Semaphorin 3E Initiates Antiangiogenic Signaling through Plexin D1 by Regulating Arf6 and R-Ras

Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.Pathological angiogenesis characterizes numerous human diseases, ranging from chronic inflammation, atherosclerosis, diabetic retinopathy, and age-related macular degeneration to cancer (5, 11, 30). Thus, elucidating the mechanisms underlying normal and aberrant blood vessel growth may provide new therapeutic options for many highly prevalent disease conditions. Ultimately, normal angiogenesis results from a precise balance between pro- and antiangiogenic mediators. Among the former, the family of vascular endothelial growth factors (VEGFs), basic fibroblastic growth factor (bFGF), sphingosine-1-phosphate (S1P), and the chemokines interleukin-8/CXCL8 and SDF-1/CXCL12 and their receptors are some of the most widely investigated (reviewed in references 3, 5, 8, and 17). The best-known angiogenesis inhibitors are proteolytic cleavage products of extracellular matrix (ECM) or serum components, such as endostatin, angiostatin, arresten, and tumstatin (reviewed in references 11 and 20). Antiangiogenic cytokines have also been described, including interferons and certain interleukins, which appear to act indirectly by limiting the expression of proangiogenic mediators or inducing antiangiogenic molecules (reviewed in references 11 and 20). In contrast, there are few known developmentally regulated, naturally occurring antiangiogenic molecules, which include platelet factor 4 (18), thrombospondin 1 (14), and pigment epithelium-derived factor (PEDF) (9). Their precise mechanism of action is not fully understood, thus limiting the ability to design new molecularly based antiangiogenic strategies.Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of the netrin, slit, eph, and semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological blood vessel development (6). For example, multiple secreted class III semaphorins, which regulate developmental axonal growth (23, 27), are now known to act through their receptors, the A family plexins (plexins A1, A2, and A3), and their coreceptors, neuropilin 1 and neuropilin 2, to initiate pro- and antiangiogenic responses (reviewed in references 6 and 19). However, neuropilins also act as coreceptors for multiple angiogenic factors, such as VEGF, thus limiting our ability to distinguish the downstream signaling events initiated by semaphorins from those resulting from their interplay with endothelial growth and motility factors (19). In this regard, recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, independently of neuropilins to control endothelial cell (EC) positioning and patterning of the developing vasculature (13, 15). These findings prompted us to explore whether Sema3E acts as a natural antiangiogenic molecule and, if so, to investigate the underlying molecular mechanism. Indeed, we show here that Sema3E is a potent inhibitor of adult and tumor-induced angiogenesis. Sema3E causes filopodial retraction in endothelial tip cells and inhibits endothelial cell adhesion by disrupting integrin-mediated adhesive structures. At the molecular level, this process involves the stimulation of plexin D1 by Sema3E, which in turn interferes with R-Ras function and leads to the rapid activation of Arf6, thus revealing a novel physiological antiangiogenic signaling route.     

Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula

Plant genes induced during early root colonization of Medicago truncatula Gaertn. J5 by a growth-promoting strain of Pseudomonas fluorescens (C7R12) have been identified by suppressive subtractive hybridization. Ten M. truncatula genes, coding proteins associated with a putative signal transduction pathway, showed an early and transient activation during initial interactions between M. truncatula and P. fluorescens, up to 8 d after root inoculation. Gene expression was not significantly enhanced, except for one gene, in P. fluorescens-inoculated roots of a Myc(-)Nod(-) genotype (TRV25) of M. truncatula mutated for the DMI3 (syn. MtSYM13) gene. This gene codes a Ca(2+) and calmodulin-dependent protein kinase, indicating a possible role of calcium in the cellular interactions between M. truncatula and P. fluorescens. When expression of the 10 plant genes was compared in early stages of root colonization by mycorrhizal and rhizobial microsymbionts, Glomus mosseae activated all 10 genes, whereas Sinorhizobium meliloti only activated one and inhibited four others. None of the genes responded to inoculation by either microsymbiont in roots of the TRV25 mutant. The similar response of the M. truncatula genes to P. fluorescens and G. mosseae points to common molecular pathways in the perception of the microbial signals by plant roots.     

Matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature

Anthrax lethal toxin (LT), a virulence factor secreted by Bacillus anthracis, is selectively toxic to human melanomas with the BRAF V600E activating mutation because of its proteolytic activities toward the mitogen-activated protein kinase kinases (MEKs). To develop LT variants with lower in vivo toxicity and high tumor specificity, and therefore greater potential for clinical use, we generated a mutated LT that requires activation by matrix metalloproteinases (MMPs). This engineered toxin was less toxic than wild-type LT to mice because of the limited expression of MMPs by normal cells. Moreover, the systemically administered toxin produced greater anti-tumor effects than wild-type LT toward human xenografted tumors. This was shown to result from its greater bioavailability, a consequence of the limited uptake and clearance of the modified toxin by normal cells. Furthermore, the MMP-activated LT had very potent anti-tumor activity not only to human melanomas containing the BRAF mutation but also to other tumor types, including lung and colon carcinomas regardless of their BRAF status. Tumor histology and in vivo angiogenesis assays showed that this anti-tumor activity is due largely to the indirect targeting of tumor vasculature and angiogenic processes. Thus, even tumors genetically deficient in anthrax toxin receptors were still susceptible to the toxin therapy in vivo. Moreover, the modified toxin also displayed lower immunogenicity compared with the wild-type toxin. All these properties suggest that this MMP-activated anti-tumor toxin has potential for use in cancer therapy.     

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.     

Development and Validation of a 20K Single Nucleotide Polymorphism (SNP) Whole Genome Genotyping Array for Apple (Malus × domestica Borkh)

High-density SNP arrays for genome-wide assessment of allelic variation have made high resolution genetic characterization of crop germplasm feasible. A medium density array for apple, the IRSC 8K SNP array, has been successfully developed and used for screens of bi-parental populations. However, the number of robust and well-distributed markers contained on this array was not sufficient to perform genome-wide association analyses in wider germplasm sets, or Pedigree-Based Analysis at high precision, because of rapid decay of linkage disequilibrium. We describe the development of an Illumina Infinium array targeting 20K SNPs. The SNPs were predicted from re-sequencing data derived from the genomes of 13 Malus × domestica apple cultivars and one accession belonging to a crab apple species (M. micromalus). A pipeline for SNP selection was devised that avoided the pitfalls associated with the inclusion of paralogous sequence variants, supported the construction of robust multi-allelic SNP haploblocks and selected up to 11 entries within narrow genomic regions of ±5 kb, termed focal points (FPs). Broad genome coverage was attained by placing FPs at 1 cM intervals on a consensus genetic map, complementing them with FPs to enrich the ends of each of the chromosomes, and by bridging physical intervals greater than 400 Kbps. The selection also included ∼3.7K validated SNPs from the IRSC 8K array. The array has already been used in other studies where ∼15.8K SNP markers were mapped with an average of ∼6.8K SNPs per full-sib family. The newly developed array with its high density of polymorphic validated SNPs is expected to be of great utility for Pedigree-Based Analysis and Genomic Selection. It will also be a valuable tool to help dissect the genetic mechanisms controlling important fruit quality traits, and to aid the identification of marker-trait associations suitable for the application of Marker Assisted Selection in apple breeding programs.     

Hepatoprotective Effect of Arctium lappa Root Extract on Cadmium Toxicity in Adult Wistar Rats

This study was performed to determine the effects of Arctium lappa (Al) to protect against cadmium damage in the rat liver. Male rats received a single i.p. dose of CdCl₂ (1.2 mg/kg body weight (BW)) with or without Al extract administered daily by gavage (300 mg/kg BW) for 7 or 56 days. After 7 days, Al caused plasma transaminase activity to diminish in groups Al (glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)) and CdAl (GPT). After 56 days, GOT and GPT plasma activities were reduced in the Cd group. No alteration in plasma levels of creatinine, total bilirubin, and total protein were observed. GOT liver activity increased in the Cd group. No alteration was observed in superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and malondialdehyde (MDA) dosage. In the Cd group, hepatocyte proportion decreased and sinusoid capillary proportion increased. In the Al and CdAl groups, the nuclear proportion increased and the cytoplasmic proportion decreased. The hepatocyte nucleus density reduced in Cd and increased in the Al group. After 56 days, there was no alteration in the Cd group. In Al and CdAl groups, the nuclear proportion increased without cytoplasmic proportion variation, but the sinusoid capillary proportion was reduced. The hepatocyte nucleus density decreased in the Cd group and increased in the Al and CdAl groups. In conclusion, the liver function indicators showed that A. lappa protected the liver against cadmium toxicity damage.     

Effect of activating and inactivating mutations of GS-and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes

Previous investigations have demonstrated that both G_s- and the G_i-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of G_sα vs. G_i2α, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-G_sα or the inactivating G226A(H21a)-G_sα point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the G_i2α wild type or the missense mutations R179E-G_i2α, Q205L-G_i2α, and G204A(H21a)-G_i2α. The activating [R201C]G_sα-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-Ll adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]G_sα-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]G_i2α or [Q205L]G_i2α mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]G_i2α slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]G_i2α or [Q205L]G_i2α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]G_i2α and [Q205L]G_i2α mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]G_i2α and [Q205L]G_i2α mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]G_i2α and [Q205L]G_i2α over differentiated controls. The inactivating [H21a]G_i2α-mutant obliterated all signs of preadipocyte differentiation. It is concluded that G_i2 plays a positive and much more important role than G_s in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process. J. Cell. Biochem. 64:242–257. © 1997 Wiley-Liss, Inc.     

Monitoring mitochondrial translation in living cells

Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we devise an imaging approach to analyze mitochondrial translation within the context of single cells, by following the incorporation of clickable non‐canonical amino acids. We apply this method to multiple cell types, including specialized cells such as cardiomyocytes and neurons, and monitor with spatial resolution mitochondrial translation in axons and dendrites. We also show that translation imaging allows to monitor mitochondrial protein expression in patient fibroblasts. Approaching mitochondrial translation with click chemistry opens new avenues to understand how mitochondrial biogenesis is integrated into the cellular context and can be used to assess mitochondrial gene expression in mitochondrial diseases.     

Epithelial-Mesenchymal Transition Induces Endoplasmic-Reticulum-Stress Response in Human Colorectal Tumor Cells

Tumor cells are stressed by unfavorable environmental conditions like hypoxia or starvation. Driven by the resulting cellular stress tumor cells undergo epithelial-mesenchymal transition. Additionally, cellular stress is accompanied by endoplasmic reticulum-stress which induces an unfolded protein response. It is unknown if epithelial-mesenchymal transition and endoplasmic reticulum-stress are occurring as independent parallel events or if an interrelationship exists between both of them. Here, we show that in colorectal cancer cells endoplasmic reticulum-stress depends on the induction of ZEB-1, which is a main factor of epithelial-mesenchymal transition. In the absence of ZEB-1 colorectal cancer cells cannot mount endoplasmic reticulum-stress as a reaction on cellular stress situations like hypoxia or starvation. Thus, our data suggest that there is a hierarchy in the development of cellular stress which starts with the presence of environmental stress that induces epithelial-mesenchymal transition which allows finally endoplasmic reticulum-stress. This finding highlights the central role of epithelial-mesenchymal transition during the process of tumorigenesis as epithelial-mesenchymal transition is also associated with chemoresistance and cancer stemness. Consequently, endoplasmic reticulum-stress might be a well suited target for chemotherapy of colorectal cancers.