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Crohn´s disease (CD) and Ulcerative colitis (UC) are idiopathic inflammatory disorders. Environmental factors, infectious microbes, ethnic origin, genetic susceptibility, and a dysregulated immune system can result in mucosal inflammation. However, the etiology of both CD and UC still remains largely unclear. Inflammatory bowel diseaserelated animal models suggest that a combination of genetic susceptibility factors and altered immune response driven by microbial factors in the enteric environment may contribute to the initiation and chronification of the disease. The intestinal immune system represents a complex network of different lymphoid and non-lymphoid cell populations as well as humoral factors. In inflammatory bowel disease, the controlled balance of the intestinal immune system is disturbed at all levels. In CD, naïve T cells preferably differentiate into Th1 or Th17 producing cells, while in UC, these cells differentiate into aberrant Th2 cells. Overall, in active inflammatory bowel disease effector T cell activity (Th1, Th17, Th2) predominates over regulatory T cells. Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of CD and UC. When chosen appropriately, these models proved to be a helpful tool to investigate pathophysiological mechanisms, as well as to test emerging therapeutic options in the preclinical phase. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) and oxazolone are the two major chemicals applied to induce Th1- and Th2-skewed intestinal inflammation, respectively. Colitis can be induced in susceptible strains of mice by intrarectal instillation of the haptenating substances TNBS or oxazolone in ethanol, which is necessary for an initial desintegration of the epithelial barrier. TNBS or oxazolone are believed to haptenize colonic autologous or microbiotic proteins rendering them immunogenic to the host immune system. While TNBS administration in the presence of ethanol results in a transmural infiltrative disease in the entire colon based on an IL-12/IL-23 driven, Th1-or Th17 mediated response, oxazolone instillation finally leads to a colitis caused by a polarized Th2 IL-13-dominated lymphocyte response. Rectal oxazolone instillation in ethanol produces a more superficial inflammation that affects the distal half of the colon rather than the whole colon. Therapeutic modulation of the disturbed immune response in patients with inflammatory bowel disease still represents a complex challenge in the clinic. Currently, none of the therapeutic measure are disease specific and they generally target the pathophysiology downstream of the driving immunpathology. So, there is still the need to develop a tailored approach to prevention of the initiation and perpetuation of the inflammatory cascade before tissue injury occurs. One important aspect of this approach might involve the induction or re-establishment of immunological tolerance. FTY720 following rapid phosphorylation to FTY-P by endogenous sphingosine kinases acts as a sphingosine-1-phosphate (S1P) receptor agonist and represents the prototype of a new generation of S1P receptor modulators. While changing currently its proposed mode of action still focus on the fact, that FTY720 effectively inhibits the egress of T-cells from lymph nodes, thereby reducing the number of antigen-primed/restimulated cells that re-circulate to peripheral inflammatory tissues. However, recent studies indicate, that its immunomodulatory properties might be more complex and exerted not only via interactions with other S1P receptor subtypes but also via a direct modulation of the inflammatory capacity of dendritic cells (DC) resulting in a modified regulation of T cell effector functions as well as in an induction of regulatory T cells and function. 1,25(OH)2D3, the active form of vitamin D, is a secosteroid hormone that has in addition to its central function in calcium and bone metabolism pronounced immune regulatory properties. The biological effects of calcitriol are mediated by the vitamin D receptor (VDR), a member of the superfamily of nuclear hormone receptors. A number of studies identified calcitriol/VDR as prominent negative regulators of Th1-type immune responses, whereas Th2 responses are not affected or even augmented. These effects have been mainly explained by direct activities on lymphocytes, subsequent studies clearly supported a role of calcitriol in modulating monocyte differentiation or DC maturation. However, to translate the immunosuppressive capacities of calcitriol into an effective immunointervention, a great challenge was the design of structural analogs of calcitriol that are devoid of adverse effects related to hypercalcemic activity. The intense study of the 25-oxa series generated a large number of calcitriol analogs exhibiting substantial dissociation between possible immunomodulatory capacities and undesired hypercalcemia. Especially, the combination of the 22-ene modification with the 25-oxa element as realized in ZK156979 yielded a very promising set of new analogs for further characterization in animal models resembling human autoimmune diseases. So, the overall aim of the studies presented here was to evaluate strategies of enhancing regulatory immunity in mouse models of Th1- and Th2-mediated colitis as a new therapeutic approach. To this end we used FTY720, 22-ene-25-oxa vitamin D (ZK156979), as well as the combination of calcitriol and dexamethasone to evaluate the respective pro-tolerogenic potential in intestinal inflammation models in mice. First, to induce Th1-mediated colitis a rectal enema of TNBS was given to Balb/c mice. FTY720 was administered i.p. from day 0-3 or 3-5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFβ and CTLA4. Moreover, we observed a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4+ T cells of FTY720-treated mice. The impact of FTY720 on regulatory T cell induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. Thus, our data provide new and strong evidence that besides its well-established migratory properties FTY720 down-regulates proinflammatory signals while simultaneously inducing the functional activity of CD4+CD25+ regulatory T cells. In a second approach, the rectal instillation of oxazolone yielded a Th2-mediated colitis. Treatment with FTY720 prominently reduced the clinical and histopathologic severity of oxazolone-induced colitis, abrogating body weight loss, diarrhea, and macroscopic and microscopic intestinal inflammation. The therapeutic effects of FTY720 were associated with a prominent reduction of the key Th2 effector cytokines IL-13, IL-4 and IL-5. Moreover, FTY720 inhibited GATA3 and T1/ST2 expression, which represent distinct markers for Th2 differentiation and Th2 effector function. Thus, our data are supportive for the view that FTY720 exhibits beneficial prophylactic as well as therapeutic effects in Th2-mediated experimental colitis by directly affecting Th2 cytokine profiles, probably by reducing GATA3 and T1/ST2. Recently, we described 22-ene-25-oxa-vitamin D (ZK156979) as a representative of a novel class of low calcemic vitamin D analogs showing prominent immunomodulative capacities. Here, we used the Th1-mediated TNBS colitis to test its anti-inflammatory properties in vivo. We found that treatment with ZK156979 clearly inhibited the severity of TNBS-induced colitis without exhibiting calcemic effects. Both early and late treatment abrogated all the clinical macroscopic and microscopic parameters of colitis severity; in addition we observed a clear down-regulation of the relevant Th1 cytokine pattern including the T-box transcription factor, T-bet. On the other hand, application of ZK156979 increased local tissue IL-10 and IL-4. Finally, as a new approach we evaluated the pro-tolerogenic potential of calcitriol and dexamethasone in acute Th1-mediated colitis. Calcitriol and/or dexamethasone were administered i.p. from day 0-3 or from day 3-5 following the instillation of the haptenating agent. The combination of these steroids most effectively reduced the clinical and histopathologic severity of TNBS colitis. Th1-related parameters were down- while Th2 markers like IL-4 and GATA3 were up-regulated. Clearly distinguishable from known steroid effects calcitriol in particular promoted regulatory T cell profiles as indicated by a marked increase of IL-10, TGFß, FoxP3 and CTLA4. Furthermore, analysis of DC mediators responsible for a pro-inflammatory differentiation of T cells revealed a clear reduction of IL-12p70, and IL23p19 as well as IL-6 and IL-17. Thus, our data suggest the concept of a steroid-sparing application of calcitriol derivatives in inflammatory bowel disease. Furthermore, the data presented suggest that early markers of inflammatory DC and Th17 differentiation might qualify as new target molecules for both calcitriol as well as for selective immune modulating vitamin D analogs. In conclusion the data of these published investigations added to the substantial progress in understanding the biology of tolerogenic DC and regulatory T cells with respect to their roles in health and disease achieved in the past years. This has led to an increasing interest in the possibility of using DC and regulatory T cells as biological therapeutics to preserve and restore tolerance to self antigens and alloantigens. Especially DC may be helpful to exert their important roles in directing tolerance and immunity by modulation of subpopulations of effector T cells and regulatory T cells. The data demonstrated in the present studies may assist to define the divergent implications of new therapeutic concepts for the treatment of inflammatory bowel disease, especially with regard to a possible auspicious impact on pro-tolerogenic DC and regulatory T cell functions. However, further studies are needed to fulfil our understanding of the complex immunomodulatory profiles of FTY720 as well as of calcitriol and its low calcemic analog ZK156979, thus accelerating their entry into the clinic as new therapeutic options for the cure of inflammatory bowel disease.
Die Identifikation neuer Hits und Leitstrukturen sind die ersten Schritte bei der Entwicklung neuer Arzneistoffe. Dieser Herausforderung wird derzeit primär mittels High-Throughput-Screening oder der gezielten Modifikationen bekannter Liganden begegnet. Eine weitere Option ist das computerbasierte virtuelle Screening, das es kostengünstig ermöglicht, in kurzer Zeit sehr viele Moleküle auf ihre potentielle biologische Aktivität hin zu untersuchen. Der in dieser Arbeit verwendete Ansatz zur Identifikation neuer Inhibitoren der 5-Lipoxygenase und der Cyclooxygenase-2 beruht auf dem Verfahren des ligandenbasierten virtuellen Screenings. Unter der Voraussetzung der Kenntnis mindestens eines Referenzliganden können so mittels einer Ähnlichkeitsanalyse potentielle neue strukturelle Grundgerüste identifiziert werden. Zu diesem Zweck wurde ein auf atomaren Partialladungen und der dreidimensionalen Struktur der Moleküle basierender Deskriptor (Charge3D/TripleCharge3D) entwickelt. In retrospektiven Studien mit Cyclooxygenase-2 Inhibitoren wurde die Effektivität der neuen Deskriptoren überprüft und mittel eines evolutionären Algorithmus optimiert. Der Charge3D Deskriptor erreicht Anreicherungsfaktoren bis zu 16,1 im ersten Perzentil der durchsuchten Datenbank, wohingegen der TripleCharge3D Deskriptor mit seiner detailierteren Ladungsauftrennung Werte von bis zu 24,8 erreichte. Ein ebensolches retrospektives Screening wurde für 5-Lipoxygenase Inhibitoren durchgeführt. Den maximalen Anreicherungsfaktor von 6,1 im ersten Prozent der Datenbank erreichte hier der Charge3D Deskriptor, der TripleCharge3D Deskriptor erreichte 5,3. Diese wesentlich geringeren Werte sind auf die Diversität der 5-LO Inhibitoren (54 Inhibitoren mit 39 verschiedenen Grundgerüsten) und deren unterschiedliche Inhibitortypen (Redox, nicht Redox und Eisen-bindende Inhibitoren) mit ihren jeweiligen Bindemodi zurückzuführen. In Screenings nach 5-LO Inhibitoren in der Naturstoffdatenbank der Firma AnalytiCon Discovery und COX-25-LO Dualinhibitoren in Datenbanken der Firma Asinex konnten unter Verwendung der beiden Deskriptoren Inhibitoren, mit für diese Targets bislang unbekannten Scaffolds identifiziert werden. Unter Verwendung des 2D Pharmakophor Deskriptors CATS wurden zuerst zwei neue Scaffolds für Inhibitoren der 5-LO identifiziert. Struktur 1 ist den in vitro Assaydaten zufolge ein direkter Inhibitor der 5-LO. Struktur 2 hingegen erreicht seine Wirkung nicht nur über die direkte Interaktion mit der 5-LO. Eine Erklärung dafür wäre die Wechselwirkung mit dem 5-LO aktivierenden Protein FLAP, der Hemmung der Translokation der 5-LO zur Kernmembran, oder die Inhibition 5-LO aktivierender bzw. inaktivierender Kinasen. In nachfolgenden Screenings mit den Strukturen 1 und 2 als Referenzstrukturen konnten mittels der Charge Deskriptoren Substanzderivate (17 Moleküle) mit 5-LO inhibitorischer Wirkung (5 Moleküle mit IC50 Werte ≤ 1 μM an partiell aufgereinigter 5-LO), identifiziert werden. Für das Screening nach COX-2/5-LO Dualinhibitoren wurden 11 Strukturen mit 7 unterschiedlichen Scaffolds unter Verwendung der Charge Deskriptoren aus gewählt. Drei Moleküle zeigten keine 5-LO Aktivität, und jeweils eines nur in intakten PMNLs bzw. im S100 Zellüberstand. Die restlichen 6 Moleküle waren in beiden 5-LO Assays aktiv (intakte PMNLs IC50 zwischen 2 und 15 μM, S100 Zellüberstand 5-LO zwischen 0.5 μM und 25 μM). Somit zeigten 7 Moleküle im S100 Assay Aktivität und konnten als direkte Inhibitoren der 5-LO identifiziert werden. Im Cyclooxygenase-2 Aktivitätsassay mit intakten MonoMac6 Zellen zeigte eine der 11 Strukturen zudem eine geringe (IC50 = 70 μM) inhibierende Aktivität. Modifikationen zur Verbesserung der COX-2 Hemmung könnten in einem potenten COX-2/5-LO Dualinhibitor resultieren, der beispielsweise in der Schmerzbehandlung eingesetzt werden könnte. Ein weiteres Projekt war die Erstellung eines Homologiemodells der 5-LO basierend auf der 15-Lipoxygenase Struktur des Kaninchens (PDB-Struktur: 1LOX). Die Sequenzidentität der beiden Strukturen (1LOX / humane 5-LO) lag bei 37 %. Das Modell wurde zum einen zur Vorhersage von zugänglichen Caspase-6 Schnittstellen an der 5-LO angewandt, und zum anderen wurden Dockingexperimente in Aktiven Zentrum und in Bereichen der C2-like Domäne der 5-LO durchgeführt. Hyperforin, ein bekannter Inhibitor d er 5-Lipoxygenase, wurde an verschiedenen Stellen des Modells für Dockingexperiment eingebracht. Die im Aktiven Zentrum erreichten Scorewerte (Chemscore = -9±1) deuteten hier auf eine unfavorisierte Bindungsstelle hin. BWA4C (ein bekannter Eisenbinder) und ZM230487 (ein nicht-redox Inhibitor) erhielten im Aktiven Zentrum Scorewerte von 27±0,1 und 22±2,5, wodurch eine Bindung als wahrscheinlich angenommen werden kann. Weitere Dockingexperimente an der C2-like Domäne, und speziell am Interface zwischen der C2-like und der katalytischen Domäne, ergaben ähnlich hohe Chemscorewerte für Hyperforin, BWA4C und ZM230487. Aus diesen Resultaten ließ sich kein eindeutiger Bindemodus für Hyperforin ableiten. Eine Positionierung im Aktiven Zentrum ist nach diesen Experimenten unwahrscheinlich, so dass die Existenz einer weiteren, experimentell noch nicht identifizierten Bindestelle vermutet werden kann. Eine solche Interaktionsfläche könnte als Ansatzpunkt für die Entwicklung weiterer 5-Lipoxygenaseinhibitoren eine zentrale Rolle einnehmen.
In this thesis I have investigated the regulation of eicosanoid synthesizing-enzymes by cannabinoid receptor agonists. Rat renal mesangial cells were used as a model system. I could show that all three (CB1, CB2, and GPR55) cannabinoid receptors are expressed on the mRNA level in rat renal mesangial cells – but with differing expression profiles. The CB1 and GPR55 receptors are expressed in comparable amounts, whereas the CB2 receptor is considerably less expressed than the CB1 and the GPR55 receptors. Furthermore I could show that stimulation of renal mesangial cells with CB1 receptor agonists, such as R(+)MA or ACEA, increased IL-1β-induced cPLA2, sPLA2-IIa, and COX2 protein and mRNA expression which subsequently led to an enhanced IL-1β-induced PGE2 formation. Additionally, the IL-1β- induced sPLA2-IIa promoter activity was also increased by CB1 receptor stimulation. Besides the modulated expression of the eicosanoid synthesizing enzymes, I could show that CB1 agonists also led to an increase of IL-1β-induced iNOS expression and subsequent NO formation. In contrast, stimulation with CB2 selective agonists led to a decrease in IL-1β- induced sPLA2-IIa protein expression and PGE2 formation. Accordingly, the IL-1β-induced sPLA2-IIa promoter activity was also reduced by CB2 receptor agonists. IL-1β-induced iNOS expression and subsequent NO formation were not influenced by CB2 recptor activation. Matching the results I obtained with CB1 receptor agonists on IL-1β-induced PGE2 formation, I could observe an increased cPLA2 protein and mRNA expression with a subsequent increase in IL-1β-induced PGE2 formation by GPR55 stimulation. Stimulation with THC, an unselective CB agonist, increased the IL-1β-induced sPLA2-IIa protein expression and subsequently led to an enhanced IL-1β-induced PGE2 formation. Subjecting the cells to higher THC concentrations surprisingly led to a reduction of the IL-1b-induced sPLA2-IIa protein expression and PGE2 formation. A possible explanation may be the differential expression of the three CB receptors. At low concentrations THC may predominantly activate CB1 and GPR55 and with increasing concentration CB2 receptors may also be activated, slightly reversing the enhancing effect. Moreover, I could show that the CB1 receptor stimulation mediated phosphorylation and hence the activation of ERK1/2 MAPK. Additionally to ERK1/2, there was also a phosphorylation and activation of NFkB observed by CB1 receptor stimulation. In my thesis I could show for the first time that PPARα was activated by IL-1β in rMC. The IL-1β-induced PPARα promoter activity was completely inhibited by addition of the CB2 receptor agonist, JWH015. These findings were confirmed by inhibition of the IL-1β-induced PGE2 formation by a PPARα antagonist (MK-886). In summary, I could show that activation of CB1 receptors in our system led to a worsening of an inflammatory condition, whereas activation of the CB2 receptors led to the complete opposite; namely a reduction of the inflammatory response by reducing the sPLA2-IIa expression and PGE2 formation. GPR55 activation did not display any alteration of inflammatory conditions, since the classical inflammatory pathway was not influenced.
Die 5-Lipoxygenase (5-LO) ist das Schlüsselenzym bei der zellulären Leukotriensynthese. Sie katalysiert zunächst die Umwandlung von AA zu 5-Hydroperoxyeicosatetraensäure (5-HPETE) und als zweiten Schritt die Dehydratisierung der 5-HPETE zum instabilen Epoxid Leukotrien A4 (LTA4). Leukotriene sind wichtige Mediatoren bei Entzündungsprozessen und allergischen Reaktionen. Die 5-LO besteht aus zwei Domänen, einer N-terminalen regulatorischen Domäne (AS 1-121) und einer C-terminalen katalytischen Domäne (AS 120-673). Die katalytische Domäne besteht hauptsächlich aus a-Helices, die regulatorische hingegen hat fast ausschließlich b-Faltblätter als Sekundärstrukturelemente. Mit ihrem aus acht Faltblättern bestehenden antiparallelen b-Sandwich hat sie Ähnlichkeit mit einer C2- Domäne. Sie besitzt eine Ca2+-Bindungsstelle und ist maßgeblich bei der Bindung des Enzyms an Membranen beteiligt. Für einige Inhibitoren und zelluläre Faktoren existieren Hinweise, dass sie an die C2-ähnliche Domäne der 5-LO binden, die direkten Beweise fehlen jedoch. Die katalytische Domäne enthält das aktive Zentrum mit einem prosthetischen Eisen und drei Phosphorylierungsstellen. In der vorliegenden Arbeit wurde zum ersten Mal erfolgreich eine Methode zur Überexpression und Aufreinigung der regulatorischen Domäne in E. coli entwickelt, die eine große Ausbeute des Zielproteins erbringt. In einer einfachen one-step Aufreinigung können bis zu 80 mg Fusionsprotein aus MBP und der regulatorischen Domäne der 5-LO (MBP-5LO1-128) pro Liter Expressionskultur gewonnen werden. Ein Verdau mit TEV-Protease führt zur effizienten Abspaltung des MBP. Die weitere Aufreinigung mit hydrophober Interaktionschromatogragphie ergibt eine Ausbeute von 3,4 mg reiner regulatorischer Domäne. Dabei weisen sowohl das Fusionsprotein, als auch die abgespaltene regulatorische Domäne eine Reinheit von über 95% auf. Leider ist die momentan erreichbare Konzentration der reinen regulatorischen Domäne noch nicht ausreichend, um damit NMR-Messungen zur Strukturaufklärung durchführen zu können. Es konnte bestätigt werden, dass die katalytische Domäne der 5-LO alleine nicht zu einer Umsetzung von Arachidonsäure in der Lage ist. Der Verlust der enzymatischen Aktivität ist vermutlich nicht auf eine Fehlfaltung der Proteindomäne zurückzuführen. Die korrekte Faltung der katalytischen Domäne konnte durch eine - wenn auch schwache - Bindung der Domäne an ATP gezeigt werden konnte. Die Zugabe von regulatorischer Domäne zu rekombinanter, aufgereinigter WT 5-LO hat unter gewissen Bedingungen (Abwesenheit von PC) einen stimulierenden Effekt auf die Aktivität des Gesamtenzyms. Dieser Effekt könnte durch eine „Dimerisierung“ der regulatorischen Domäne mit WT 5-LO hervorgerufen werden. In Gegenwart von PC ruft die regulatorische Domäne eine Hemmung der 5-LO-Aktivität hervor. Außerdem ist MBP-5LO1-128 (im Gegensatz zu MBP alleine) in der Lage, das Zwischenprodukt der 5-LO, 5-HPETE, in einer 1:1 Reaktion zu reduzieren. Diese Fähigkeit könnte zur Unterdrückung einer Selbstaktivierung der 5-LO physiologisch bedeutend sein. Interessant ist, dass die Reduktion in Anwesenheit von Ca2+, PC oder PC und Ca2+ deutlich verringert war. Ca2+ führte vor allem in Abwesenheit von PC zu einer deutlichen Verminderung der 5-HPETE-Reduktion. In Gegenwart von PC zeigt Ca2+ nur einen minimalen Einfluss auf die 5-HPETE-Reduktion. Es existieren deutliche Hinweise für eine Konformationsänderung von C2-Domänen durch Ca2+-Bindung [105; 106]. Geht man davon aus, dass dies auch für die C2-ähnliche Domäne der 5-LO zutrifft, erklärt sich damit die Verminderung der 5-HPETE-Menge in Gegenwart von Ca2+. Es ist gut vorstellbar, dass eine Ca2+-induzierte Konformationsänderung zu einer schlechteren Zugänglichkeit der für die Reduktion wichtigen Aminosäureseitenketten führt. Die Anwesenheit von PC führte zu einer noch stärkeren Hemmung der 5-HPETEReduktion, als die Zugabe von Ca2+. Die Beobachtung, dass 5-HPETE in Anwesenheit von PC teilweise vor einer Reduktion geschützt wird, ist in Übereinstimmung mit Ergebnissen von Rakonjac [62]. Vermutlich werden die, für die Reduktion wichtigen Aminosäureseitenketten bei Bindung der Domäne an die PC-Micellen teilweise verdeckt. Cystein 99 könnte eine Rolle bei dieser Reaktion spielen. Es konnte jedoch gezeigt werden, dass es bei der Reduktion von 5-HPETE nicht zu einer maßgeblichen Ausbildung von Disulfidbrücken kommt. Vorstellbar wäre die Ausbildung einer Hydroxysulfonylgruppe am Cystein. Eine Beteiligung von Cystein 99 wird durch die Ergebnisse des Dockings von 5-HPETE an das Homologiemodell der 5-LO und experimentelle Daten aus anderen Arbeiten (M. Hörnig, O. Rådmark) unterstützt. Um den genauen Mechanismus aufzuklären und die physiologische Relevanz zu zeigen sind weitere umfassende Untersuchungen nötig. Um eine Bindung von Hyperforin, LP 121, C06 und B02 an die regulatorische Domäne zu untersuchen wurde ein Kompetitionsassay entwickelt. Dabei wurde rekombinante, aufgereinigte 5-LO mit dem entsprechenden Inhibitor versetzt und dann versucht, die Hemmung mit Hilfe der regulatorischen Domäne aufzuheben. Greift der Inhibitor an der regulatorischen Domäne an, so sollte die Zugabe von MBP-5LO1-128 bewirken, dass ein Teil des Inhibitors an die regulatorische Domäne bindet, er sich somit nicht mehr an der 5-LO befindet und seine Wirkung vermindert wird. Um sicher zu gehen, dass die beobachteten Effekte nicht artifiziell sind, wurde ein Inhibitor, der an der katalytischen Domäne der 5-LO angreift als Negativkontrolle für den Assay benutzt. Für diesen Zweck wurde der Eisenligandinhibitor Zileuton ausgewählt. Es ist bekannt, dass Zileuton das Eisen im aktiven Zentrum der 5-LO chelatiert und nicht an die regulatorische Domäne bindet. Die Hemmung von Zileuton konnte erwartungsgemäß durch Zugabe der regulatorischen Domäne nicht aufgehoben werden. Hyperforin und B02 zeigten eine deutlich verminderte 5-LOHemmung bei steigender Menge an MBP-5LO1-128. Die Hemmung durch LP 121 und C06 wurde nicht von der regulatorischen Domäne beeinflusst. Es konnte also ein effektives und einfaches Screeningverfahren zur Untersuchung der Bindung von 5-LO-Inhibitoren an die regulatorische Domäne entwickelt werden, mit der Einschränkung, dass der Assay nur für den Nachweis einer reversiblen Bindung von Inhibitoren an die regulatorische Domäne tauglich ist. Dieser Assay wird sich in Zukunft als wichtiges Tool bei der Suche nach neuen 5-LO-Inhibitoren erweisen, da die Substanzen mit einem direkten Angriff des aktiven Zentrums bisher nicht zu effektiven und nebenwirkungsarmen Arzneimitteln geführt haben.
In the first part of this study, we have identified the two steroid hormones progesterone and norgestimate as novel TRPC channel blockers. Both substances blocked TRPC-mediated Ca2+ influx with micromolar activities in fluorometric measurements. TRPC channel inhibition did not seem to be a general steroid effect since another progestin, the norgestimate metabolite levonorgestrel, was not effective. Norgestimate was 4- to 5-fold more active on the TRPC3/6/7 subfamily compared to TRPC4/5, whereas progesterone was similarly potent. This selectivity of norgestimate was confirmed by patch clamp recordings. As norgestimate blocked channels directly gated by DAG with a fast kinetic, we assume the compound acts on the channel protein itself. This view was further substantiated by the lack of effects on IP3R-mediated Ca2+ release from the endoplasmic reticulum, which is activated in parallel with TRPCs by Gq/11-coupled receptor stimulation. Norgestimate did not only block ectopically expressed TRPC channels but also native, TRPC-mediated currents in rat aortic smooth muscle cells with similar activity. The usefulness of norgestimate as a tool compound for the investigation of physiological TRPC functions was tested in isolated vessel rings. Consistent with TRPC6 being an essential component of the alpha-1-adrenoceptor-activated cation channel, we demonstrated a direct vasorelaxant, endothelium-independent effect of norgestimate on rat aortic rings precontracted with phenylephrine. Thus, our results provide further experimental support for a role of TRPC6 in alpha-1-adrenergic vessel constriction. In the second part of this study, we screened a human aorta cDNA-library for novel TRPC4-interacting proteins with a modified yeast two-hybrid (Y2H) system in which the TRPC4-C-terminus was expressed as tetrameric bait protein, thereby mimicking the native channel conformation. Of the eleven interacting proteins found SESTD1 was chosen for further analyses since it contains a phospholipid-binding Sec14p-like domain and thus could be involved in regulation of TRPC channels by phospholipids. After the biochemical validation of the found interaction, the first spectrin domain of SESTD1 was then identified to interact with the CIRB domain of TRPC4 in directed Y2H tests. SESTD1 also co-immunoprecipitated with the closely related TRPC5 protein in which the SESTD1-binding domain is highly conserved. Independent of the CIRB site, co-immunoprecipitation with TRPC6 and the distantly related TRPM8 channel was observed indicating the existence of other sites in these channel proteins that mediate interaction with SESTD1. Analysis of SESTD1 gene expression in human tissues showed that its transcripts are ubiquitously expressed and tissues with significant coexpression with TRPC4 and -5 were identified. We have generated two polyclonal antisera directed against SESTD1 that consistently detected SESTD1 protein in brain, aorta, heart, and in smooth muscle and endothelial cells. The functional consequences of the found interaction were investigated by examination of the TRPC5-mediated Ca2+ influx in a clonal HM1 cell line stably expressing the channel. Since SESTD1 overexpression had no detectable effects on TRPC5-mediated Ca2+ influx, most likely due to expression of endogenous SESTD1, we knocked-down the native protein with specific siRNA. This procedure reduced TRPC5-mediated Ca2+ influx following receptor stimulation by 50%. Parallel biotinylation experiments did not reveal any differences in cell surface expressed TRPC5-protein, suggesting that reduction of TRPC5 activity resulted from a loss of a direct SESTD1 effect on the channel. In addition, in immunofluorescence experiments we observed that reduced SESTD1 protein levels resulted in a redistribution of the multifunctional protein ß-catenin from the plasma membrane to the cytosol. This result may point to an involvement of SESTD1 in formation and maintenance of adherens junctions. SESTD1 contains a phospholipid-binding Sec14p-like domain and we were the first to demonstrate its Ca2+-dependent binding to phosphatidic acid and all physiological phosphatidylinositol mono- and bisphosphates in vitro. The physiological function of this binding activity is not known at present, but it could play a role in regulation of associated TRPC channels. TRPC4 and -5 channels are activated by phospholipid hydrolysis and also bind phospholipids directly. The identification of SESTD1 as novel TRPC-interacting protein could thus be an important step forward in the investigation and better comprehension of the complex molecular mechanisms of TRP channel regulation by lipids.
The µ-opioid receptor is the primary target structure of most opioid analgesics and thus responsible for the predominant part of their wanted and unwanted effects. Carriers of the frequent genetic µ-opioid receptor variant N40D (allelic frequency 8.2 - 17 %), coded by the single nucleotide polymorphism A>G at position 118 of the µ-opioid receptor coding gene OPRM1 (OPRM1 118A>G SNP), suffer from a decreased opioid potency and from a higher need of opioid analgesics to reach adequate analgesia. The aim of the present work was to identify the mechanism by which the OPRM1 118A>G SNP decreases the opioid potency and to quantify its effects on the analgesic potency and therapeutic range of opioid analgesics.
To elucidate the consequences of the OPRM1 118A>G SNP for the effects of opioid analgesics, brain regions of healthy homozygous carriers of the OPRM1 118A>G SNP were identified by means of functional magnetic resonace imaging (fMRI), where the variant alters the response to opioid analgesics after painful stimulation. Afterwards, the µ-opioid receptor function was analyzed on a molecular level in post mortem samples of these brain regions. Finally, the consequences of the OPRM1 118A>G SNP for the analgesic and respiratory depressive effects of opioids were quantified in healthy carriers and non-carriers of OPRM1 118A>G SNP by means of experimental pain- and respiratory depression-models.
To identify pain processing brain regions, where the variant alters the response to opioid analgesics after painful stimulation, we investigated the effects of different alfentanil concentration levels (0, 25, 50 and 75 ng/ml) on pain-related brain activation achieved by short pulses (300 msec) of gaseous CO2 (66% v/v) delivered to the nasal mucosa using a 3.0 T magnetic head scanner in 16 non-carriers and nine homozygous carriers of the µ-opioid receptor gene variant OPRM1 118A>G. In brain regions associated with the processing of the sensory dimension of pain (pain intensity), such as the primary and secondary somatosensory cortices and the posterior insular cortex, the activation decreased linearly in relation to alfentanil concentrations, which was significantly less pronounced in OPRM1 118G carriers. In contrast, in brain regions known to process the affective dimension of pain (emotional dimension), such as the parahippocampal gyrus, amygdala and anterior insula, the pain-related activation disappeared already at the lowest alfentanil dose, without genotype differences.
Subsequently, we investigated the µ-opioid receptor-expression ([3H]-DAMGO saturation experiments, OPRM1 mRNA analysis by means of RT-PCR), the µ-opioid receptor affinity ([3H]-DAMGO saturation and competition experiments) and µ-opioid receptor signaling ([35S]- GTPγS binding experiments) in post mortem samples of the human SII-region, as a cortical projection region coding for pain intensity, and lateral thalamus, as an important region for nociceptive transmission. Samples of 22 non-carriers, 21 heterozygous and three homozygous carriers of OPRM1 118A>G SNP were included into the analysis. The receptor expression and receptor affinity of both brain regions did not differ between non-carriers and carriers of the variant N40D. In non-carriers, the µ-opioid receptors of the SII-region activated the receptor bound G-protein more efficiently than those of the thalamus (factor 1.55-2.27). This regional difference was missing in heterozygous (factor 0.78-1.66) and homozygous (factor 0.66-1.15) carriers of the N40D variant indicating a reduced receptor-G-protein-coupling in the SII-region.
Finally, the consequences of the alteration of µ-opioid receptor function in carriers and noncarriers of the genetic variant was investigated using pain- and respiratory depression-models. Therefore, 10 healthy non-carriers, four heterozygous and six homozygous carriers of the µ- opioid receptor variant N40D received an infusion of four different concentrations of alfentanil (0, 33.33, 66.66 and 100 ng/ml). At each concentration level, analgesia was assessed by means of electrically (5 Hz sinus 0 to 20 mA) and chemically (200 ms gaseous CO2 pulses applied to the nasal mucosa) induced pain, and respiratory depression was quantified by means of hypercapnic challenge according to Read and recording of the breathing frequency. The results showed that depending on the used pain model, both heterozygous and homozygous carriers of the variant N40D needed 2 – 4 times higher alfentanil concentrations to achieve the same analgesia as non-carriers. This increase seems to be at least for homozygous carriers unproblematic, because to reach a comparable respiratory depression as non-carriers, they needed 10-12 times higher alfentanil concentrations.
The results of this work demonstrate that the µ-opioid receptor variant N40D causes a regionally limited reduction of the signal transduction efficiency of µ-opioid receptors in brain regions involved in pain processing. Thus, the painful activation of sensory brain regions coding for pain intensity is not sufficiently suppressed by opioid analgesics in carriers of the variant N40D. Due to the insufficient suppression in hetero- and homozygous carriers of the variant N40D, the concentration of opioids has to be increased by a factor 2 - 4, in order to achieve the same analgesia as in non-carriers. At the same time, the respiratory depressive effects are decreased to a greater extent in homozygous carriers of the N40D variant as they need a 10 - 12 times higher opioid concentration to suffer from the same degree of respiratory depression as non-carriers. Due to the increased therapeutic range of opioid analgesics, an increase of the opioid dose seems to be harmless, at least for homozygous carriers of the N40D variant.
Eine weltweite Veränderung der Lebensweise hat eine Zunahme der Anzahl adipöser Menschen und damit ein verstärktes Auftreten von Typ 2 Diabetes mellitus zur Folge. Eine häufig auftretende Komplikation dieser Erkrankung ist das diabetische Fußulkus, dessen molekularen und zellbiologischen Grundlagen weitestgehend unbekannt sind. Für einen normalen Wundheilungsverlauf ist ein Zusammenspiel vieler Wachstumsfaktoren und Zytokine essentiell. Auch die Insulinsensitivität der Haut scheint von großer Bedeutung zu sein. Die Funktionen von Insulin im Wundheilungsprozess und in der Haut sind weitestgehend unerforscht. Um ein besseres Verständnis für die Bedeutung von Insulin in der Wundheilung zu erhalten, bestand das Ziel dieser Arbeit in der Analyse eines Insulin-regulierten Enzyms in der Haut, der HMG-CoA-Reduktase, während des Heilungsprozesses normaler sowie diabetisch chronischer Wunden. Die HMG-CoA-Reduktase katalysiert den geschwindigkeitsbestimmenden Schritt im Mevalonat-Stoffwechselweg und ist somit indirekt an vielen zellulären Ereignissen beteiligt. Die Verletzung von murinem Hautgewebe führte zu einem Anstieg der HMG-CoA-Reduktase-mRNA-Expression an Tag 3 und an Tag 13 nach Verletzung. Die Lokalisation der HMG-CoA-Reduktase im Wundgewebe zeigte, dass insbesondere die am Wundrand gelegenen Keratinozyten durch eine besonders starke mRNA-Expression des Enzyms charakterisiert waren. Im Gegensatz dazu konnte im diabetischen, chronischen Wundgewebe keine Regulation der mRNA-Expression der HMG-CoA-Reduktase detektiert werden. Wundheilungsrelevante Faktoren, wie Insulin und EGF, induzierten die mRNA-Expression der HMG-CoA-Reduktase in HaCaT-Keratinozyten (in vitro). Für die Insulin-vermittelte mRNA-Induktion konnte gezeigt werden, dass der Transkriptionsfaktor SREBP2 für die Transkription des Gens essentiell war. Neben einer erhöhten Transkription der HMG-CoA-Reduktase wurde ebenfalls eine gesteigerte Enzymaktivität nach Insulin- oder EGF-Stimulation detektiert. Die Induktion der Insulin-vermittelten HMG-CoA-Reduktase-Aktivität stand in einem funktionellen Zusammenhang zur Biosynthese des angiogenen Faktors VEGF. Der Einfluss der HMG-CoA-Reduktase auf die VEGF-Biosynthese war posttranskriptionell über die Phosphorylierung des eIF4E-BP1 vermittelt. Auch im tierexperimentellen Modell (in vivo) konnte durch eine Statin-Behandlung bei Mäusen gezeigt werden, dass die Enzymaktivität der HMG-CoA-Reduktase in Keratinozyten für eine normale VEGF-Expression essentiell war. Die VEGF-Synthese wurde von der Aktivität der HMG-CoA-Reduktase in vivo, wie in vitro nach Insulin-Stimulation, posttranskriptionell beeinflusst. Die Analyse weiterer wundheilungsrelevanter Vorgänge ergab, dass eine Inhibierung der HMG-CoA-Reduktase in vivo wie in vitro eine Verringerung der Keratinozytenproliferation zur Folge hatte. Die Keratinozytenproliferation ist ein wichtiger Vorgang bei der Reepithelisierung einer Wunde. Ist das Wundareal durch Keratinozyten geschlossen, beginnt die Differenzierung der Zellen. Die Ergebnisse dieser Arbeit konnten zeigen, dass der Differenzierungsprozess von Keratinozyten in vitro mit einer Induktion der HMG-CoA Redukase Aktivität assoziiert war. Eine Hemmung der Enzymaktivität hatte eine Inhibierung der mRNA-Expression von Keratinozyten-Differenzierungsmarkern, wie Involucrin, Filaggrin und Keratin 1 zur Folge. Zusammenfassend weisen die Ergebnisse dieser Arbeit darauf hin, dass die HMG-CoA-Reduktase wichtige Prozesse, wie Wundangiogenese und Keratinozytenproliferation, im kutanen Wundheilungsverlauf beeinflusst.
Disruption of the complex gastrointestinal ecosystem between the resident microflora and the colonic epithelial cells has been associated with increased inflammation and altered cell growth. Possible endpoints of this disturbance are IBD and CRC. The data presented in this thesis, entitled "PPARgamma as molecular target of epithelial functions in the gastrointestinal tract", shed further light on the underlying molecular mechanisms contributing to the well ordered homeostasis of this gastrointestinal ecosystem. Except for elucidating important roles for mesalazine and the dietary HDAC inhibitors butyrate and SFN in a) the modulation of cellular growth, b) the induction of APs, and c) the control of NFkappaB signalling in CRC cells, the involvement of the nuclear hormone receptors PPARgamma und VDR as "gatekeepers" in these intricate regulatory mechanisms were established. Future work will be engaged in analysing whether these in vitro findings are also physiologically relevant in regard to prevention and therapy of gastrointestinal diseases. Within the scope of this work, in Paper I and II it could be demonstrated that butyrate and mesalazine act via PPARgamma to induce their anti-proliferative and pro-apoptotic actions along the caspase signalling pathway. Activation of the intrinsic and extrinsic signalling trail and the down-regulation of anti-apoptotic proteins are responsible for increased caspase-3 activity caused by butyrate. In contrast, mesalazine merely activates this cascade via the extrinsic trail and the IAPs. Moreover, a signal transduction pathway leading to increased cell death via p38 MAPK - PPARgamma - caspase-3 in response to butyrate was unveiled. In addition, there is strong evidence that mesalazine-mediated pro-apoptotic and growth-inhibitory abilities are controlled by PPARgamma-dependent and -independent mechanisms which appear to be triggered at least in part by the modulation of the tumor suppressor gene PTEN and the oncoprotein c-myc, respectively. In Paper III and IV the induction of the APs HBD-2 and LL-37 in response to the dietary HDAC inhibitors butyrate and SFN was pinpointed. Regarding the molecular events of this regulation, the data presented in this thesis provide strong evidence for the involvement of VDR in HBD-2- and LL-37-induced gene expression, while the participation of PPARgamma was excluded. Moreover, the role for p38 MAPK and TGF-beta1 in the up-regulation of LL-37 caused by butyrate was established. In contrast, SFN-mediated induction of HBD-2 is modulated via ERK1/2 signalling. The findings in Paper V clearly refer to the involvement of the nuclear hormone receptors PPARgamma and VDR in butyrate-mediated suppression of inducible NFkappaB activation dependent on the stimulated signalling pathway caused by LPS or TNFalpha. Moreover, an inhibitory role for VDR in the regulation of basal NFkappaB activation was revealed. On the contrary, a modulating role for PPARgamma on basal NFkappaB could be debarred. Altogether the data presented in this thesis not only provide new insights in the understanding of the fundamental gastrointestinal physiology regulated by nuclear hormone receptors, but also may offer opportunities for the development of potential drug targets and therapeutic strategies in the treatment of IBD and CRC.
5-LO is the key enzyme in the biosynthesis of proinflammatory leukotrienes. It catalyses the conversion of arachidonic acid to the hydroperoxy intermediate 5(S)-hydroperoxy-6- trans-8,11,14-cis-eicosatetraenoic acid (5-HpETE). In a second step 5-LO catalyses a dehydration reaction forming the unstable epoxide intermediate 5(S)-trans-5,6-oxido-7,9- trans-11,14-cis-eicosatetraenoic acid (leukotriene A4 , LTA4). The 5-LO gene is subjected to versatile regulation mechanisms. Apart from regulation by DNA-methylation and histone acetylation / deacetylation 5-LO gene expression can be regulated by the differentiation inducers calcitriol (1,25-dihydroxyvitamin D3) and transforming growth factor beta (TGFβ) 5-LO gene expression. In the myeloid cell lines Mono Mac 6 (MM6) and HL-60, differentiation with both agents caused a prominent upregulation of 5-LO mRNA level, of 5-LO protein expression and of 5-LO activity. Treatment with calcitriol alone already has an impact on 5-LO gene expression which is additionally potentiated by TGFβ treatment. Previous nuclear run-off analysis and reporter gene analysis could not associate the 5-LO promoter with the induction of 5-LO mRNA expression mediated by calcitriol and TGFβ. Inclusion of the 5-LO coding sequence (cds) and inclusion of the 5-LO cds plus the last four introns of the gene (J to M) in the 5-LO promoter construct pN10 led to an enhanced reporter gene activity. The inductions were dependent on vitamin D receptor (VDR) and retinoid x receptor (RXR) cotransfection. Therefore the work was concentrated on identifying elements outside the 5-LO promoter region which contribute to the calcitriol / TGFβ effect on 5-LO mRNA expression. Insertion of the LTA4 hydrolase coding sequence – a coding sequence of similar size - instead of the 5-LO cds led to a loss of the calcitriol / TGFβ effect (pN10LTA4Hcds 1-fold induction). Therewith, it was proven that the presence of the 5-LO cds is crucial for the upregulating effect of calcitriol / TGFβ on 5-LO mRNA level. Cloning of the SV40 promoter instead of pN10 upstream of the 5-LO cds still showed inducibility by treatment with the inducers which argues for a promoter unspecific effect. Insertion of the 5-LO cds in a promoterless basic vector (pGL3cds) displayed same inductions by calcitriol / TGFβ treatment as the 5-LO promoter 5-LO cds construct (pN10cds). Thus, the effect of the inducers is not dependent on the 5-LO promoter under the in vitro conditions of the reporter gene assay. Hence, further cloning was done with promoterless constructs. Through 5-LO cds deletion constructs a positive regulating region in exon 10 to 14 was discovered. To adapt the natural gene context the last four introns (J-M) of the 5-LO gene were inserted in a promoterless construct containing exon 10 to 14 (pGL3cdsΔABInJM). 5end deletion constructs of it revealed putative vitamin D responsive elements (VDREs) in exon 12 and intron M. Mutation of the putative VDREs led to a reduced calcitriol effect –more prominent when the putative VDRE in intron M was mutated (reduction of 40%). Moreover another putative VDRE in exon 10 with an adjacent SMAD binding element (SBE) was detected. SMAD proteins are effector proteins of TGFβ signalling. Gelshift experiments demonstrated in vitro binding of the VDR-RXR heterodimer to those three putative VDREs. By chromatin immunoprecipitation (ChIP) assay in vivo binding of VDR and RXR was shown to the VDRE in the region of exon 10, exon 12 and intron M. 8h and 24h incubation with calcitriol / TGFβ resulted in enhanced expression of VDR in each of the examined regions. The VDR is able to bind to the VDRE without its ligand, whereas this goes along with corepressor recruitment and thus the VDR has a repressive effect on transcription. Histone H4 acetylation was increased when MM6 cells were treated for 8h or 24h with calcitriol or the combination of calcitriol / TGFβ. This finding implies that at that point of time corepressors associated with the VDR are replaced by coactivators. It seems convincing that 5-LO transcription is mainly promoted by calcitriol alone which leads to a more accessible chromatin structure. Previous data indicated that calcitriol and TGFβ upregulate 5-LO RNA maturation and 5- LO transcript elongation. Thus several elongation markers were investigated by ChIP analysis: Histone H3 lysine 36 (H3K36) trimethylation and H4K20 monomethylation were detected in the analysed regions in exon 10, exon 12 and intron M. In region exon 10 the H3K36 trimethylation status was enhanced after 24h calcitriol or calcitriol / TGFβ treatment. An increased H4K20 monomethylation status in all regions was observed when MM6 cells were treated for 24h with calcitriol / TGFβ. 24h treatment with both agents also enhanced the recruitment of the elongation form of RNA polymerase II, which is phosphorylated at serine 2 of the carboxyterminal domain, to the investigated regions. These findings prove the positive regulating role for calcitriol and TGFβ on 5-LO transcript elongation. A putative mechanism of the effect of calcitriol and TGFβ on 5-LO RNA maturation might be the elevated phosphorylation of serine 2 of the RNA Polymerase II which is known to be followed by recruiting polyadenylating factors.
Extracts of Boswellia serrata, also known as Indian frankincense, have been used to treat inflammatory diseases in the Indian ayurvedic medicine or Chinese traditional medicine (TCM) for over 3000 years, but the molecular mechanisms of the anti-inflammatory effects are still not well understood. It is obvious that the boswellic acids, the major compounds in the extracts, are responsible for the efficacy. This work employed a protein fishing technique to identify putative targets of boswellic acids at different stages within the inflammatory cascade. For fishing experiments, boswellic acids were immobilized to sepharose and incubated with cell lysates. After washing and boiling, fished proteins were separated by SDS-PAGE and analysed by MALDI-TOF-MS. CatG, DNA-PK and the protein kinase Akt were identified by protein pulldowns with immobilised BAs and characterised as selective and important targets for BAs with an IC50 in the range of physiologically achievable plasma levels up to 5 microM. In addition, the influence on several signal transductions by BAs was tested. Calcium influx, arachidonic acid release, platelet aggregation and TNFalpha-release were assayed to reveal further pharmacological effects of BAs. Celecoxib is a well-known selective COX-2 inhibitor that is in clinical use. In this work, it is demonstrated that celecoxib is also a highly potent direct 5-LO inhibitor. Celecoxib is used in arthritis and its gastro-intestinal side effects are reduced compared to non-selective NSAIDs. In patients with a familiar disposition to polyp forming, celecoxib reduced polyps and the incidence of colon cancer. Because of lowered leukotriene levels in patients under celecoxib therapy it was plausible to test whether celecoxib interferes with 5-LO. Here it is shown that the activity of 5-LO is inhibited in PMNL and cell-free assays with IC50 of 8 microM in intact cells, 20 microM with supplemented arachidonic acid and 30 microM in cell-free systems. Thus, celecoxib is a dual inhibitor of COX-2 and 5-LO. Since 2006, celecoxib has been approved as an orphan drug for the treatment of familial adenomatous polyposis. Aside from this indication, it could be useful for treatment of asthma and other diseases where 5-LO is implicated.