RNA-dependent association with myosin IIA promotes F-actin-guided trafficking of the ELAV-like protein HuR to polysomes

The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase Cδ (PKCδ)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKCδ remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes

CXCL16 and oxLDL are induced in the onset of diabetic nephropathy

Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated α3-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored α3 integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN

Studies on the structure and function of the multienzyme enniatin synthetase

Doller, Anke: Untersuchungen zur Struktur und Funktion des Multienzyms Enniatinsynthetase Enniatine haben vielfältige pharmakologische Eigenschaften und sind daher von hohem biotechnologischen Interesse. Diese Verbindungen gehören zur Klasse der Cyclodepsipeptide und werden nichtribosomal von einem Multienzym, der Enniatinsynthetase (ESYN) gebildet. Enniatine bestehen aus jeweils drei Molekülen der D-2-Hydroxyisovaleriansäure und einer N-methylierten L-Aminosäure. Diese Grundbausteine sind alternierend durch Peptid- und Esterbindungen verknüpft. Im Gegensatz zu anderen Peptidsynthetasen besitzt das Enzym nur zwei Reaktionsmodule (EA und EB), die Zweierbausteine synthetisieren, die in einem iterativen Prozess verknüpft werden. Ziel der Arbeit war es, durch den Einsatz biochemischer und molekularbiologischer Methoden weitere Erkenntnisse über die Struktur und Funktion dieses Enzyms zu erhalten. Ein Hauptpunkt der Untersuchungen bildete die funktionale Expression der Enniatinsynthetase. Als Expressionsorganismus wurde Streptomyces gewählt, da in diesem Organismus bereits eine ganze Reihe von Enzymen des Sekundärmetabolismus exprimiert wurden. Dazu wurde ein Konstrukt aufgebaut, welches das gesamte Gen der ESYN enthält. Da der Codongebrauch der Streptomyceten unterschiedlich zu dem von Fusarien ist, musste dieser N-terminal angeglichen werden. Der Aufbau des Konstrukts erfolgte in 7 Stufen in einem E. coli/ Streptomyces Shuttlevektor. Expressionsversuche in S. lividans blieben trotz intensiven Screenings erfolglos, ebenso in dem Depsipeptidproduzenten S. tsusimaensis. Der Grund könnte darin liegen, dass die positiven Transformanden sensitiv auf das entstehende Enniatin sind und dadurch nicht überleben. Deswegen wurden nur Transformanden gefunden, bei denen das Insert aus dem Vektor eliminiert wurde. Weiter wurde die molekulare Basis der Substraterkennung an zwei Enniatinsynthetasen unterschiedlicher Substratspezifität untersucht. Interessanterweise lässt sich die Spezifität durch die von Stachelhaus (1999, Chem. & Biol. 6: 493-505) und Challis (2000, Chem. & Biol. 7: 211-224) vorgeschlagenen Modelle nicht vorhersagen. Dies zeigt, dass die von bakteriellen Systemen abgeleiteten Modelle nicht ohne weiteres auf pilzliche Systeme übertragbar sind. Eine weitere Analyse des Enniatinsynthetasegens ergab im Gegensatz zu den bisher bekannten Peptidsynthetasen drei Kondensationsdomänen, die in einem iterativen Synthesemechanismus auch gefordert werden. Eine Kondensation wird für die Ausbildung der Peptidbindung benötigt, zwei weitere für die Elongation und finale Zyklisierungsreaktion. Dies ist eine weitere Bestätigung für den postulierten Mechanismus. Es konnte durch Cross-Linking-Experimente gezeigt werden, dass in der Enniatinsynthetase N- und C-Terminus dicht beieinander liegen, was darauf hinweist, dass die beiden Reaktionsmodule in unmittelbarer Nachbarschaft zueinander liegen müssen. Dies konnte bereits durch die Ergebnisse des Epitop-Mapping mit monoklonalen Antikörpern postuliert werden.Doller, Anke: Studies on the structure and function of the multienzyme enniatin synthetase Enniatins are cyclohexadepsipeptides and constitute a class of pharmacologically interesting compounds and are of high biotechnological interest. They are synthesized nonribosomally by the large multifunctional enzyme enniatin synthetase (ESYN) following a thiotemplate mechanism. Enniatins are composed of alternating residues of D-hydroxyisovaleric acid and a branched-chain N-methyl-L-amino acid linked by peptide and ester bonds. In contrast to other peptide synthetases ESYN is a two-module (EA and EB) enzyme, which catalyzes biosynthesis in an interative manner. The goal of the present work was to get more insight into the structure and function of ESYN by the use of biochemical and molecular biolological methods. The main focus of the studies was the functional expression of ESYN. Streptomyces was used as the host for expression, because in this organism several enzymes of secondary metabolism could already be expressed. Therefore a construct was made representing the whole ESYN gene. The codon usage of Streptomyces differs from that of Fusaria, so it had to be adapted at the N-terminus. The construction of the plasmid was done in 7 steps in a Escherichia coli/Streptomyces shuttle vector. Despite intensive screening, expression in S. lividans and in the depsipeptide producer S. tsusimaensis was unsuccessful. The reason for that might be the toxic effect of enniatin on positive tranformants. Thus, only transformants could be detected, where the insert had been eliminated. In a second part the molecular basis of substrate recognition on two different enniatin synthetases was examined. Interestingly , the value of the methods of Stachelhaus et al. (1999, Chem. & Biol. 6: 493-505) and Challis et al. (2000, Chem. & Biol. 7: 211-224) is limited for the identification of the amino acids activated. This shows that results obtained from bacterial models cannot be transferred to fungal systems. Further analysis of the enniatin synthetase gene revealed, in contrast to other known peptide synthetases, the existence of three condensation domains, necessary in an iterative mechanism of biosynthesis. One condensation domain is required for peptide bond formation, the two other domains are necessary for elongation and final cyclization, respectively. This is a further confirmation for the postulated iterative mechanism. Furthermore, it could be shown by cross-linking experiments, that the C- and N-terminus of ESYN as well as modules EA and EB are located close to each other. This was also postulated by the results of epitope-mapping using monoclonal antibodies

Tandem Phosphorylation of Serines 221 and 318 by Protein Kinase Cδ Coordinates mRNA Binding and Nucleocytoplasmic Shuttling of HuR▿

Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cδ (PKCδ)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCδ mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D1, and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCδ coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions

Lymphotoxin α, a novel target of posttranscriptional gene regulation by HuR in HepG2 cells

AbstractThe role of the RNA-binding protein human antigen R (HuR) in hepatocarcinogenesis is still elusive. By employing short hairpin (sh)RNA-dependent knockdown approach, we demonstrate that lymphotoxin α (LTα) is a target of posttranscriptional gene regulation by HuR in hepatocellular carcinoma (HepG2) cells. Consequently, the increased mRNA decay upon HuR depletion significantly affects lymphotoxin expression at both, the mRNA and protein level. Biotin-pulldown assay showed that HuR specifically interacts with the 3′-untranslated region (3′-UTR) of the LTα mRNA. Furthermore, electrophoretic mobility shift assay (EMSA) implicates that the RNA-binding critically depends on the RNA recognition motif 2 (RRM2) and the hinge region of HuR

Posttranslational Modification of the AU-Rich Element Binding Protein HuR by Protein Kinase Cδ Elicits Angiotensin II-Induced Stabilization and Nuclear Export of Cyclooxygenase 2 mRNA▿

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3′ untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cδ (PKCδ), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCδ-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCδ represents an important novel mode of HuR activation implied in renal COX-2 regulation

Nitric oxide induces TIMP-1 expression by activating the transforming growth factor beta-Smad signaling pathway

Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression