ntn-1, EGFR, and their negative controls, that GLN-mediated preservation of FN, Ntn-1, and EGFR levels is critical in GLN’s protection against hyperthermia in IEC-6 cells. By using a selective FN-Integrin interaction inhibitor GRGDSP, its negative control peptide GRGESP, and Src-kinase inhibitor PP2, I showed that FN-Integrin signaling and Src-kinase activation are essential in GLN-mediated protection in the intestine. This applied to EGFR signaling as demonstrated using the EGFR tyrosine kinase inhibitor AG1478. In addition to GRGDSP and AG1478, ERK1/2 inhibitors PD98059 and UO126 as well as the p38MAPK inhibitor SB203580 revealed that GLN is protective by activating ERK1/2 and dephosphorylating p38MAPK via FN-Integrin and EGFR signaling. However, GLN-mediated PI3-K/Akt/Hsp70 activation seems to occur independently of FN-Integrin and EGFR signaling as indicated by Western blots as well as experiments using the PI3-K inhibitor LY294002, GRGDSP, and AG1478. The results showed that GLN activates cell survival signaling pathways via integrins as well as EGFRs after hyperthermia. Moreover, I found that GLN-mediated preservation of FN expression after HS is regulated via PI3-K signaling. Whether GLN-mediated PI3-K signaling happens simultaneously to FN-Integrin and EGFR signaling or whether PI3-K signaling coordinates FN-Integrin and EGFR signaling needs to be investigated in future studies. Further, experiments with PD98059 and GRGDSP revealed that ERK1/2 assists in mediating transactivation of HSF-1 following HS. This leads to increases in Hsp70 expression via FN-Integrin signaling, which is known to attenuate apoptosis after thermal injury. Fluorescence microscopy results indicated that HS and GLN regulate cell are size changes and the morphology of F-actin via FN-Integrin signaling. Experiments using GRGDSP and GRGESP showed that GLN enhances cellular survival via FN-Integrin signaling in a manner that does not require increased intracellular GLN concentrations (as quantified using LC-MS/MS). In summary, my thesis work gives new and potentially clinically relevant mechanistic insights into GLN-mediated molecular cell survival pathways. These results warrant clinical translation to assess if clinical outcome of critically ill patients suffering from gastrointestinal diseases can be improved by GLN treatment and/or by targeting the molecular pathways found in my studies.
N2  - Kritische Erkrankungen wie Sepsis, Schock und entzündliche Darmerkrankungen sind eine der häufigsten Todesursachen in den Vereinigten Staaten und dem Rest der Welt. Von großer Bedeutung sind deshalb Studien, die darauf abzielen, neue therapeutische Ansätze zu finden, die Gewebe und Zellen vor Verletzungen schützen und Entzündungen verhindern. Im letzten Jahrzehnt hat sich herausgestellt, dass Glutamin Schutz gegen die pathophysiologischen und pathobiochemischen Entgleisungen während solcher Erkrankungen vermitteln kann. Trotzdem ist es bisher nur sehr unvollständig gelungen, die zugrundeliegenden molekularen Mechanismen, die für diese zellulären Schutzfunktionen von Glutamin verantwortlich sind, aufzuklären. In diesem Zusammenhang gab es erste Hinweise darauf, dass die Stabilisierung von extrazellulären Matrixproteinen und die Aktivierung der Signalwege extrazellulärer Matrixproteinrezeptoren eine wichtige Rolle spielen könnten. Hierauf basierend habe ich die Hypothesen aufgestellt, dass die Expression von extrazellulären Matrixproteingehalten und von epidermalen Wachstumsfaktorrezeptoren (EGFR), sowie die Aktivierung von Integrin- und EGFR-Signalwegen Schlüsselrollen in den protektiven Mechanismen von Glutamin spielen. Meine Arbeiten zeigten anhand von Western Blots und durch den Einsatz von Silencing RNA (gegen Fibronektin, Netrin-1, EGFR und deren Negativkontrollen), dass Glutamin extrazelluläre Matrixproteingehalte, wie Fibronektin und Netrin-1, und EGFR-Levels vor Hitzestress in Darmepithelzellen bewahrt, wodurch Apoptose verhindert wird. Außerdem konnte ich mit Hilfe des selektiven Inhibitors GRGDSP, seiner Negativkontrolle GRGESP und des Src-Kinase-Inhibitors PP2 nachweisen, dass auch die Aktivierung von Fibronektin-Integrin-Signalwegen und von Src-Kinasen an den Schutzmechanismen von Glutamin im Darm beteiligt sind. Wie mit Hilfe des spezifischen EGFR-Tyrosinkinase-Inhibitors AG1478 nachgewiesen werden konnte, trifft dies auch für die EGFR-Signalwege zu. Zusätzlich zu GRGDSP und AG1478 wurden auch die ERK1/2-Kinase-Inhibitoren PD98059 und UO126 und der p38MAPK-Inhibitor verwendet, um zu zeigen, dass Glutamin Darmepithelzellen durch die Aktivität von Fibronektin-Integrin- und EGFR-Signalwegen vor Hitzestress schützt, indem es EKR1/2 phosphoryliert und p38MAPK dephosphoryliert. PI-3K/Akt/Hsp70-Signalwege werden ebenfalls von Glutamin aktiviert, jedoch unabhängig von Fibronektin-Integrin- und EGFR-Signalwegen. Dies konnte anhand von Western Blot Experimenten und mit Hilfe von LY294002 (PI-3K-Inhibitor), GRGDSP und AG1478 bewiesen werden. Glutamin scheint die gleichen Signalwege sowohl mit Hilfe von Integrinen als auch von EGFR zu beeinflussen, um so den Zelltod in Darmepithelzellen nach Hitzestress zu verhindern. Desweiteren konnte gezeigt werden, dass Glutamin die Erhaltung von Fibronektinkonzentrationen nach Hitzestress durch den PI-3K-Signalweg reguliert. Ob Glutamin den PI-3K-Signalweg parallel zu den Integrin- und EGFR-Signalwegen aktiviert oder ob der von Glutamin ausgelöste PI-3K-Signalweg die Integrin- und EGFR-Signalwege steuert, muss noch weiter untersucht werden. Experimente mit PD98059 und GRGDSP zeigten, dass ERK1/2 in der Gegenwart von Glutamin den Transkriptionsfaktor HSF-1 durch den Fibronektin-Integrin-Signalweg nach Hitzestress transaktiviert. Dadurch nimmt die Expression von Hsp70, welches den Zelltod nach Hitzeschock verhindert, zu. Mit Hilfe der Fluoreszenzmikroskopie konnte gezeigt werden, dass Hitzestress und Glutamin die Zellgröße und die Aktinmorphologie von Darmepithelzellen durch Fibronektin-Integrin-Signalwege steuern können. Außerdem ergaben Versuche mit GRGDSP und GRGESP, dass Glutamin unabhänging von seinen intrazellulären Konzentrationen das Überleben von Darmepithelzellen durch die Aktivierung von Fibronektin-Integrin-Signalwegen erhöhen kann. Hierbei wurden die Glutaminkonzentrationen mit LC-MS/MS gemessen. Die Versuche im Rahmen meiner Dissertation konnten neue und potentiell klinisch relevante Signalwege der molekularen Schutzmechanismen von Glutamin aufdecken. Diese Ergebnisse können die Grundlage für translationale Studien darstellen, die weiter untersuchen, ob die Überlebensrate von Patienten mit entzündlichen Darmerkrankungen durch die Behandlung mit Glutamin oder durch gezielte Beeinflussung der hier untersuchten molekularen Mechanismen verbessert werden kann.
KW  - Glutamin
KW  - Integrine
KW  - Epidermaler Wachstumsfaktor-Rezeptor
KW  - Apoptosis
KW  - glutamine
KW  - integrins
KW  - epidermal growth factor receptor
KW  - cell death
KW  - cell survival
KW  - EGFR
KW  - ERK1/2
KW  - p38MAPK
KW  - PI3-K
KW  - Akt
KW  - Apoptose
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-77399
ER  - 
TY  - JOUR
A1  - Rauert-Wunderlich, Hilka
A1  - Siegmund, Daniela
A1  - Maier, Eduard
A1  - Giner, Tina
A1  - Bargou, Ralf C.
A1  - Wajant, Harald
A1  - Stühmer, Thorsten
T1  - The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NF kappa B Transcription Factors
JF  - PLoS ONE
N2  - Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.
KW  - signal inhibition
KW  - necrotic cell death
KW  - cell viability testing
KW  - cell death
KW  - small interfering RNAs
KW  - HT29 cells
KW  - phosphorylation
KW  - multiple myeloma
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130140
VL  - 8
IS  - 3
ER  - 
TY  - JOUR
A1  - Reddy, C. E.
A1  - Albanito, L.
A1  - De Marco, P.
A1  - Aiello, D.
A1  - Maggiolini, M.
A1  - Napoli, A.
A1  - Musti, A. M.
T1  - Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons
JF  - Cell Death & Disease
N2  - Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.
KW  - c-Jun
KW  - JNK
KW  - cell death
KW  - neurons
KW  - trophic/potassium deprivation
KW  - lithium
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128793
VL  - 4
IS  - e852
ER  -