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.Constant availability of molecular oxygen is crucial for the structure and function of any mammalian cell. Therefore, cellular responses to reduced oxygen tensions (hypoxia) play an important role in development and many aspects of physiological homeostasis. Many important disease processes, including ischemic vascular diseases and cancer, involve reduced tissue oxygenation, and cellular adaptation to this is implicated in disease progression and clinical outcome. The hypoxia-inducible transcription factor (HIF) 2 is a central mechanism responding to low cellular oxygenation and mediates a variety of systemic and local adaptive responses, including the control of red cell production, regulation of angiogenesis, modulation of vascular tone, enhancement of glycolysis, and cellular glucose uptake (for a review, see Refs. 1-3). HIF consists of a heterodimer of ␣-and ␤-subunits, both being basic helix-loop-helix-Per Arnt Sim domain proteins. Whereas HIF␤ is constitutively expressed, HIF␣ subunits are unstable and inversely correlated to the availability of molecular oxygen. At least two oxygen-regulated isoforms of HIF␣ have been identified, HIF-1␣ and HIF-2␣, sharing a high degree of sequence homology and a similar domain structure (4). Regulation of HIF is primarily governed by oxygen-dependent hydroxylation of its HIF␣ subunits, which influences protein stability and transcriptional activity.Growth and behavior of tumor cells is strongly dependent on their microenvironment, where hypoxia is both a stress factor and an important signal (for a review, see Refs. 5 and 6). Dating back to 1927, Otto Warburg had already described that tumor cells have a much increased utilization of the glycolytic pathway (7). Since then, a number of studies have established that indeed HIF is necessary to activate glycolysis in tumor cells in order to maintain energy homeosta...

Bacillus subtilis synthesizes glutamate from 2-oxoglutarate and glutamine using the glutamate synthase, encoded by the gltAB operon. Glutamate degradation involves the catabolic glutamate dehydrogenase (GDH) RocG. Expression of both gltAB and rocG is controlled by the carbon and nitrogen sources. In the absence of glucose or other well-metabolizable carbon sources, B. subtilis is unable to grow unless provided with external glutamate. In this work, we isolated mutations that suppressed this growth defect of B. subtilis on minimal media (sgd mutants). All mutations enabled the cells to express the gltAB operon even in the absence of glucose. The mutations were all identified in the rocG gene suggesting that the catabolic GDH is essential for controlling gltAB expression in response to the availability of sugars.

The regulatory link between carbon and nitrogen metabolism in Bacillus subtilis: regulation of the gltAB operon by the catabolite control protein CcpA Bacillus subtilis assimilates ammonium by the concerted action of glutamine synthetase and glutamate synthase. The expression of the gltAB operon encoding the latter enzyme is impaired in B. subtilis ccpA mutant strains. CcpA is a pleiotropic transcriptional regulator that is the key factor in the regulation of carbon metabolism. However, in addition to their defect in catabolite repression ccpA mutants are unable to grow on minimal media with glucose and ammonium as the single sources of carbon and nitrogen, respectively. In this work, the expression of the gltAB operon was analysed and its role in growth on minimal sugar/ammonium media was studied. Expression of gltAB requires induction by glucose or other glycolytically catabolized carbon sources. In ccpA mutants, gltAB cannot be induced by glucose due to the low activity of the phosphotransferase sugar transport system in these mutants. A mutation that allowed phosphotransferase system activity in a ccpA background simultaneously restored glucose induction of gltAB and growth on glucose/ammonium medium. Moreover, artificial induction of the gltAB operon in the ccpA mutant allowed the mutant strain to grow on minimal medium with glucose and ammonium. It may be concluded that expression of the gltAB operon depends on the accumulation of glycolytic intermediates which cannot occur in the ccpA mutant. The lack of gltAB induction is the bottleneck that prevents growth of the ccpA mutant on glucose/ammonium media. The control of expression of the gltAB operon by CcpA provides a major regulatory link between carbon and amino acid metabolism. INTRODUCTIONBacillus subtilis utilizes glucose and glutamine as the preferred sources of carbon and nitrogen, respectively. If glucose or glutamine is present in the growth medium, the genes encoding enzymes involved in the utilization of secondary sources of carbon and nitrogen, respectively, are not expressed. This phenomenon is called catabolite repression.The global control of carbon catabolism in B. subtilis is exerted by a pleiotropic regulatory protein, CcpA. In the presence of glucose, CcpA can interact with regulatory sites in the control regions of regulated operons to either repress or activate transcription. DNA-binding activity of CcpA is triggered by interaction with a protein of the phosphotransferase system (PTS), HPr or its regulatory paralogue Crh. In the presence of glucose, HPr and Crh are phosphorylated by a HPr kinase/phosphorylase (HPrK/P) on a regulatory seryl residue. HPr(Ser-P) and Crh(Ser-P) act as cofactors for CcpA (Deutscher et al., 1995(Deutscher et al., , 2002 Galinier et al., 1997;Henkin, 1996; Stülke & Hillen, 2000). Recent proteome and transcriptome studies have demonstrated that about 250 and 85 genes are subject to CcpA-dependent repression and activation, respectively. Among the genes repressed by CcpA are those encoding enzymes required for...

Hypoxia leads to the upregulation of a variety of genes mediated largely via the hypoxia inducible transcription factor (HIF). Prominent HIF-regulated target genes such as the vascular endothelial growth factor (VEGF), the glucose transporter 1 (Glut-1), or erythropoietin (EPO) help to assure survival of cells and organisms in a low oxygenated environment. Here, we are the first to report the hypoxic regulation of the sperm associated antigen 4 (SPAG4). SPAG4 is a member of the cancer testis (CT) gene family and to date little is known about its physiological function or its involvement in tumor biology. A number of CT family candidate genes are therefore currently being investigated as potential cancer markers, due to their predominant testicular expression pattern. We analyzed RNA and protein expression by RNAse protection assay, immunofluorescent as well as immunohistological stainings. To evaluate the influence of SPAG4 on migration and invasion capabilities, siRNA knockdown as well as transient overexpression was performed prior to scratch or invasion assay analysis. The hypoxic regulation of SPAG4 is clearly mediated in a HIF-1 and VHL dependent manner. We furthermore show upregulation of SPAG4 expression in human renal clear cell carcinoma (RCC) and co-localization within the nucleolus in physiological human testis tissue. SPAG4 knockdown reduces the invasion capability of RCC cells in vitro and overexpression leads to enhancement of tumor cell migration. Together, SPAG4 could possibly play a role in the invasion capability and growth of renal tumors and could represent an interesting target for clinical intervention.

The tricarboxylic acid (TCA) cycle is one of the major routes of carbon catabolism in Bacillus subtilis. The syntheses of the enzymes performing the initial reactions of the cycle, citrate synthase, and aconitase, are synergistically repressed by rapidly metabolizable carbon sources and glutamine. This regulation involves the general transcription factor CcpA and the specific repressor CcpC. In this study, we analyzed the expression and intracellular localization of CcpC. The synthesis of citrate, the effector of CcpC, requires acetyl-CoA. This metabolite is located at a branching point in metabolism. It can be converted to acetate in overflow metabolism or to citrate. Manipulations of the fate of acetyl-CoA revealed that efficient citrate synthesis is required for the expression of the citB gene encoding aconitase and that control of the two pathways utilizing acetyl-CoA converges in the control of citrate synthesis for the induction of the TCA cycle. The citrate pool seems also to be controlled by arginine catabolism. The presence of arginine results in a severe CcpC-dependent repression of citB. In addition to regulators involved in sensing the carbon status of the cell, the pleiotropic nitrogen-related transcription factor, TnrA, activates citB transcription in the absence of glutamine.