Influence of environmental factors on the regulation of metalloproteases ADAM10 and ADAM17

Die Metalloproteasen ADAM10 und ADAM17 sind für die Proteolyse diverser Zelladhäsionsmoleküle, Wachstumsfaktoren, Zytokine und auch von deren Rezeptoren verantwortlich, welche in vielen physiologischen und pathologischen Prozessen eine wichtige Rolle spielen. Aus diesem Grund steht die Regulation der Proteasen in einem besonderen Fokus vieler Forschergruppen. Gleichzeitig rückte in den letzten Jahren auch der Einfluss von Umweltfaktoren auf verschiedene zelluläre Prozesse und die Entstehung von beispielsweise inflammatorischen Erkrankungen zunehmend in den Mittelpunkt. Inwiefern diese Faktoren auch die Metalloproteasen beeinflussen könnten, ist bislang jedoch noch kaum untersucht. Im Hauptteil der vorliegenden Arbeit wurde daher der Frage nachgegangen, inwieweit Fettsäuren aus der Nahrung ADAM10 und ADAM17 in einem Mausmodell der Atherosklerose (low density lipoprotein-Rezeptor-Knock-out, LDLR-/-) regulieren. Eine wichtige Rolle beider Proteasen in der Atherogenese ist wahrscheinlich, da sie für die Prozessierung einer Reihe von Entzündungsmediatoren und Zelladhäsionsmolekülen verantwortlich sind, die in die Entstehung der Atherosklerose involviert sind. Zahlreiche Studien zeigten in der Vergangenheit, dass Fischöl, welches reich an mehrfach ungesättigten Omega-3-Fettsäuren ist, protektiv auf die Atherogenese wirkt. Dies konnte im Rahmen dieser Arbeit bestätigt werden. Hier wurden Wildtyp- und LDLR-/--Mäuse mit einer fettreichen Diät gefüttert, deren Fettanteil entweder hauptsächlich aus gesättigten Fettsäuren (Schweineschmalz) bestand oder mit Fischöl angereichert war. Es konnte gezeigt werden, dass Fischöl-reiche Nahrung zu weniger atherosklerotischen Plaques in den Aorten der LDLR-/--Mäuse führte, als eine Schweineschmalz-angereicherte Diät. Dieser anti-atherogene Effekt von Fischöl konnte nicht auf eine verminderte systemische Inflammation zurückgeführt werden, da die Konzentration zirkulierender inflammatorischer Marker nicht beeinflusst wurde. Die Reduktion atherosklerotischer Läsionen korrelierte jedoch mit einer signifikant verringerten Konzentration zirkulierender endothelialer Zelladhäsionsmoleküle, die Substrate von ADAM10 und ADAM17 sind. Da sich die Proteinexpression der Proteasen nicht veränderte, kann spekuliert werden, dass eine reduzierte Aktivität hierfür verantwortlich ist. Im Ergebnis zeigen diese Daten, dass Fischöl aus der Nahrung die proteolytische Aktivität von ADAM10 und ADAM17 in vivo beeinflussen kann. Eine reduzierte Prozessierung endothelialer Zelladhäsionsproteine könnte die Barrierefunktion des Endothels verbessern und so zu einer verminderten Lipoproteininsudation und Makrophagen-akkumulation in der Intima führen, was letztlich der Atherogenese entgegen wirkt. In einem weiteren Teil dieser Arbeit wurde eine mögliche ernährungsspezifische Regulation von ADAMs durch Isothiocyanate untersucht. Diese sekundären Pflanzenstoffe sind in vielen Kreuzblütlergewächsen wie Senf oder Brokkoli zu finden und Gegenstand der Forschung aufgrund ihrer potenziellen antioxidativen, antiinflammatorischen und antikanzerogenen Eigenschaften. Die hier gewonnenen Daten lassen vermuten, dass Isothiocyanate in der Lage sind, die Phosphorylierung der MAP-Kinasen ERK1/2 in einer epidermalen Keratinozyten-Zelllinie zu stimulieren. Der Einsatz verschiedener Inhibitoren, ließ den Schluss zu, dass dies auf eine Metalloprotease-abhängige Aktivierung des epidermal growth factor receptor EGFR zurückzuführen ist. Letztlich scheiterten weitere Versuche diesen Mechanismus zu reproduzieren und im Detail zu untersuchen. Es konnte daher nicht abschließend geklärt werden, inwiefern Isothiocyanate die Aktivität von ADAMs in diesem Zellsystem modulieren können. Neben der Ernährung ist die Beeinflussung der ADAMs durch zahlreiche weitere Umweltfaktoren denkbar. Im dritten Teil der Arbeit wurde daher untersucht, ob eine hypoxische Umgebung der Zellen die Proteasen beeinflusst. Eine verminderte Sauerstoffverfügbarkeit tritt beispielsweise sehr häufig in Tumoren und Entzündungsherden auf und eine erhöhte Expression von ADAM17 wird mit diversen Tumorarten assoziiert. Das Mikro-Milieu neoplastischer Zellen ist für die Tumorgenese von entscheidender Bedeutung, da es Eigenschaften wie Proliferation, Invasivität und die Metastasierung enorm beeinflussen kann. Es konnte hier gezeigt werden, dass eine 48-stündige Langzeitstimulation der epithelialen Karzinoma-Zelllinie HEp-2 mit Interferon γ zu einer erhöhten mRNA-Expression von ADAM17 führte, was mit einer erhöhten Prozessierung des Substrates TNFR1 korrelierte. Eine Kultivierung der Zellen unter Hypoxie verhinderte diese Stimulation jedoch. Die molekularen Mechanismen dieser differentiellen Regulation von ADAM17 konnten im Rahmen dieser Arbeit zwar nicht geklärt werden, die hier gewonnenen Erkenntnisse weisen dennoch darauf hin, dass die Sauerstoffverfügbarkeit bei Experimenten, die sich mit der Regulation von ADAM17 in Tumoren beschäftigen, eine größere Beachtung finden sollte. Zusammenfassend lieferte diese Arbeit neue Erkenntnisse zur Regulation der Aktivität von ADAM10 und ADAM17 durch verschiedene Umweltfaktoren. Ein besseres Verständnis der Regulationsmechanismen könnte zur Entwicklung neuer therapeutischer Strategien für ADAM-assoziierte Krankheiten wie kardiovaskulären Erkrankungen, Entzündungen und verschiedenen Tumorarten beitragen. Zusätzlich zu pharmazeutischen Interventionen, könnte dies beispielsweise auch eine Änderung von Lebensgewohnheiten wie der Ernährung umfassen.The metalloproteases ADAM10 and ADAM17 are responsible for the proteolysis of a number of cell adhesion molecules, growth factors, cytokines and even their receptors. Such molecules play important roles in physiological and pathological processes. The regulation of ADAM proteases is therefore of major importance for many research groups. In this respect, the influence of environmental factors on several cellular processes has become an emerging field of interest due to the potential impact of these factors on the development of for example inflammatory diseases. However, so far little is known about a potential regulation of ADAM10 and ADAM17 by environmental factors. Therefore, the main part of this thesis deals with the question whether dietary fatty acids might regulate ADAM10 and ADAM17 in an atherosclerosis mouse model (low density lipoprotein receptor knockout, LDLR-/-). An important role for both proteases in this disease can be estimated, since a number of inflammatory mediators and cell adhesion molecules, who are involved in atherogenesis, are actual substrates for ADAM10 and -17. It has already been shown that polyunsaturated omega-3 fatty acids of fish oil exert protective effects in atherosclerosis. This could be confirmed in the present work. In this study, wild-type and LDLR-/- mice were fed a high fat diet supplemented with either lard (rich in saturated fatty acids) or fish oil. The fish oil-supplemented diet led to less atherosclerotic lesion development in the aorta of LDLR-/- animals than the lard-rich diet. This anti-atherogenic effect of fish oil was not due to a reduction of systemic inflammation, since the plasma concentrations of several classical inflammatory molecules were not reduced. In contrast, the reduction of atherosclerotic lesions in fish oil-fed animals correlated with a significant reduction of a couple of circulating endothelial cell adhesion molecules that are substrates for ADAM10 and ADAM17. This was not accompanied by changes in the protein expression of the proteases, suggesting that this can be rather ascribed to a reduced proteolytic activity. Taken together, evidence is provided here, that fish oil exerted an anti-atherogenic effect in LDLR-/- mice which was accompanied by a reduced release of ADAM17 and ADAM10 substrates from endothelial cells. It is suggested that fish oil-decreased ADAM activity contributes to improved endothelial barrier function and thus counteracts intimal lipoprotein insudation and macrophage accumulation. A second part of this thesis dealt with another possible dietary regulation of ADAMs by isothiocyanates. These secondary plant metabolites are present in cruciferious vegetables such as mustard and broccoli and are of particular interest thanks to their antioxidative, anti-inflammatory and anti-cancerous properties. Results obtained here indicate, that isothiocyanates are able to stimulate the phosphorylation of the MAP-kinases ERK1/2 in an epidermal keratinocyte cell line. The use of different inhibitors suggested, that this is due to a metalloprotease-mediated activation of the epidermal growth factor receptor (EGFR). However, other experiments failed to reproduce these data and analyse the mechanisms in detail. In this respect, it cannot be concluded with certainty if isothiocyanates regulate the activity of ADAMs. Apart from nutrition, it is possible that ADAMs can be influenced by a number of other environmental factors. Hence, a third part of this work analysed whether a hypoxic environment has an impact on ADAM10 or ADAM17 activity. Reduced oxygen availability is present in many cancers and at sites of inflammation and ADAM17 is associated with several types of cancer. The micro-environment of neoplastic cells is of uppermost importance for tumorigenesis, since it can greatly influence processes such as proliferation, invasiveness and metastasis. In this work, a long-term (48 hrs) stimulation of the epithelial carcinoma cell line HEp-2 with interferon γ led to an increased mRNA expression of ADAM17, which correlated with an increase in the processing of tumor necrosis factor α receptor 1. Surprisingly, incubation of the cells in a hypoxic environment completely abrogated this stimulation. While the molecular mechanisms underlying this effect could not be addressed in this work, it nevertheless points out, that the oxygen availability has to be carefully taken into account in experiments that analyse the regulation of ADAM17 in cancer tissue. Taken together, the results of this thesis provide new insights into the regulation of ADAM activity by several environmental factors. A detailed understanding of the regulatory mechanisms could possibly contribute to the development of new therapeutic strategies for ADAM-associated diseases such as cardiovascular diseases, inflammation or certain types of cancer. Apart from pharmaceutical intervention, one such strategy could be a change in lifestyle like nutrition

Two Factors that Bind to Highly Conserved Sequences in Mammalian Type C Retroviral Enhancers.

The transcriptional enhancers of the Moloney and Friend murine leukemia viruses (MLV) are important determinants of viral pathogenicity. We used electrophoretic mobility shift and methylation interference assays to study nuclear factors which bind to a region of these enhancers whose sequence is identical between Moloney and Friend viruses and particularly highly conserved among 35 mammalian type C retroviruses whose enhancer sequences have been aligned (E. Golemis, N. A. Speck, and N. Hopkins, J. Virol. 64:534-542, 1990). Previous studies identified sites for the leukemia virus factor b (LVb) and core proteins in this region (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987) as well as a site, overlapping those for LVb and core, for a third factor (N. R. Manley, M. A. O\u27Connell, P. A. Sharp, and N. Hopkins, J. Virol. 63:4210-4223, 1989). Surprisingly, the latter factor appeared to also bind two sites identified in the Friend MLV enhancer, Friend virus factor a and b1 (FVa and FVb1) sites, although the sequence basis for the ability of the protein to bind these diverse sites was not apparent. Here we describe the further characterization of this binding activity, termed MCREF-1 (for mammalian type C retrovirus enhancer factor 1), and the identification of a consensus sequence for its binding, GGN8GG. We also identify a factor, abundant in mouse T-cell lines and designated LVt, which binds to two sites in the Moloney MLV enhancer, overlapping the previously identified LVb and LVc binding sites. These sites contain the consensus binding site for the Ets family of proteins. We speculate on how distinct arrays of these factors may influence the disease-inducing phenotype

The Leukemic Protein Core Binding Factor Beta (CBFbeta)-Smooth-Muscle Myosin Heavy Chain Sequesters CBFalpha2 into Cytoskeletal Filaments and Aggregates

The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor β (CBFβ) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFβ-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFα1 to 3) and a non-DNA binding subunit (CBFβ). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBFβ-SMMHC, CBFβ, and CBFα2 (also known as AML1 or PEBP2αB). When expressed individually, CBFα2 was located in the nuclei of transfected cells, whereas CBFβ was distributed throughout the cell. On the other hand, CBFβ-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFα2 was able to drive localization of CBFβ into the nucleus in a dose-dependent manner. In contrast, CBFα2 colocalized with CBFβ-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFα-interacting domain within CBFβ-SMMHC abolished this CBFα2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFβ-SMMHC when coexpressed with CBFα2. CBFα2 sequestration by CBFβ-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFβ-SMMHC plays a dominant negative role by sequestering CBFα2 into cytoskeletal filaments and aggregates, thereby disrupting CBFα2-mediated regulation of gene expression. The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a cytogenetic abnormality consistently associated with acute myeloid leukemia (AML) subtype M4Eo (2, 21), a variant of subtype M4 with abnormal eosinophils in the bone marrow and sometimes in the peripheral blood. The inversion results in the reciprocal fusions of two genes: theMYH11 gene (16p13), which encodes the smooth-muscle myosin heavy chain (SMMHC), and the CBFB gene (16q22), which encodes the β subunit of the core binding factor (CBFβ) (24). The chimeric gene CBFB-MYH11 fuses most of the 5′ coding region of CBFB in frame with the 3′ portion of MYH11, resulting in the production of the chimeric protein CBFβ-SMMHC. The reciprocal fusion,MYH11-CBFB, is not believed to be important since its expression is below detectable levels in leukemic cells and it is deleted in some patients with an unbalanced inversion (24, 25,29). CBFβ is the heterodimeric partner of CBFα proteins, and together they constitute the core binding factors (CBF). CBF was initially identified as a transcriptional regulator of Moloney murine leukemia virus (50, 51) and polyomavirus (4, 18, 36, 37,43) in mice, and it was subsequently shown to be an important transcriptional activator of genes involved in mammalian hematopoiesis and bone development (5, 12, 14, 15, 20, 32, 40, 46). Monomeric CBFα proteins bind DNA, albeit weakly (3, 50). Although CBFβ does not make any detectable direct contact with DNA (50), it enhances the DNA binding affinity of the CBFα proteins (4, 36). While CBFβ is expressed from a single gene in the human and mouse, there are three CBFα genes, all of which encode the so-called runt domain (3, 22,54), which is required for both DNA binding and interaction with CBFβ. One of the three genes, CBFA2, also known as AML1 or PEBP2αB, is located on human chromosome 21 and is involved in several different leukemias as a result of translocations (16, 31, 34, 35, 41). Chromosomal inversions and translocations involving either CBFB orCBFA2 are the most frequent cytogenetic abnormalities in human AMLs (27). Gene-targeting experiments in mice have demonstrated that the CBFα2 and CBFβ subunits are likely to function together as a complex in vivo. Homozygous disruption of either Cbfa2 (39,48) or Cbfb (42, 49) in mice produces an identical phenotype: both Cbfa2 −/− andCbfb −/− embryos demonstrate a failure of definitive hematopoiesis in the liver, and in both cases the embryos die at around day 12.5 due to extensive hemorrhages. The inv(16) chimeric gene CBFB-MYH11 has been shown to exert a dominant negative effect in vivo by a mouse knock-in experiment (8). CBFB-MYH11was introduced into the mouse genome to replace one copy of theCbfb gene. The expression of this chimeric gene was controlled by the endogenous Cbfb promoter, thus simulating the condition in leukemic patients. CBFβ-SMMHC was found to dominantly suppress the function of the CBFα2:CBFβ heterodimer, since mouse embryos heterozygous for the knock-inCbfb-MYH11 gene (CbfbCBFB-MYH11/+ ) displayed a phenotype similar to that of Cbfb−/− andCbfa2−/− embryos, i.e., failure of definitive hematopoiesis and midgestation lethality. In vitro, the chimeric protein was shown to retain its ability to interact with CBFα proteins and participate in the formation of protein-DNA complexes (23). Although presence of the chimeric protein reduces CBF DNA-binding activity in cultured Ba/F3 lymphoid and 32D c13 myeloid cells (6), it is not clear how this reduction was achieved. Unlike wild-type CBFβ, CBFβ-SMMHC can potentially form dimers and multimers via the rod-like domain of the myosin chain (23, 25). Two possible mechanisms could explain the dominant negative effect of the chimeric CBFβ-SMMHC protein. One is that CBFβ-SMMHC, via heterodimerization with CBFα2, can assemble into a ternary complex at the core sites within promoters of target genes and interfere with the regulation of gene expression. The second possibility is that CBFβ-SMMHC, with its capacity to form multimers, can sequester CBFα2 into nonfunctional complexes, thus preventing it from regulating transcription of target genes. Previous studies by our group demonstrated that CBFβ-SMMHC can form rod-like nuclear structures as well as cytoplasmic stress fibers in NIH 3T3 cells stably transfected with a CBFB-MYH11cDNA construct (53). However, the effect on CBFα2 subcellular localization by CBFβ-SMMHC has not been fully examined. In this study, we used transient-transfection assays in combination with immunofluorescence and green fluorescent protein (GFP) tags to demonstrate that CBFβ-SMMHC does, in fact, sequester CBFα2 in abnormal locations. We also demonstrated that the sequestration requires the abilities of CBFβ-SMMHC to interact with CBFα2 and to multimerize. This observed sequestration can at least partially explain the dominant negative effect of the CBFβ-SMMHC protein on CBF function in leukemogenesis

Cloning and Characterization of Subunits of the T-Cell Receptor and Murine Leukemia Virus Enhancer Core-Binding Factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly

Cbfa2 is Required for the Formation of Intra-Aortic Hematopoietic Clusters

Cbfa2 (AML1) encodes the DNA-binding subunit of a transcription factor in the small family of core-binding factors (CBFs). Cbfa2 is required for the differentiation of all definitive hematopoietic cells, but not for primitive erythropoiesis. Here we show that Cbfa2 is expressed in definitive hematopoietic progenitor cells, and in endothelial cells in sites from which these hematopoietic cells are thought to emerge. Endothelial cells expressing Cbfa2 are in the yolk sac, the vitelline and umbilical arteries, and in the ventral aspect of the dorsal aorta in the aorta/genital ridge/mesonephros (AGM) region. Endothelial cells lining the dorsal aspect of the aorta, and elsewhere in the embryo, do not express Cbfa2. Cbfa2 appears to be required for maintenance of Cbfa2 expression in the endothelium, and for the formation of intra-aortic hematopoietic clusters from the endothelium

Runx1 Expression Marks Long-Term Repopulating Hematopoietic Stem Cells in the Midgestation Mouse Embryo

AbstractHematopoietic stem cells (HSCs) are first found in the aorta-gonad-mesonephros region and vitelline and umbilical arteries of the midgestation mouse embryo. Runx1 (AML1), the DNA binding subunit of a core binding factor, is required for the emergence and/or subsequent function of HSCs. We show that all HSCs in the embryo express Runx1. Furthermore, HSCs in Runx1+/− embryos are heterogeneous and include CD45+ cells, endothelial cells, and mesenchymal cells. Comparison with wild-type embryos showed that the distribution of HSCs among these various cell populations is sensitive to Runx1 dosage. These data provide the first morphological description of embryonic HSCs and contribute new insight into their cellular origin

Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d

Endothelio-Mesenchymal Interaction Controls runx1 Expression and Modulates the notch Pathway to Initiate Aortic Hematopoiesis

SummaryHematopoietic stem cells (HSCs) are produced by a small cohort of hemogenic endothelial cells (ECs) during development through the formation of intra-aortic hematopoietic cell (HC) clusters. The Runx1 transcription factor plays a key role in the EC-to-HC and -HSC transition. We show that Runx1 expression in hemogenic ECs and the subsequent initiation of HC formation are tightly controlled by the subaortic mesenchyme, although the mesenchyme is not a source of HCs. Runx1 and Notch signaling are involved in this process, with Notch signaling decreasing with time in HCs. Inhibiting Notch signaling readily increases HC production in mouse and chicken embryos. In the mouse, however, this increase is transient. Collectively, we show complementary roles of hemogenic ECs and mesenchymal compartments in triggering aortic hematopoiesis. The subaortic mesenchyme induces Runx1 expression in hemogenic-primed ECs and collaborates with Notch dynamics to control aortic hematopoiesis