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Der Einfluss molekularer Mechanismen auf die Expression des NO-sensitiven Rezeptors lösliche Guanylatzyklase (sGC)

By Rashi Srivastava

Abstract

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO) which leads to the activation of GMP dependent protein kinases and to vasodilation. NO signaling may be affected by altered expression of sGC subunits, as has been shown in different pathological and physiological conditions and developmental stages. The molecular mechanisms underlying altered sGC expression in these and other conditions have not yet been revealed. Gene expression can also be regulated at the level of mRNA through alterations in translational efficiency and in mRNA stability. HuR (Human R) is a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins. Among other RNAs, there has been recent evidence that the expression of sGC is subject to post-transcriptional regulation by HuR. It has been shown that chronic hypertension induces changes in HuR expression and activity, which account for decreased sGC expression and activity in the aorta of hypertensive rats. This thesis should study was performed in an effort to provide some insight to the transcriptional and post-transcriptional regulation of sGC expression in a mammal, the rat. We investigated rat sGC alpha-1 transcriptional regulation in rat lung fibroblast (RLF-6) cells. The 3000bp 5' upstream region of the alpha-1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs- Alpha3000 (with -2794 bp), Alpha1100 (-1092 bp), Alpha350 (-346 bp) and Alpha200 (-200 bp). The promoter activity was the highest in the 200bp construct (about 6-fold higher than Alpha3000) suggesting that this fragment contains all the crucial elements necessary to support basal transcription of the alpha-1 sGC gene. Analysis of the 200 bp of the 5’ UTR of the alpha-1 gene was performed using the MATINSPECTOR V2.2 software for putative transcription factors. The constructs containing the deleted sites for NFY and Sp1 showed a significant decrease in constitutive promoter activity by almost 80% and 60% respectively, implying that these transcription factors are crucial elements in the basal expression of the of sGC alpha-1 subunit. Treatment of RLF-6 cells with genistein 50 microM and mithramycinA 100 nM, known to inhibit the NFY and Sp1 binding to DNA respectively, reflected the same effects. Furthermore the cGMP content of the cells was significantly reduced by both inhibitors, almost completely by genistein, and by about 40 % by mithramycinA. Electrophoretic mobility-shift assay (EMSA) clearly showed the formation of multiple complexes with the biotinylated ODN (decoy oligodeoxynucleotide) probes for NFY and Sp1 when incubated with RLF-6 nuclear extract. A “supershift” observed in the presence of antibodies to the individual transcription factors confirmed that these factors were present in the shifted band, indeed. NFY and Sp1 are instrumental in several physiological and pathophysiological effects mediated by several growth factors in smooth muscle cells. Thus the regulation of the promoter, in response to serum, was also analysed. 10% foetal calf serum led to decreased alpha-1 sGC level as shown by western blots performed with rat aorta. Decreased sGC alpha-1 mRNA expression was observed in RLF-6 cells and cultured rat aortic smooth muscle cells incubated with FCS for 24 hours. This decrease was reflected in the promoter activity in RLF-6 cells using both Alpha3000 and Alpha200 constructs confirming that the regulation took place at promoter level. EMSA performed with nuclear extracts from FCS treated RLF-6 cells led to diminished binding to NFY, but to an enhanced binding to Sp1 site. We concluded that the factors Sp1 and NFY (the sites overlapping) compete for binding, and in the presence of FCS, it is Sp1 that binds stronger, and hence results in diminishing promoter activity. In order to delineate the post-transcriptional regulation of sGC alpha-1 subunit, studies were performed to demonstrate the regulation of expression of the mRNA stabilizing protein HuR. It has been observed that exposure of isolated rat aortic segments to the activator of adenylyl cyclase, forskolin, strongly reduced sGC alpha-1/beta-1 and HuR protein and mRNA expression in a time-dependent and actinomycin D-sensitive fashion. Transcription factor decoy approach proved that the cAMP-induced down-regulation of HuR is mediated by the activation of AP-1. It has been established that HuR stabilises the sGC alpha-1 and beta-1 mRNA. However the pathway underlying this regulation remains unknown. In order to identify the mechanism of this regulation, we looked for HuR interacting proteins employing the yeast two hybrid assay. The enzyme of the polyamine catabolic pathway spermidine/spermine N1-acetyltransferase (SSAT) was found to interact with the hinge region of HuR. This interaction was confirmed by performing immunoprecipitation and GST-pulldown experiments. A direct effect of these proteins on each other’s biological activity was not visible as tested through the SSAT activity assay and HuR gel shift. It might be possible that SSAT-mediated modulation of local polyamine concentrations enhances/reduces HuR activity and sGC expression to affect cell proliferation. In summary, this study represents an analysis of the rat sGC alpha-1 promoter regulation in rat fibroblast cells and identifies NFY and Sp1 as important factors in sGC alpha-1 expression. It also gives first evidence of sGC regulation at the transcriptional level in response to an external stimulus, and proposes the possible mechanism. It also identifies SSAT as a HuR interacting protein. These might have implications in the various pathophysiological conditions where sGC plays an important role.Die lösliche Guanylatzyklase (soluble guanylyl cyclase, sGC) ist ein Schlüsselenzym der NO/cGMP Signaltransduktion im kardiovaskulären System. Nach Aktivierung der sGC durch Stickstoffmonoxid (NO) kommt es in glatten Muskelzellen zur Produktion von zyklischem 3’,5’-Guanosinmonophosphat (cGMP), das als intrazelluläres Signaltransduktionsmolekül die Aktivierung von cGMP-abhängigen Proteinkinasen vermittelt und zur Vasodilatation führt. Die NO/cGMP-Signaltransduktion kann auch auf der Ebene der Gen-Expression beeinflusst werden. So konnte in verschiedenen physiologischen bzw. pathophysiologischen Modellen gezeigt werden, dass die unterschiedliche Expression von sGC-Untereinheiten die NO/cGMP-Signaltransduktion beeinflusst. Die molekularen Mechanismen, die auf der unterschiedlichen Expression der sGC-Untereinheiten basieren, wurden bisher noch nicht ausreichend aufgeklärt. Die Gen-Expression kann auf mRNA-Ebene durch Veränderung der Translation und der mRNA- (messenger ribonucleic acid, Boten-Ribonnukleinsäure) Stabilität reguliert werden. HuR (Human R) ist ist ein ubiquitär vorkommendes Mitglied der embryonic lethal abnormal vision (ELAV-) Familie der RNA-bindenden Proteine. Neben der Regulierung anderer Gene gibt es Hinweise darauf, dass auch die Expression der sGC einer post-translationalen Regulation durch HuR unterliegt. So wurde gezeigt, dass chronischer Bluthochdruck Veränderungen in der HuR-Expression und -Aktivität verursacht, was zu einer verminderten sGC-Expression und -Aktivität in der Aorta von hypertensiven Ratten führt. Diese Arbeit soll einen Einblick in die transkriptionale und post-transkriptionale Regulation der sGC-Expression im Rattenmodell liefern. Im Rahmen dieser Arbeit wurde die translationale Regulation der alpha-1 sGC-Untereinheit der Ratte in Rattenlungen-Fibroblastenzellen (rat lung fibroblast cells, RLA-6) untersucht. Es wurden die 3000 bp der 5´ upstream Region des Gens der alpha-1 sGC-Untereinheit isoliert und mit Hilfe von Luziferase-Reporter-Konstrukten - Alpha3000 (mit -2794 bp), Alpha1100 (-1092 bp), Alpha350 (-346 bp) und Alpha200 (-200 bp) - auf Promotor-Aktivität untersucht. Dabei war die Promotor-Aktivität des 200 bp Konstrukts am höchsten (ca. 6-fach im Vergleich zu Alpha3000). Dies deutete daraufhin, dass dieses Fragment entscheidende Elemente enthält, die für die basale Transkription des alpha-1 sGC Gens nötig sind. Um nach vermeintlichen Transkriptionsfaktoren zu suchen, wurde die Analyse der 200 bp am 5´ UTR des alpha-1 Gens mit der MATINSPECTOR V2.2 Software durchgeführt. Die Konstrukte mit den entfernten Stellen für NFY und Sp1 zeigten eine signifikante Verminderung in der konstitutiven Promotor-Aktivität zu fast 80 und 60%. Dies deutete daraufhin, dass diese Transkriptionsfaktoren entscheidend sind für die basale Expression der alpha-1 sGC-Untereinheit. Die Behandlung der RLF-6 Zellen mit Genistein (50 mikroM) und Mithramycin A (100 nM), die die Anbindung von NFY und Sp1 an die DNA inhibieren, führte ebenfalls zu einer Reduktion der Promotor-Aktivität. Außerdem war der Gehalt von cGMP in den Zellen durch die beiden Inhibitoren signifikant reduziert - mit Genistein wurde eine fast komplette Inhibition erreicht und mit Mithramycin A eine Reduktion um ca. 40%. Der EMSA (electrophoretic mobility-shift assay) zeigte deutlich die Bildung von multiplen Komplexen mit biotinylierten ODN (decoy oligodeoxynucleotide) Proben für NFY und Sp1, wenn diese mit RLF-6 Zellkern-Extrakt inkubiert wurden. In einem „supershift“ konnten die einzelnen Transkriptionsfaktoren mit Antikörpern nachgewiesen werden. NFY und Sp1 sind an zahlreichen physiologischen und pathophysiologischen Prozessen in glatten Muskelzellen beteiligt, die durch Wachstumsfaktoren gesteuert werden. Aus diesem Grund wurde die Regulation des Promotors in Gegenwart von Serum analysiert. Mit Western-Blot Analysen konnte gezeigt werden, dass 10 % fötales Kälber-Serum (foetal calf serum, FCS) zu einer verminderten Expression von alpha-1 sGC in Rattenaortenringen führte. Eine verminderte sGC alpha-1 mRNA-Expression wurde in RLF-6 Zellen und in kultivierten glatten Muskelzellen der Rattenaorta, die mit FCS über 24 Stunden inkubiert wurden, demonstriert. Diese Verminderung ging einher mit einer verminderten Promotor-Aktivität der Konstrukte Alpha3000 und Alpha200 in RLF-6 Zellen, was eine Regulation auf Promotor-Ebene aufzeigte. EMSA Studien mit Zellkern-Extrakt aus FCS-vorbehandelten RLF-6 Zellen zeigten eine verminderte NFY-, aber eine gesteigerte Sp1-Bindung. Daraus wurde gefolgert, dass die Faktoren Sp1 und NFY um eine Bindungsstelle am Promotor konkurrieren und dass die Gegenwart von FCS zu einer verstärkten Sp1-Bindung und damit zu einer verminderten Promotor-Aktivität führt. Um die post-transkriptionale Regulation der alpha-1 sGC-Untereinheit zu demonstrieren, wurden Versuche zur Regulation der Expression des mRNA-stablisierenden Proteins HuR durchgeführt. Es konnte gezeigt werden, dass die Inkubation von isolierten Rattengefäßringen mit Forskolin, einem Aktivator der Adenylatzyklase, die sGC alpha-1/beta-1 und die HuR Protein- und mRNA-Expression zeitabhängig und Actinomycin D-sensitiv reduziert. Der Transkriptionsfaktor-Abbau bewies, dass die cAMP-induzierte Runterregulation von HuR durch die Aktivierung von AP-1 vermittelt wird. Es konnte festgestellt werden, dass HuR die alpha-1 und beta-1 mRNA der sGC stabilisiert. Aber die Regulation dieser Signalwege war bisher unbekannt. Um den Mechanismus der Regulation zu identifizieren, haben wir nach HuR-interagierenden Proteinen durch Anwendung des Yeast-two-hybrid assays gesucht. Die Enzyme des Polyamin-katabolischen Signalwegs Spermidin/ Spermin N1-Acetyltransferase (SSAT) wurden als eine hinge Region von HuR entdeckt. Diese Interaktion wurde bestätigt durch Immunopräzipitation und GST-pulldown Experimente. Einen direkten gegenseitigen Effekt auf die Aktivität dieser Proteine war nicht sichtbar, was durch SSAT activity assays und HuR gel shifts getested wurde. Es könnte möglich sein, dass die SSAT-vermittelte Modulation der lokalen Polyamin-Konzentration die HuR-Aktivität und die sGC-Expression fördert bzw. vermindert bei der Zellproliferation. Diese Arbeit zeigt eine Analyse der Promotor-Regulation der alpha-1 sGC-Untereinheit der Ratte in Fibroblastenzellen der Ratte und identifiziert NFY und Sp1 als wichtige Faktoren in der alpha-1 sGC-Expression. Die sGC-Regulation auf transkriptionaler Ebene unter Beteiligung der Transkriptionsfaktoren NFY und Sp1, ausgelöst durch Serum als externen Stimulus, weist auf einen möglichen Mechanismus hin. Außerdem wurde SSAT als HuR-Interaktionsprotein identifiziert. Die in dieser Arbeit dargestellte Regulation der sGC-Expression könnte Einfluss haben auf verschiedene pathophysiologische Zustände

Topics: Guanylatcyclase, Genexpression, Stickstoffmonoxid, RNS, RNS-Bindungsproteine, Yeast-Two-Hybrid-System, ddc:570
Year: 2006
OAI identifier: oai:publikationen.ub.uni-frankfurt.de:1929

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