Margarete Goppelt-Struebe

The Lysyl Oxidases LOX and LOXL2 Are Necessary and Sufficient to Repress E-cadherin in Hypoxia INSIGHTS INTO CELLULAR TRANSFORMATION PROCESSES MEDIATED BY HIF-1

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

Coordinate Expression of Cyclooxygenase-2 and Renin in the Rat Kidney in Renovascular Hypertension

Prostaglandins contribute to the regulation of renin synthesis and secretion. We tested the hypothesis that the inducible isoform of prostaglandin G/H synthase, cyclooxygenase-2, contributes to the stimulation of renin synthesis in renovascular hypertension. The expression of cyclooxygenase-2 and renin was investigated in the kidneys of rats with two-kidney, one-clip renovascular hypertension or sham operation. Systolic blood pressure was increased 2 weeks after clipping (153+/-7 versus 112+/-4 mmHg in controls, n=6 each, P<.05) and continued to rise until 4 weeks. Cyclooxygenase-2 mRNA levels were increased in clipped kidneys but remained unchanged or slightly decreased in nonclipped kidneys. Cyclooxygenase-2 protein was expressed mainly in the macula densa and occasionally in distal tubular cells not associated with the macula densa. Two weeks after clipping, the percentage of juxtaglomerular apparatus staining positive for cyclooxygenase-2 was 27.8+/-3.6 in clipped kidneys, 3.1+/-0.4 in contralateral kidneys, and 8.0+/-1.3 in controls; the percentages for immunoreactive renin staining in the afferent arteriole were 33.6+/-2 in clipped kidneys, 1.9+/-0.5 in contralateral kidneys, and 12.4+/-4.0 in controls, respectively. Similar parallel changes in renin and cyclooxygenase-2 staining were observed 4 weeks after clipping. The percentage of cyclooxygenase-2-positive juxtaglomerular apparatus correlated positively with the percentage that was renin positive (r=0.78, P<.05). Double immunostaining showed coexpression of cyclooxygenase-2 and renin protein in the same juxtaglomerular apparatus. Our data are consistent with a role for macula densa cyclooxygenase-2 in the regulation of renin in renovascular hypertension.

Molecular mechanisms involved in the regulation of prostaglandin biosynthesis by glucocorticoids.

The anti-inflammatory properties of glucocorticoids are attributed in part, to their interference with prostaglandin synthesis. Phospholipases A2 and cyclooxygenases, the key enzymes of prostaglandin biosynthesis, are targets of glucocorticoid action; the molecular mechanisms, however, are not yet understood in detail. Obviously, glucocorticoids can act at different levels of gene regulation depending on cell type and inducing stimulus. The current knowledge of glucocorticoid interference with phospholipase A2 and cyclooxygenase expression is summarized. In comparison with other nonsteroidal anti-inflammatory drugs, glucocorticoids are unique inasmuch as they also inhibit cytokine synthesis and expression of other inflammation-related enzymes. Based on a more detailed understanding of glucocorticoid action, it may be possible to therapeutically exploit the anti-inflammatory effects and at the same time avoid the unwanted metabolic actions of these steroids.

Connective tissue growth factor: Context-dependent functions and mechanisms of regulation

Connective tissue growth factor (CTGF, CCN2) is a secreted matricellular protein, the functions of which depend on the interactions with other molecules in the microcellular environment. As an example of context‐dependent activity of CTGF, this review will outline different aspects of CTGF function in relation to angiogenesis. CTGF is barely expressed in normal adult tissue, but is strongly upregulated in fibrotic tissue and is also increased during development, in wound healing, or in certain types of cancer. Accordingly, gene expression of CTGF is tightly regulated. To highlight the complexity of the regulation of CTGF gene expression, we discuss here the mechanisms involved in CTGF regulation by TGFbeta in different cell types, and the mechanisms related to CTGF gene expression in cells exposed to mechanical forces. Finally, we will touch upon novel aspects of epigenetic regulation of CTGF gene expression.

Expression of cyclooxygenase isoforms in the rat spinal cord and their regulation during adjuvant-induced arthritis

Abstract.Objective and Design: Spinal regulation of cyclooxygenase (COX) isoforms was investigated in the animal model of peripheral inflammation induced by injection of complete Freunds-type adjuvant (CFA) in the rat hindpaw.¶Subjects and Treatment: Peripheral inflammation was induced by intraplantar injection of CFA in one hind footpad of male Sprague Dawley rats (n = 3 per time point).¶Methods: Spinal cord was removed after different times (3 h to 22 d). mRNA and protein were isolated and analyzed by comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively.¶Results: Under the acute inflammatory stimulus 6 h after CFA application, RT-PCR revealed a twofold increase in COX-2 mRNA that reached baseline again at day 3. This transient increase occurred in the lumbar spinal cord, but changes in COX-2 mRNA expression were also registered in RNA preparations from cervical sections, spinal COX-2 induction thus not being a spatially confirmed phenomenon. Western blot analysis of spinal membrane preparations reflected the transient COX-2 mRNA induction at protein levels. During the chronic phase of arthritis at day 22, COX-2 levels were again raised significantly (1.6 fold) over baseline. Spinal levels of COX-1 were not altered at any time point of the peripheral inflammation.¶Conclusion: These data imply a regulatory role for COX-2 but not COX-1 in the spinal modulation under acute and chronic peripheral inflammation.

Rho-dependent inhibition of the induction of connective tissue growth factor (CTGF) by HMG CoA reductase inhibitors (statins)

It was supposed that inhibitors of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG CoA) reductase (statins) might inhibit the expression of the fibrosis‐related factor CTGF (connective tissue growth factor) by interfering with the isoprenylation of Rho proteins. The human renal fibroblast cell line TK173 was used as an in vitro model system to study the statin‐mediated modulation of the structure of the actin cytoskeleton and of the expression of CTGF mRNA. Incubation of the cells with simvastatin or lovastatin time‐dependently and reversibly changed cell morphology and the actin cytoskeleton with maximal effects observed after about 18 h. Within the same time period, statins reduced the basal expression of CTGF and interfered with CTGF induction by lysophosphatidic acid (LPA) or transforming growth factor beta. Simvastatin and lovastatin proved to be much more potent than pravastatin (IC50 1–3 μM compared to 500 μM). The inhibition of CTGF expression was prevented when the cells were incubated with mevalonate or geranylgeranylpyrophosphate (GGPP) but not by farnesylpyrophosphate (FPP). Specific inhibition of geranylgeranyltransferase‐I by GTI‐286 inhibited LPA‐mediated CTGF expression whereas an inhibitor of farnesyltransferases FTI‐276 was ineffective. Simvastatin reduced the binding of the small GTPase RhoA to cellular membranes. The effect was prevented by mevalonate and GGPP, but not FPP. These data are in agreement with the hypothesis that interference of statins with the expression of CTGF mRNA is primarily due to interference with the isoprenylation of RhoA, in line with previous studies, which have shown that RhoA is an essential mediator of CTGF induction. The direct interference of statins with the synthesis of CTGF, a protein functionally related to the development of fibrosis, may thus be a novel mechanism underlying the beneficial effects of statins observed in renal diseases.

Connective Tissue Growth Factor Is Overexpressed in Complicated Atherosclerotic Plaques and Induces Mononuclear Cell Chemotaxis In Vitro

Objective—Atherosclerotic blood vessels overexpress connective tissue growth factor (CTGF) mRNA, but the role of CTGF in atherosclerosis remains controversial. To assess the hypothesis that CTGF is involved in atherosclerotic plaque progression, we investigated CTGF protein expression and distribution in the different types of plaque morphology. Methods and Results—Serial cross-sections of 45 human carotid plaques were immunohistochemically analyzed for the presence of CTGF protein, neovascularization (von Willebrand factor), macrophages (CD68), and T cells (CD3). The lesions were categorized according to American Heart Association (AHA) classification as fibrous (type IV and V) or complicated plaques (type VI). The levels of CTGF were significantly higher in complicated compared with fibrous plaques (P=0.002). CTGF accumulated particularly in the rupture-prone plaque shoulder and in the areas of neovascularization or infiltration with inflammatory cells. Macrophage-like cells stained positive for CTGF protein in plaques. Subsequent in vitro studies showed that although monocyte-derived macrophages do not produce CTGF on stimulation with transforming growth factor-β, lipopolysaccharide, or thrombin, they take it up from culture medium. Furthermore, CTGF induces mononuclear cell chemotaxis in a dose-dependent manner. ConclusionCTGF protein is significantly increased in complicated compared with fibrous plaques and may enhance monocyte migration into atherosclerotic lesions, thus contributing to atherogenesis.

LOCALIZATION OF CYCLOOXYGENASE-2 AND PROSTAGLANDIN E2 RECEPTOR EP3 IN THE RAT LUMBAR SPINAL CORD

Cyclooxygenase-2 (COX-2) is now considered to be the major constitutively expressed COX isozyme in the central nervous system. The present immunocytochemical study details localization of COX-2 immunoreactivity in rat spinal cord along with the expression of prostaglandin E2 receptor subtype EP3. Prominent COX-2 staining was observed in the nuclear envelope of neurons throughout the spinal cord, especially in the superficial dorsal horn laminae and motoneurons of lamina IX, as well as in glial cells of the white matter. Expression of EP3 receptor was strictly confined to afferent terminal areas in the superficial dorsal horns.

Differentiation-associated expression of prostaglandin G/H synthase in monocytic cells

Significant progress in the investigation of the regulation of prostanoid formation has recently been made by cloning a second gene coding for prostaglandin G/H synthase (PGHS; EC 1.14.99.1). In this study we examined the expression of the two PGHS isoforms during phorbol ester induced monocytic differentiation of human myeloid leukemia cells (U937). Murine and ovine PGHS‐1 probes hybridized to 2.8‐ and 5.5‐kb mRNA species, whereas the murine PGHS‐2 probe hybridized to a 5.3‐kb species. Western blot analysis using antisera to mouse PGHS‐1 and to a synthetic peptide derived from a mouse PGHS‐2‐specific region revealed a band of 70 kDa for PGHS‐1 and a doublet of about 85 kDa for PGHS‐2. Unlike PGHS‐2, which was not expressed in U937 control cells, both PGHS‐1 protein and mRNA were detected in untreated U937 cells. TPA strongly induced PGHS‐2 protein and also increased the amount of PGHS‐1 protein. Correspondingly, a marked induction of PGHS‐2 mRNA was found, but virtually no change in the expression of the PGHS‐1 2.8‐kb mRNA occurred. The induction of both PGHS isoforms turned out to be dexamethasone‐sensitive. The suppression of PGHS‐2 induction was more pronounced. These results suggest that both PGHS‐1 and to a larger extent PGHS‐2 contribute to the upregulation of prostanoid synthesis during monocytic differentiation.

Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton.

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.