Angela Graness

Universität Ulm, Ulm, Baden-Wuerttemberg, Germany

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Publications (6)27.87 Total impact

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    ABSTRACT: Thrombospondin-1 (TSP-1), which is synthesized by mesangial cells, is known for its anti-angiogenic activity and its ability to activate latent TGF-beta. TSP-1 is upregulated in renal diseases associated with tissue remodeling. Therefore, we hypothesized that the expression of TSP-1 might be modulated by changes in cell morphology involving proteins of the Rho family. Spreading of mesangial cells after detachment and reseeding was characterized by the formation of lamellipodia and focal adhesions, pointing toward a Rac-1-mediated rearrangement of actin structures. Clustering of focal adhesion proteins was also observed in a model system of nocodazole-induced disruption of microtubules. These morphological alterations were impeded by pharmacological inhibition of Src family kinases, of the small GTPase Rac-1, or by downregulation of Rac-1 by siRNA. Upon cell spreading, TSP-1 was upregulated in the absence and much more prominently in the presence of serum, but also after nocodazole treatment. TSP-1 upregulation was controlled by activation of Src family kinases, ERK 1/2 and Rac-1, whereas activation of RhoA-ROCK signaling was not linked to TSP-1 induction. We thus provide evidence that TSP-1 expression is induced by common signaling pathways, which are activated by morphological alterations of renal mesangial cells or by soluble factors as contained in serum, and these pathways include Src family kinases, ERK 1/2 and Rac-1. Our data suggest that tissue remodeling activates gene expression of pathophysiologically relevant proteins such as TSP-1.
    American journal of physiology. Renal physiology 03/2008; 294(2):F407-13. · 3.61 Impact Factor
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    ABSTRACT: Cucurbitacins are recognised as anti-tumour agents because of their interference with STAT3 signalling, but may also affect the integrity of the actin cytoskeleton. In the present study the effect of cucurbitacin I was investigated in fibroblasts. In these cells, cucurbitacin I interfered with lysophosphatidic acid (LPA) signalling. It inhibited tyrosine phosphorylation of focal adhesion proteins and induction of connective tissue growth factor (CTGF), a potent profibrotic protein. Inhibition of Src family kinases with PP2, but not the inactive analogue PP3, also interfered with LPA-mediated tyrosine phosphorylation and induction of CTGF. Jak2-STAT3 signalling seemed to be the connecting link, because CTGF induction was sensitive to AG490, an inhibitor of Jak2, and cucurbitacin I, an inhibitor of Jak2 and STAT3. However, LPA did not activate tyrosine phosphorylation of STAT3. Furthermore, cucurbitacin I was as effective in STAT3 knock out cells as in control cells. Therefore, the inhibitory effect of cucurbitacin I was not related to inhibition of STAT3. Immunocytochemical analysis of cucurbitacin I-treated cells revealed disassembly of F-actin fibres, reorganisation into F-actin patches and resolution of focal adhesions. The phenotypic changes resembled changes observed after treatment of the cells with cytochalasin D, which has been shown to interfere with CTGF induction. Concentrations of cucurbitacin I, which have been shown to target Jak2-STAT3 signalling, thus, profoundly affect the actin cytoskeleton and may therefore modulate cell morphology, migration, adherence and gene expression also in non-tumour cells.
    Biochemical Pharmacology 07/2006; 72(1):32-41. · 4.58 Impact Factor
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    ABSTRACT: The integrin-linked kinase (ILK) serves as an adapter protein to link the cytoplasmic domains of integrins with cytoskeletal components. Organization of the actin cytoskeleton is strongly influenced by the small GTPase RhoA, which also regulates gene expression. To investigate the impact of ILK deficiency on RhoA-mediated signaling we used ILK-deficient fibroblasts. The cytoskeleton of ILK (-/-) cells was characterized by less organized F-actin fibers, compared to wild type mouse fibroblasts. Stimulation of the cells with lysophosphatidic acid (LPA) or the microtubule disrupting agent colchicine increased polymerization of F-actin stress fibers in ILK (+/+) cells, whereas ILK (-/-) cells showed a network of short thin cortical actin fibers, cell rounding and finally detachment from the surface of the culture plates. The structural changes were primarily attributable to the activation of RhoA in both cell types. ILK deficiency also affected gene expression. The basal levels of several proteins related to fibroblast differentiation, such as connective tissue growth factor (CTGF), thrombospondin 1 and alpha smooth muscle actin, were reduced in ILK (-/-) cells. However, induction of CTGF expression by LPA or colchicine was comparable in ILK (+/+) and ILK (-/-) cells. Furthermore, stimulation of CTGF or thrombospondin by TGFbeta was not reduced by ILK deficiency. Inhibition of the RhoA-associated kinase or overexpression of dominant negative RhoA reduced the stimulated CTGF expression indicative of a role for RhoA signaling in CTGF expression. Taken together, ILK is involved in RhoA-dependent reorganization of the actin cytoskeleton, whereas activation of RhoA and RhoA-mediated gene expression is independent of ILK.
    Cellular Signalling 05/2006; 18(4):433-40. · 4.47 Impact Factor
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    ABSTRACT: Expression of connective tissue growth factor (CTGF) is sensitive to reorganization of the actin cytoskeleton, but also to alterations in cell morphology due to extracellular forces, for example, cyclic stretching or mechanical loading. Dynamic alterations of focal adhesion proteins were thus proposed to modulate CTGF induction. Immortalized human renal fibroblasts were cultured in or on top of preformed collagen-1 gels. Proteins were detected by immunofluorescence and quantified by Western blotting. Fibroblasts cultured in/on collagen gels resembled cells in vivo by their spindle-like morphology, absence of actin stress fibers, small punctiform focal contacts, and low levels of CTGF expression. Disassembly of microtubules by short-term treatment with colchicine induced cell rounding, cortical recruitment of patchy F-actin, reorganization of focal contacts into strong clusters, and upregulation of CTGF, all of which were dependent on RhoA-Rho-kinase signaling. Clustering of focal adhesion sites activated Src-family kinases and focal adhesion kinase (FAK). Interference with Src activity by PP2 had no effect on the morphological alterations but decreased tyrosine phosphorylation of focal adhesion proteins and almost completely prevented upregulation of CTGF. Furthermore, inhibition of phosphatidylinositol 3-kinase reduced CTGF expression. On the other hand, when the fibroblasts were cultured on a rigid matrix, that is collagen-coated plates, strong focal complexes prevented the dynamic alterations, and RhoA-mediated upregulation of CTGF expression was independent of Src-FAK signaling. Assembly of focal adhesion proteins regulates CTGF expression, providing a link between actin network, adhesion receptors, and CTGF-mediated functions such as synthesis of extracellular matrix proteins.
    Kidney International 05/2006; 69(8):1341-9. · 8.52 Impact Factor
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    ABSTRACT: Introduction In closing wounds or developing fibrotic tissue, fibroblasts are exposed to mechanical stress, which leads to alterations in cell morphology and reorganization of the cytoskeleton. The impact of morphological changes on gene expression was investigated in the present study. As a model system, we used a human renal fibroblast cell line and studied the expression of connective tissue growth factor (CTGF). CTGF is a downstream mediator of TGF-β mediating many of the pro-fibrotic actions of this growth factor and has also been shown to be up-regulated by static or dynamic pressure. The molecular mechanisms regulating CTGF expression related to stress are not yet known.Materials and methods A human renal fibroblast cell line was kindly provided by Dr Müller, Göttingen, Germany. These cells express CTGF upon treatment with soluble stimuli (Heusinger-Ribeiro et al. 2001; Eberlein et al. 2001). CTGF expression was detected by northern and Western blot analysis. The actin cytoskeleton was visualized by rhodamine phalloidin staining, and microtubules were detected by immunocytochemistry.Results Low concentrations of the microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF mRNA and protein expression in the human renal fibroblast cell line TK173. The up-regulation was prevented by stabilization of the microtubules by paclitaxel (taxol). As a consequence of microtubule disruption, the small GTPase RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction: interference with RhoA signalling by simvastatin, toxin B and Y27632 prevented up-regulation of CTGF. The important role of RhoA was supported by an increased CTGF expression upon overexpression of constitutively active RhoA. Direct disassembly of the actin cytoskeleton by latrunculin B interfered with colchicine-mediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D unexpectedly increased CTGF expression. This indicated that the content of F-actin per se was not the major determinant for CTGF gene expression. It has been shown, however, that cytochalasin D sequesters G-actin, whereas latrunculin increases the level of G-actin. Our data are thus in accordance with an inverse correlation between G-actin levels and CTGF expression.Discussion These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF. Recently, decreased levels of G-actin were observed in vascular smooth muscle cells in response to increased vascular pressure (Cipolla et al. 2002). Our findings thus provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.
    International Journal of Experimental Pathology 01/2004; 85(1). · 2.04 Impact Factor
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    ABSTRACT: Modulation of the cytoskeletal architecture was shown to regulate the expression of CTGF (connective tissue growth factor, CCN2). The microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF expression, which was prevented upon stabilization of the microtubules by paclitaxel. As a consequence of microtubule disruption, RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction. Overexpression of constitutively active RhoA induced CTGF synthesis. Interference with RhoA signaling by simvastatin, toxinB, C3 toxin, and Y27632 prevented up-regulation of CTGF. Likewise, direct disintegration of the actin cytoskeleton by latrunculin B interfered with nocodazole-mediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D, however, unexpectedly increased CTGF expression indicating that the content of F-actin per se was not the major determinant for CTGF gene expression. Given the fact that cytochalasin D sequesters G-actin, a decrease in G-actin increased CTGF, while increased levels of G-actin corresponded to reduced CTGF expression. These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF and provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.
    Journal of Biological Chemistry 12/2003; 278(45):44305-11. · 4.65 Impact Factor