ß-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression.

Increased endothelial cell (EC) apoptosis is associated with the development of atherosclerotic plaques that develop predominantly at sites exposed to disturbed flow (DF). Strategies to promote EC survival may therefore represent a novel therapeutic approach in cardiovascular disease. Nitric oxide (NO) and ß-catenin have both been shown to promote cell survival and they interact in ECs as we previously demonstrated. Here we investigated the physiological role of ß-catenin as a mediator of NO-induced cell survival in ECs. We found that ß-catenin depleted human umbilical vein ECs (HUVEC) stimulated with pharmacological activators of endothelial NO synthase (eNOS) showed a reduction in eNOS phosphorylation (Ser1177) as well as reduced intracellular cyclic guanosine monophosphate levels compared to control cells in static cultures. In addition, ß-catenin depletion abrogated the protective effects of the NO donor, S-nitroso-N-acetylpenicillamine, during TNFα- and H2O2-induced apoptosis. Using an orbital shaker to generate shear stress, we confirmed eNOS and ß-catenin interaction in HUVEC exposed to undisturbed flow and DF and showed that ß-catenin depletion reduced eNOS phosphorylation. ß-catenin depletion promoted apoptosis exclusively in HUVEC exposed to DF as did inhibition of soluble guanylate cyclase (sGC) or ß-catenin transcriptional activity. The expression of the pro-survival genes, Bcl-2 and survivin was also reduced following inhibition of ß-catenin transcriptional activity, as was the expression of eNOS. In conclusion, our data demonstrate that ß-catenin is a positive regulator of eNOS activity and cell survival in human ECs. sGC activity and ß-catenin-dependent transcription of Bcl-2, survivin, BIRC3 and eNOS are essential to maintain cell survival in ECs under DF.

Two E-selectin ligands, BST-2 and LGALS3BP, predict metastasis and poor survival of ER-negative breast cancer.

Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.

Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, cell morphology and migration.

BACKGROUND: Cell migration is essential during development and in human disease progression including cancer. Most cell migration studies concentrate on known or predicted components of migration pathways. RESULTS: Here we use data from a genome-wide RNAi morphology screen in Drosophila melanogaster cells together with bioinformatics to identify 26 new regulators of morphology and cytoskeletal organization in human cells. These include genes previously implicated in a wide range of functions, from mental retardation, Down syndrome and Huntington's disease to RNA and DNA-binding genes. We classify these genes into seven groups according to phenotype and identify those that affect cell migration. We further characterize a subset of seven genes, FAM40A, FAM40B, ARC, FMNL3, FNBP3/FBP11, LIMD1 and ZRANB1, each of which has a different effect on cell shape, actin filament distribution and cell migration. Interestingly, in several instances closely related isoforms with a single Drosophila homologue have distinct phenotypes. For example, FAM40B depletion induces cell elongation and tail retraction defects, whereas FAM40A depletion reduces cell spreading. CONCLUSIONS: Our results identify multiple regulators of cell migration and cytoskeletal signalling that are highly conserved between Drosophila and humans, and show that closely related paralogues can have very different functions in these processes.

Transcriptional regulation of the small GTPase RhoB gene by TGF{beta}-induced signaling pathways.

The purpose of the present study was to investigate the mechanism of transcriptional induction of the small GTPase RhoB gene by the transforming growth factor beta (TGFbeta) signaling pathway and the role of this regulation in TGFbeta-induced cell migration. To achieve our goals, we utilized a combination of siRNA-mediated gene silencing, adenovirus-mediated gene transfer receptor and MAPK inhibition, transactivation assays, and DNA-protein interaction assays in human HaCaT keratinocytes. We found that the RhoB gene is a direct transcriptional target of TGFbeta. We show that TGFbeta activates an early MEK/ERK pathway and that this activation is required for the recruitment of Smad3 to a novel, nonclassical, Smad binding element in the proximal RhoB promoter, in a p53-dependent manner. This element is overlapping with a CCAAT box that constitutively binds nuclear factor Y. Mutagenesis of this site abolished the Smad-mediated transactivation of the RhoB promoter. Finally, silencing of RhoB gene expression via siRNA or utilization of a dominant negative form of RhoB significantly inhibited TGFbeta-induced migration of HaCaT keratinocytes and DU145 prostate cancer cells. Our findings establish RhoB as a direct transcriptional target of TGFbeta in human keratinocytes and identify an important role of RhoB in TGFbeta-induced cell migration.-Vasilaki, E., Papadimitriou, E., Tajadura, V., Ridley, A. J., Stournaras, C., Kardassis, D. Transcriptional regulation of the small GTPase RhoB gene by TGFbeta-induced signaling pathways.

The Rho1p exchange factor Rgf1p signals upstream from the Pmk1 mitogen-activated protein kinase pathway in fission yeast.

The Schizosaccharomyces pombe exchange factor Rgf1p specifically regulates Rho1p during polarized growth. Rgf1p activates the beta-glucan synthase (GS) complex containing the catalytic subunit Bgs4p and is involved in the activation of growth at the second end, a transition that requires actin reorganization. In this work, we investigated Rgf1p signaling and observed that Rgf1p acted upstream from the Pck2p-Pmk1p MAPK signaling pathway. We noted that Rgf1p and calcineurin play antagonistic roles in Cl(-) homeostasis; rgf1Delta cells showed the vic phenotype (viable in the presence of immunosuppressant and chlorine ion) and were unable to grow in the presence of high salt concentrations, both phenotypes being characteristic of knockouts of the MAPK components. In addition, mutations that perturb signaling through the MAPK pathway resulted in defective cell integrity (hypersensitivity to caspofungin and beta-glucanase). Rgf1p acts by positively regulating a subset of stimuli toward the Pmk1p-cell integrity pathway. After osmotic shock and cell wall damage HA-tagged Pmk1p was phosphorylated in wild-type cells but not in rgf1Delta cells. Finally, we provide evidence to show that Rgf1p regulates Pmk1p activation in a process that involves the activation of Rho1p and Pck2p, and we demonstrate that Rgf1p is unique in this signaling process, because Pmk1p activation was largely independent of the other two Rho1p-specific GEFs, Rgf2p and Rgf3p.

Role of Rho GTPases and Rho-GEFs in the regulation of cell shape and integrity in fission yeast.

The Rho family of GTPases are highly conserved molecular switches that control some of the most fundamental processes of cell biology, including morphogenesis, vesicular transport, cell division and motility. Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to extracellular stimuli. In fission yeast, there are seven Dbl-related GEFs and they activate six Rho-type GTPases within a particular spatio-temporal context. The failure to do so might have consequences reflected in aberrant phenotypes and in some cases lead to cell death. In this review, we briefly summarize the role of Rho GTPases and Rho-GEFs in the establishment and maintenance of cell polarity and cell integrity in Schizosaccharomyces pombe.

Rgf1p is a specific Rho1-GEF that coordinates cell polarization with cell wall biogenesis in fission yeast.

Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. We have identified a new GEF, designated Rgf1p, which specifically regulates Rho1p during polarized growth. The phenotype of rgf1 null cells was very similar to that seen after depletion of Rho1p, 30% of cells being lysed. In addition, rgf1(+) deletion caused hypersensitivity to the antifungal drug Caspofungin and defects in the establishment of bipolar growth. rho1(+), but none of the other GTPases of the Rho-family, suppressed the rgf1Delta phenotypes. Moreover, deletion of rgf1(+) suppressed the severe growth defect in rga1(+) null mutants (a Rho1-GAP, negative regulator). Rgf1p and Rho1p coimmunoprecipitated and overexpression of rgf1(+) specifically increased the GTP-bound Rho1p; it caused changes in cell morphology, and a large increase in beta(1,3)-glucan synthase activity. These effects were similar to those elicited when the hyperactive rho1-G15V allele was expressed. A genetic relationship was observed between Rgf1p, Bgs4p (beta[1,3]-glucan synthase), and Pck1p (protein kinase C [PKC] homologue); Bgs4p and Pck1p suppressed the hypersensitivity to Caspofungin in rgf1Delta mutants. Rgf1p localized to the growing ends and the septum, where Rho1, Pck1p, and Bgs4p are known to function. Our results suggest that Rgf1p probably activates the Rho functions necessary for coordinating actin deposition with cell wall biosynthesis during bipolar growth, allowing the cells to remodel their wall without risk of rupture.

Synthesis of alpha-glucans in fission yeast spores is carried out by three alpha-glucan synthase paralogues, Mok12p, Mok13p and Mok14p.

Fission yeast possesses a family of (1,3)-alpha-glucan synthase-related genes; one of them, mok1+/ags1+, plays an essential function in morphogenesis during vegetative growth. Here we show that three mok1+ paralogues -mok12+, mok13+ and mok14+- are required for sporulation to succeed, acting at different stages of the spore wall maturation process. Mutation of mok12+ affected the efficiency of spore formation and spore viability. Deletion of mok13+ does not affect spore viability but the spores showed reduced resistance to stress conditions. mok14Delta mutant spores failed to accumulate the amylose-like spore wall-specific polymer. mok12+, mok13+ and mok14+ expression was restricted to sporulating cells and the proteins localized to the spore envelope but with different timing. mok11+ was also induced during the sporulation process although its deletion did not show apparently a sporulation defect. In vegetative cells, beta-glucans are more abundant than alpha-glucans (55% versus 28%). In spores, the situation was the opposite, alpha-glucans accounted for 46% while beta-glucans were approximately 38% of the total polysaccharides. We found at least two types of alpha-glucan polymers, Mok12p and Mok13p, were involved in the synthesis of the greater part of alpha-glucan in the spores envelope, a polymer that is mainly digested with alpha-1,3 glucanase, while Mok14p, homologous to starch synthases, was required for the synthesis of the iodine-reactive polymer that is made of alpha-1,4 glucose residues.

Schizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that regulates cell wall beta-glucan biosynthesis through the GTPase Rho1p.

Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell-wall synthesis. Here, we describe the cloning and characterization of rgf3+, a member of the Rho family of guanine nucleotide exchange factors (Rho GEFs). The rgf3+ gene was cloned by complementation of a mutant (ehs2-1) hypersensitive to drugs that interfere with cell-wall biosynthesis. The rgf3+ gene was found to be essential for cell viability and depletion of Rgf3p afforded phenotypes similar to those obtained following depletion of Rho1p. However, the cell death caused by Rgf3p depletion could be rescued by the presence of 1.2 M sorbitol, whereas depletion of Rho1 was lethal under the same conditions. We show that Rgf3p is a specific Rho1-GEF. The hypersensitivity to drugs affecting the cell wall of the ehs2-1 mutant was suppressed by overexpression of rho1+ but not by any of the other GTPases of the Rho family. Rgf3p interacted with the GDP-bound form of Rho1p and promoted the GDP-GTP exchange. In addition, we show that overexpression of Rgf3p produces multiseptated cells and increases beta-1,3-glucan synthase activity and the amount of cell wall beta-1,3-glucan. Rgf3p localized to the septum and the mRNA level was regulated in a cell-cycle-dependent manner peaking during septation. Our results suggest that Rgf3p acts as a positive activator of Rho1p, probably activating the Rho functions that coordinate cell-wall biosynthesis to maintain cell integrity during septation.