Heregulin-mediated ErbB2-ERK signaling activates hyaluronan synthases leading to CD44-dependent ovarian tumor cell growth and migration

Heregulin (HRG)-induced cell responses are mediated by the ErbB family of tyrosine kinase receptors. In this study we have investigated HRG activation of ErbB2, extracellular signal-regulated kinase (ERK) signaling, and their role in regulating hyaluronan synthase (HAS) activity in human ovarian tumor cells (SK-OV-3.ipl cells). Immunological and biochemical analyses indicate that ErbB2, ErbB3, and ErbB4 are all expressed in SK-OV-3.ipl cells and that ErbB4 (but not ErbB3) is physically linked to ErbB2 following HRG stimulation. Furthermore, our data indicate that the HRG-induced ErbB2.ErbB4 complexes stimulate ErbB2 tyrosine kinase, which induces both ERK phosphorylation and kinase activity. The activated ERK then increases the phosphorylation of HAS1, HAS2, and HAS3. Consequently, all three HAS isozymes are activated resulting in hyaluronan (HA) production. Because HRG-mediated HAS isozyme phosphorylation/activation can be effectively blocked by either AG825 (an ErbB2 inhibitor) or thiazolidinedione compound (an ERK blocker), we conclude that ErbB2-ERK signaling and HAS isozyme phosphorylation/HA production are functionally coupled in SK-OV-3.ipl cells. HRG also promotes HA- and CD44-dependent oncogenic events (e.g. CD44-Cdc42 association, p21-activated kinase 1 activation, and p21-activated kinase 1-filamin complex formation) and tumor cell-specific behaviors in an ErbB2-ERK signaling-dependent manner. Finally, we have found that the down-regulation of HAS isozyme expression (by transfecting cells with HAS1/HAS2/HAS3-specific small interfering RNAs) not only inhibits HRG-mediated HAS phosphorylation/activation and HA production but also impairs CD44-specific Cdc42-PAK1/filamin signaling, cytoskeleton activation and tumor cell behaviors. Taken together, these findings clearly indicate that HRG activation of ErbB2-ERK signaling modulates HAS phosphorylation/activation and HA production leading to CD44-mediated oncogenic events and ovarian cancer progression.     

Keratometry device for surgical support

Background  

High astigmatisms are usually induced during corneal suturing subsequent to tissue transplantation or any other surgery which involves corneal suturing. One of the reasons is that the procedure is intimately dependent on the surgeon's skill for suturing identical stitches. In order to evaluate the influence of the irregularity on suturing for the residual astigmatism, a prototype for ophthalmic surgical support has been developed. The final intention of this prototype is to be an evaluation tool for guided suture and as an outcome diminish the postoperative astigmatism.     

Arabidopsis Putative Selenium-Binding Protein1 Expression Is Tightly Linked to Cellular Sulfur Demand and Can Reduce Sensitivity to Stresses Requiring Glutathione for Tolerance

Selenium-Binding Protein1 (SBP1) gene expression was studied in Arabidopsis (Arabidopsis thaliana) seedlings challenged with several stresses, including cadmium (Cd), selenium {selenate [Se(VI)] and selenite [Se(IV)]}, copper (Cu), zinc (Zn), and hydrogen peroxide (H₂O₂) using transgenic lines expressing the luciferase (LUC) reporter gene under the control of the SBP1 promoter. In roots and shoots of SBP1∷LUC lines, LUC activity increased in response to Cd, Se(VI), Cu, and H₂O₂ but not in response to Se(IV) or Zn. The pattern of expression of SBP1 was similar to that of PRH43, which encodes the 5′-Adenylylphosphosulfate Reductase2, a marker for the induction of the sulfur assimilation pathway, suggesting that an enhanced sulfur demand triggers SBP1 up-regulation. Correlated to these results, SBP1 promoter showed enhanced activity in response to sulfur starvation. The sulfur starvation induction of SBP1 was abolished by feeding the plants with glutathione (GSH) and was enhanced when seedlings were treated simultaneously with buthionine sulfoxide, which inhibits GSH synthesis, indicating that GSH level participates in the regulation of SBP1 expression. Changes in total GSH level were observed in seedlings challenged with Cd, Se(VI), and H₂O₂. Accordingly, cad2-1 seedlings, affected in GSH synthesis, were more sensitive than wild-type plants to these three stresses. Moreover, wild-type and cad2-1 seedlings overexpressing SBP1 showed a significant enhanced tolerance to Se(VI) and H₂O₂ in addition to the previously described resistance to Cd, highlighting that SBP1 expression decreases sensitivity to stress requiring GSH for tolerance. These results are discussed with regard to the potential regulation and function of SBP1 in plants.The highly conserved sequences of selenium-binding proteins (SBPs) among diverse species and kingdoms suggest that SBPs share a fundamental biological role (Agalou et al., 2005). SBP1 was first characterized in mouse liver as a ⁷⁵selenium (Se)-binding protein in experiments designed to identify selenoproteins (Bansal et al., 1990). In mammals, Se is an essential nutrient. It is incorporated in the selenoamino acid Se-Cys, which is required for the translation of several proteins involved in cell defense or hormone regulation (Behne and Kyriakopoulos, 2001; Papp et al., 2007), and can as well be bound by binding proteins such as SBP1 (Bansal et al., 1990). Although a physiological function has not yet been assigned to SBP1, its involvement in detoxification mechanisms is largely suggested. Down-regulation of SBP1 expression is correlated with rapid tumor development in many organs (Kim et al., 2006; Stammer et al., 2008), and recently, SBP1 was characterized as a biomarker for schizophrenia (Glatt et al., 2005). Moreover, its homolog SBP2 is suggested to play a protective role as a scavenger of toxic electrophiles or oxidant species (Lanfear et al., 1993; Cohen et al., 1997; Mattow et al., 2006). Other functions, such as intra-Golgi protein transport, have been assigned to mammalian SBP (Porat et al., 2000).To date, Se has not been demonstrated to be essential in land plants, but a Se-containing glutathione (GSH) peroxidase has been isolated from Chlamydomonas reinhardtii (Fu et al., 2002). Except at low concentration, where it can have a positive effect on plant growth, Se is highly toxic. Se toxicity results from its chemical similarity with sulfur (S), leading to nonspecific replacement of S by Se in proteins and other S compounds (White et al., 2004; Sors et al., 2005). Se is taken up by roots from the soil mostly in inorganic forms {selenate [Se(VI)] or selenite [Se(IV)]} and then converted to organic forms that can accumulate in plant tissues or be volatilized in the atmosphere (Terry et al., 2000; Ellis and Salt, 2003). Mechanisms of Se tolerance in plants could be achieved by the conversion of selenoamino acids into their methylated forms, nonincorporable into proteins, or by compartmentalization into vacuoles in organic or inorganic forms (Lauchli, 1993; Nakamuro et al., 2000). SBP may participate in Se tolerance, as Arabidopsis (Arabidopsis thaliana) plants overexpressing SBP1 have increased resistance to Se(IV) (Agalou et al., 2005).In a previous study, we showed that SBP1 protein accumulated in response to cadmium (Cd) in Arabidopsis cultured cells and plants (Sarry et al., 2006; Dutilleul et al., 2008). SBP1 overexpression led to enhanced tolerance to Cd in Arabidopsis, suggesting that SBP1 may represent a new detoxification mechanism that plants use to face heavy metal toxicity, possibly through direct binding to the metal (Dutilleul et al., 2008). Besides its involvement in response to metal and metalloid stresses, data suggest other functions. For example, in Lotus japonicus, SBP1 was reported to be involved in nodule formation and function (Flemetakis et al., 2002). In rice (Oryza sativa), overexpression of SBP1 led to enhanced tolerance against different pathogens (Sawada et al., 2004).In the Arabidopsis genome, three SBP genes are present. SBP1 appeared to be the most expressed gene in healthy plants and in response to stress (Dutilleul et al., 2008). We showed that SBP1 was ubiquitously expressed in nonstressed conditions, notably in actively growing tissues and during seed development, highlighting the dynamic regulation of SBP1 expression during development as well (Dutilleul et al., 2008). To get a better understanding of SBP1 protein function in plants, the main goal of this article was to analyze SBP1 promoter activity in response to different stresses and to highlight signals that may regulate its expression. Expression analysis using luciferase (LUC) imaging showed that SBP1 and PRH43, which encodes 5′-Adenylylphosphosulfate Reductase2, a marker for the induction of the S assimilation pathway, were similarly regulated by the different stresses and highly induced in response to S starvation (−S). In addition, SBP1-overexpressing plants showed increased tolerance to stress affecting the GSH level. These results are discussed together with the potential function of SBP1 in plants.     

Out of the North Sea: the Zeeland Ridges Neandertal

In 2001, a portion of human frontal bone was discovered in sediments extracted from the bottom of the North Sea, 15 km off the coast of the Netherlands. The extraction zone is located in the so-called Zeeland Ridges area located at 51°40′ northern latitude and 3°20′ eastern longitude. The specimen was dredged up from sediments containing Late Pleistocene faunal remains and Middle Palaeolithic artefacts, including well-finished small handaxes and Levallois flakes. The details of the supraorbital morphology, as well as the quantitative assessment of the shape of the external surface of the squama using traditional and 3D geometric morphometrics, unambiguously assign the Zeeland Ridges frontal bone to Homo neanderthalensis. Carbon and nitrogen isotopic analysis indicate that the Zeeland Ridges hominin, like other Neandertals, was highly carnivorous and does not show evidence for the consumption of aquatic foods. A lesion on the outer table and diploic layer of the bone in the area of the supratoral sulcus can be interpreted as the result of an intradiploic epidermoid cyst, a type of neoplasm diagnosed for the first time in Neandertal remains. So far, the Zeeland Ridges Neandertal is the first Pleistocene fossil hominin found under seawater and the first recorded in the Netherlands.     

The Monothiol Single-Domain Glutaredoxin Is Conserved in the Highly Reduced Mitochondria of Giardia intestinalis

The highly reduced mitochondria (mitosomes) of Giardia intestinalis are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of G. intestinalis thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.Giardia intestinalis is a parasitic protist that was considered amitochondrial until recently (7). Although typical ATP-producing mitochondria are not present, related organelles, named mitosomes, were discovered in this organism (29). Mitosomes and mitochondria have a number of similarities; most notably, both are surrounded by a double membrane (29), they have a common mode of protein import and maturation (6, 24), and both harbor key components of the FeS cluster assembly machinery (29). These similarities indicate that mitosomes are highly reduced forms of mitochondria (7, 30), even though alternative evolutionary scenarios are still being discussed (17).FeS clusters are cofactors of a number of FeS proteins involved mainly in electron transport, energetic metabolism, synthetic pathways, and biological sensing (12). Most importantly, the FeS protein Rli1 is indispensable for rRNA processing and ribosome biogenesis (15, 31). Consequently, the biogenesis of FeS clusters is an essential process for all cells from bacteria to human cells (15). In most nonplant eukaryotes, the crucial part of this biosynthetic pathway occurs in the mitochondrion or mitochondrion-related organelles (14, 24, 28). Studies of Saccharomyces cerevisiae mitochondria showed that the FeS cluster assembly is centered on IscU, a metallochaperone that serves as a scaffold for a new FeS cluster. The cysteine desulfurase IscS (in S. cerevisiae named Nfs1) forms a heterodimer with Isd11. This heterodimer catalyzes the mobilization of sulfur for the FeS cluster. The delivery of iron is most likely regulated by frataxin (1). Reducing equivalents, which are required during FeS cluster biogenesis, are provided by a short electron transport chain, including the mitochondrial [2Fe2S] ferredoxin and ferredoxin:NADH reductase. Finally, a transient FeS cluster is transferred from IscU (Isu1/2) to apoproteins by the action of the Hsp70 (Ssq1) and Hsp40 (Jac1) chaperones and the proteins IscA (Isa1/2) and Iba57 (15). This last step also requires a monothiol class glutaredoxin (Grx5) with a characteristic CGFS active site motif (20). This class of glutaredoxins catalyzes the reduction of disulfide bonds in proteins converting glutathione (GSH) to GSH disulfide. It has been recently demonstrated that dimeric monothiol glutaredoxins can coordinate a [2Fe2S] cluster via the cysteine residue of the active site of each monomer and the cysteines of two GSH molecules (20). Although the exact role of Grx5 remains to be elucidated, it was hypothesized that, in the final step of iron sulfur cluster biogenesis, the FeS cluster that is transiently formed on an IscU scaffold protein is transferred to a Grx5 dimer in a GSH-dependent manner and is subsequently passed on to the target apoproteins (20).The ability of Giardia mitosomes to assemble FeS clusters on apoferredoxin has been demonstrated (29), indicating that all necessary components of the FeS cluster assembly machinery are present in these organelles. However, only three components known from S. cerevisiae mitochondria have been shown to be localized to mitosomes so far (IscS, IscU, and [2Fe2S] ferredoxin), while some others, including Isd11, frataxin, and GSH, were reported to be absent in Giardia (4, 5, 21). In this study we identified monothiol glutaredoxin in a mitosome-enriched fraction of G. intestinalis (Gigrx) using a proteomic approach. The mitosomal localization of Gigrx was confirmed by expression of the tagged protein in Giardia cells. The protein contains an unusually long N-terminal extension, which is involved in targeting the protein to the organelle, while the C-terminal part contains conserved residues required for the coordination of an FeS cluster. Phylogenetic analysis showed the close relationship between Gigrx and its mitochondrial homologues, which is consistent with the proposed mitochondrial origin of the iron-sulfur cluster assembly machinery in G. intestinalis.     

ADP Ribosylation Factor Like 2 (Arl2) Regulates Breast Tumor Aggressivity in Immunodeficient Mice

We have previously reported that ADP ribosylation factor like 2 (Arl2), a small GTPase, content influences microtubule dynamics and cell cycle distribution in breast tumor cells, as well as the degree and distribution of phosphorylated P53. Here we show, in two different human breast adenocarcinoma models, that Arl2 content has a major impact on breast tumor cell aggressivity both in vitro and in vivo. Cells with reduced content of Arl2 displayed reduced contact inhibition, increased clonogenic or cluster formation as well as a proliferative advantage over control cells in an in vitro competition assay. These cells also caused larger tumors in SCID mice, a phenotype which was mimicked by the in vivo administration of siRNA directed against Arl2. Cells with increased Arl2 content displayed reduced aggressivity, both in vitro and in vivo, with enhanced necrosis and were also found to contain increased PP2A phosphatase activity. A rt-PCR analysis of fresh human tumor breast samples suggested that low Arl2 expression was associated with larger tumor size and greater risk of lymph node involvement at diagnosis. These data underline the role of Arl2, a small GTPase, as an important regulator of breast tumor cell aggressivity, both in vitro and in vivo.     

The Warburg Effect Is Genetically Determined in Inherited Pheochromocytomas

The Warburg effect describes how cancer cells down-regulate their aerobic respiration and preferentially use glycolysis to generate energy. To evaluate the link between hypoxia and Warburg effect, we studied mitochondrial electron transport, angiogenesis and glycolysis in pheochromocytomas induced by germ-line mutations in VHL, RET, NF1 and SDH genes. SDH and VHL gene mutations have been shown to lead to the activation of hypoxic response, even in normoxic conditions, a process now referred to as pseudohypoxia. We observed a decrease in electron transport protein expression and activity, associated with increased angiogenesis in SDH- and VHL-related, pseudohypoxic tumors, while stimulation of glycolysis was solely observed in VHL tumors. Moreover, microarray analyses revealed that expression of genes involved in these metabolic pathways is an efficient tool for classification of pheochromocytomas in accordance with the predisposition gene mutated. Our data suggest an unexpected association between pseudohypoxia and loss of p53, which leads to a distinct Warburg effect in VHL-related pheochromocytomas.