Einfluss mechanischer Kräfte auf die Genexpression humaner dermaler Fibroblasten

Mechanischer Stress spielt in der Haut eine bedeutende Rolle bei biologischen Prozessen wie der Wundheilung und Narbenbildung, sowie in pathologischen Situationen wie Keloiden, hypertrophen Narben und Fibrosen. Zur Untersuchung des Einflusses von mechanischem Stress auf Hautfibroblasten wurden in dieser Arbeit zwei unterschiedliche Zellkulturmodellsysteme verwendet, die als Bound Lattice (BL) und Free Lattice (FL) bezeichnet werden. Während im FL-System primäre Hautfibroblasten eine Kollagen-I-Matrix frei kontrahieren können, wird im BL-System durch Anlegen eines Nylonrings an die Peripherie der Zellkulturschale die Kontraktion verhindert und folglich mechanischer Stress induziert. Im Rahmen der Untersuchung der beiden Kollagengelsysteme BL und FL wurden Analysen der Genexpressionprofile zu frühen Zeitpunkten im Zeitraum von 1 bis 8 h mit den beiden Methoden SAGE und PIQOR� durchgeführt. Dabei wurden mehr als 50 Transkripte aus den Bereichen Transkriptionsfaktoren, Wachstumsfaktoren, Signaltransduktionen, Komponenten des Zytoskeletts und der EZM sowie Zellrezeptoren identifiziert, die in den Systemen differentiell exprimiert waren. Zusätzlich wurden durch Northernblot-Technik und mittels Q-PCR ausgewählte Kandidaten validiert. Durch eine Literaturrecherche konnten funktionelle Beziehungen der identifizierten Transkripte dargestellt werden. Dabei reagieren die mechanisch belasteten Fibroblasten zeitabhängig in mehreren Phasen durch die Expression geeigneter Transkripte. In der ersten Phase werden Mechanosensoren an der Oberfläche der Zelle stärker exprimiert, die eine bessere Übertragung der mechanischen Signale ins Zellinnere erlaubt. Anschließend werden Komponenten des Zytoskeletts differentiell exprimiert, die über Signaltransduktionswege die Expression von Wachstumsfaktoren initiieren. Der dadurch gestartete autokrine parakrine Loop führt dann zu der Expression weiterer Transkripte, die den nach 20 h enstehenden �aktivierten Fibroblasten� initiieren. Im Zusammenhang mit der Entstehung des �aktivierten Fibroblasten� konnte aufgezeigt werden, dass der Myofibroblasten ähnliche Phänotyp nicht nur im BL-System aktiviert wird, sondern auch im FL-System suppremiert zu werden scheint. Weiterführende Untersuchungen der in diesem Fall identifizierten Kandidatengene kann helfen, die Entstehung bzw. Supprimierung des �aktivierten Phänotyps� besser zu verstehen. Die im SAGE erstellen Expressionsprofile wurden des Weiteren dazu genutzt, charakteristische Transkripte für primäre Hautfibroblasten, Vorhautfibroblasten und transfizierte Fibroblasten zu identifizieren. Die bioinformatische Analyse dieser Fibroblastentypen führte zu der Identifizierung zahlreicher differentiell exprimierter Gene. Es konnte aufgezeigt werden, dass Fibroblasten unterschiedlichen Ursprungs unterschiedliche Expressionsprofile besitzen. Des Weiteren konnten durch die Auswertung der im NCBI publizierten SAGE-Datenbanken potentielle Fibroblastenmarker identifiziert werden

A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor

BACKGROUND: Bone morphogenetic proteins (BMPs) are key regulators in the embryonic development and postnatal tissue homeostasis in all animals. Loss of function or dysregulation of BMPs results in severe diseases or even lethality. Like transforming growth factors β (TGF-βs), activins, growth and differentiation factors (GDFs) and other members of the TGF-β superfamily, BMPs signal by assembling two types of serine/threonine-kinase receptor chains to form a hetero-oligomeric ligand-receptor complex. BMP ligand receptor interaction is highly promiscuous, i.e. BMPs bind more than one receptor of each subtype, and a receptor bind various ligands. The activin type II receptors are of particular interest, since they bind a large number of diverse ligands. In addition they act as high-affinity receptors for activins but are also low-affinity receptors for BMPs. ActR-II and ActR-IIB therefore represent an interesting example how affinity and specificity might be generated in a promiscuous background. RESULTS: Here we present the high-resolution structures of the ternary complexes of wildtype and a variant BMP-2 bound to its high-affinity type I receptor BMPR-IA and its low-affinity type II receptor ActR-IIB and compare them with the known structures of binary and ternary ligand-receptor complexes of BMP-2. In contrast to activin or TGF-β3 no changes in the dimer architecture of the BMP-2 ligand occur upon complex formation. Functional analysis of the ActR-IIB binding epitope shows that hydrophobic interactions dominate in low-affinity binding of BMPs; polar interactions contribute only little to binding affinity. However, a conserved H-bond in the center of the type II ligand-receptor interface, which does not contribute to binding in the BMP-2 – ActR-IIB interaction can be mutationally activated resulting in a BMP-2 variant with high-affinity for ActR-IIB. Further mutagenesis studies were performed to elucidate the binding mechanism allowing us to construct BMP-2 variants with defined type II receptor binding properties. CONCLUSION: Binding specificity of BMP-2 for its three type II receptors BMPR-II, Act-RII and ActR-IIB is encoded on single amino acid level. Exchange of only one or two residues results in BMP-2 variants with a dramatically altered type II receptor specificity profile, possibly allowing construction of BMP-2 variants that address a single type II receptor. The structure-/function studies presented here revealed a new mechanism, in which the energy contribution of a conserved H-bond is modulated by surrounding intramolecular interactions to achieve a switch between low- and high-affinity binding

Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro

Purpose: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix (TM) oligonucleotide array technology to identify genes that are regulated by hCTGF. Methods: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix (TM) oligonucleotide array technology. Results were validated by real time RT-PCR. Results: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology. Conclusions: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye

Receptor oligomerization and beyond: a case study in bone morphogenetic proteins

BACKGROUND: Transforming growth factor (TGF)β superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. RESULTS: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. CONCLUSION: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system

TNF Receptor-Associated Factor 1 is a Major Target of Soluble TWEAK

Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB pathway using the IKK2 inhibitor, TPCA1. In contrast, in some cell lines, TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924, however, which inhibits classical and alternative NFκB signaling, blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NFκB signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction, but potentiated the ability of soluble TWEAK to trigger production of the classical NFκB-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NFκB pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets, which can be equally well induced by soluble and membrane TWEAK, remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies

Crystal structure analysis reveals a spring-loaded latch as molecular mechanism for GDF-5–type I receptor specificity

Dysregulation of growth and differentiation factor 5 (GDF-5) signalling, a member of the TGF-β superfamily, is strongly linked to skeletal malformation. GDF-5-mediated signal transduction involves both BMP type I receptors, BMPR-IA and BMPR-IB. However, mutations in either GDF-5 or BMPR-IB lead to similar phenotypes, indicating that in chondrogenesis GDF-5 signalling seems to be exclusively mediated through BMPR-IB. Here, we present structural insights into the GDF-5:BMPR-IB complex revealing how binding specificity for BMPR-IB is generated on a molecular level. In BMPR-IB, a loop within the ligand-binding epitope functions similar to a latch allowing high-affinity binding of GDF-5. In BMPR-IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF-5 might impair receptor binding and activation

Metabolic Fingerprinting of Murine L929 Fibroblasts as a Cell-Based Tumour Suppressor Model System for Methionine Restriction

Since Otto Warburg reported in 1924 that cancer cells address their increased energy requirement through a massive intake of glucose, the cellular energy level has offered a therapeutic anticancer strategy. Methionine restriction (MetR) is one of the most effective approaches for inducing low-energy metabolism (LEM) due to the central position in metabolism of this amino acid. However, no simple in vitro system for the rapid analysis of MetR is currently available, and this study establishes the murine cell line L929 as such a model system. L929 cells react rapidly and efficiently to MetR, and the analysis of more than 150 different metabolites belonging to different classes (amino acids, urea and tricarboxylic acid cycle (TCA) cycles, carbohydrates, etc.) by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. The system facilitates the rapid and efficient testing of potential cancer therapeutic metabolic targets. To date, MS studies of MetR have been performed using organisms and yeast, and the current LC/MS analysis of the intra- and extracellular metabolites in the murine cell line L929 over a period of 5 days thus provides new insights into the effects of MetR at the cellular metabolic level

Association between pulse width and health-related quality of life after electroconvulsive therapy in patients with unipolar or bipolar depression: an observational register-based study

To examine the association between pulse width and HRQoL measured within one week after electroconvulsive therapy (ECT) and at six-month follow-up in patients with unipolar or bipolar depression. This was an observational register study using data from the Swedish National Quality Registry for ECT (2011–2019). Inclusion criteria were: age ≥18 years; index treatment for unipolar/bipolar depression; unilateral electrode placement; information on pulse width; EQ-5D measurements before and after ECT. Multiple linear regressions were performed to investigate the association between pulse width (0.5 ms) and HRQoL (EQ-5D-3L index; EQ VAS) one week after ECT (primary outcome) and six months after ECT (secondary outcome). The sample included 5,046 patients with unipolar (82%) or bipolar (18%) depression. At first ECT session, 741 patients (14.7%) had pulse width 0.5 ms. There were no statistically significant associations between pulse width and HRQoL one week after ECT. In the subsample of patients with an EQ-5D index recorded six months after ECT (n = 730), patients receiving 0.5 ms had significantly lower HRQoL (−0.089) compared to p = .011). The corresponding analysis for EQ VAS did not show any statistically significant associations. No robust associations were observed between pulse width and HRQoL after ECT. On average, significant improvements in HRQoL were observed one week and six months after ECT for patients with unipolar or bipolar disease, independent of the pulse width received.</p

The Selection of NFκB Inhibitors to Block Inflammation and Induce Sensitisation to FasL-Induced Apoptosis in HNSCC Cell Lines Is Critical for Their Use as a Prospective Cancer Therapy

Inflammation is a central aspect of tumour biology and can contribute significantly to both the origination and progression of tumours. The NF&kappa;B pathway is one of the most important signal transduction pathways in inflammation and is, therefore, an excellent target for cancer therapy. In this work, we examined the influence of four NF&kappa;B inhibitors&mdash;Cortisol, MLN4924, QNZ and TPCA1&mdash;on proliferation, inflammation and sensitisation to apoptosis mediated by the death ligand FasL in the HNSCC cell lines PCI1, PCI9, PCI13, PCI52 and SCC25 and in the human dermal keratinocyte cell line HaCaT. We found that the selection of the inhibitor is critical to ensure that cells do not respond by inducing counteracting activities in the context of cancer therapy, e.g., the extreme IL-8 induction mediated by MLN4924 or FasL resistance mediated by Cortisol. However, TPCA1 was qualified by this in vitro study as an excellent therapeutic mediator in HNSCC by four positive qualities: (1) proliferation was inhibited at low &mu;M-range concentrations; (2) TNF&alpha;-induced IL-8 secretion was blocked; (3) HNSCC cells were sensitized to TNF&alpha;-induced cell death; and (4) FasL-mediated apoptosis was not disrupted