Specific Circular RNA Signature of Endothelial Cells: Potential Implications in Vascular Pathophysiology.

Circular RNAs (circRNAs) are a recently characterized family of gene transcripts forming a covalently closed loop of single-stranded RNA. The extent of their potential for fine-tuning gene expression is still being discovered. Several studies have implicated certain circular RNAs in pathophysiological processes within vascular endothelial cells and cancer cells independently. However, to date, no comparative study of circular RNA expression in different types of endothelial cells has been performed and analysed through the lens of their central role in vascular physiology and pathology. In this work, we analysed publicly available and original RNA sequencing datasets from arterial, veinous, and lymphatic endothelial cells to identify common and distinct circRNA expression profiles. We identified 4713 distinct circRNAs in the compared endothelial cell types, 95% of which originated from exons. Interestingly, the results show that the expression profile of circular RNAs is much more specific to each cell type than linear RNAs, and therefore appears to be more suitable for distinguishing between them. As a result, we have discovered a specific circRNA signature for each given endothelial cell type. Furthermore, we identified a specific endothelial cell circRNA signature that is composed four circRNAs: circCARD6, circPLXNA2, circCASC15 and circEPHB4. These circular RNAs are produced by genes that are related to endothelial cell migration pathways and cancer progression. More detailed studies of their functions could lead to a better understanding of the mechanisms involved in physiological and pathological (lymph)angiogenesis and might open new ways to tackle tumour spread through the vascular system.

Long non-coding RNA expression profile in cytogenetically normal acute myeloid leukemia identifies a distinct signature and a new biomarker in NPM1-mutated patients.

Long non-coding RNAs are defined as transcripts larger than 200 nucleotides but without protein-coding potential. There is growing evidence of the important role of long non-coding RNAs in cancer initiation, development and progression. In this study, we sought to evaluate the long non-coding RNA expression profile of patients with cytogenetically normal acute myeloid leukemia (AML). RNA-sequencing of 40 cytogenetically normal AML patients allowed us to quantify 11,036 long non-coding RNAs. Among these, more than 8000 were previously undescribed long non-coding RNAs. Using unsupervised analysis, we observed a specific long non-coding RNA expression profile dependent on the mutational status of the NPM1 gene. Statistical analysis allowed us to identify a minimal set of 12 long non-coding RNAs capable of discriminating NPM1-mutated from NPM1-wild-type patients. These results were validated by qRT-PCR on an independent cohort composed of 134 cytogenetically normal AML patients. Furthermore, we have identified one putative biomarker, the long non-coding RNA XLOC_109948 whose expression pattern predicts clinical outcome. Interestingly, low XLOC_109948 expression indicates a good prognosis especially for NPM1-mutated patients. Transient transfection of GapmeR against XLOC_109948 in NPM1-mutated OCI-AML3 cell line treated with Ara-C or ATRA enhances apoptosis suggesting XLOC_109948 plays a role in drug sensitivity. This study improves our knowledge of the long non-coding RNA transcriptome in cytogenetically normal AML patients. We observed a distinct long non-coding RNA expression profile in patients with the NPM1 mutation. The newly identified XLOC_109948 long non-coding RNA emerged as a strong prognostic factor able to better stratify NPM1-mutated patients.

Hypoxia and ER stress promote Staufen1 expression through an alternative translation mechanism.

Under physiological stress conditions the cell protects itself through a global blockade on cap-dependent translation of mRNA. This allows cap-independent mechanisms such as internal ribosome entry site (IRES)-mediated translation to take over and initiate the translation of a specific pool of mRNAs that encode proteins involved in protecting the cell from stress. Staufen 1 (Stau1) is an RNA-binding protein that has been previously implicated in the regulation of stress granule formation and therefore could play a key role in protecting the cell against stress stimuli such as oxidative and endoplasmic reticulum (ER) stress. We hypothesized that Stau1 mRNA could, like many stress response genes, contain an IRES in its 5'UTR. Here we describe that a bona fide IRES element is present in the 5'UTR of Stau1 mRNA, which is activated under hypoxic and ER stress conditions. Further, we show that the activity of PERK kinase, a major effector of the ER stress response, is required for Stau1 IRES-mediated translation during ER stress. These results suggest that Stau1 is a stress response gene that remains efficiently translated during hypoxia and ER stress despite the substantial global inhibition of cap-dependent protein translation, promoting cell recovery following stress.

Stabilization of the G-quadruplex at the VEGF IRES represses cap-independent translation.

The activation of translation contributes to malignant transformation and is an emerging target for cancer therapies. RNA G-quadruplex structures are general inhibitors of cap-dependent mRNA translation and were recently shown to be targeted for oncoprotein translational activation. In contrast however, the G-quadruplex within the 5'UTR of the human vascular endothelial growth factor A (VEGF) has been shown to be essential for IRES-mediated translation. Since VEGF has a pivotal role in tumor angiogenesis and is a major target of anti-tumoral therapies, we investigated the structure/function relationship of the VEGF G-quadruplex and defined whether it could have a therapeutic potential. We found that the G-quadruplex within the VEGF IRES is dispensable for cap-independent function and activation in stress conditions. However, stabilization of the VEGF G-quadruplex by increasing the G-stretches length or by replacing it with the one of NRAS results in strong inhibition of IRES-mediated translation of VEGF. We also demonstrate that G-quadruplex ligands stabilize the VEGF G-quadruplex and inhibit cap-independent translation in vitro. Importantly, the amount of human VEGF mRNA associated with polysomes decreases in the presence of a highly selective stabilizing G-quadruplex ligand, resulting in reduced VEGF protein expression. Together, our results uncover the existence of functionally silent G-quadruplex structures that are susceptible to conversion into efficient repressors of cap-independent mRNA translation. These findings have implications for the in vivo applications of G-quadruplex-targeting compounds and for anti-angiogenic therapies.

VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level.

Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3'-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3' untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation.

The nonlysosomal ß-glucosidase GBA2 promotes endoplasmic reticulum stress and impairs tumorigenicity of human melanoma cells.

Glycosphingolipids, which are abundant at the surface of melanoma cells, play crucial roles in tumor progression. We investigated whether a newly described glycosphingolipid hydrolase, encoded by the GBA2 gene, can modulate human melanoma cell growth and death. GBA2 expression was quantified on melanoma cells by RT-qPCR. The antiproliferative effects of GBA2 were assessed in tumor cells expressing inducible GBA2 and in established melanoma xenografts. As a control an inducible catalytically inactive GBA2 mutant was generated. Sphingolipid levels were monitored by mass spectrometry; unfolded protein response (UPR) and apoptosis were assessed by Western blot and flow cytometry analyses, respectively. We report that GBA2 is down-regulated in melanoma; inducible expression of GBA2 affects endogenous sphingolipid metabolism by promoting glucosylceramide degradation (decrease by 78%) and ceramide generation; this is followed by a UPR that causes apoptosis, subsequent decreased anchorage-independent cell growth, and reduced in vivo tumor growth (by 40%); and all these events are abrogated when expressing a catalytically inactive GBA2. This study documents for the first time the antitumor activity of GBA2 and provides evidence for the role of nonlysosomal glucosylceramide breakdown as a source of bioactive ceramide and a mechanistic link between glycolipid catabolism and the UPR/death response of melanoma cells.

Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy.

Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell's viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.

Differential prognostic values of the three AKT isoforms in acute myeloid leukemia.

The PI3K-AKT-mTOR pathway lies at the confluence of signaling pathways in which various components are subjected to activating genetic alterations in acute myeloid leukemia (AML), thus contributing to oncogenesis. Three AKT isoforms exist in humans. However, whether one isoform predominates in AML remains unknown. This study reveals that AKT3 behaves very distinctly than AKT1 or AKT2 in both normal myeloid differentiation and AML. During normal differentiation, AKT3 is preferentially expressed in hematopoietic stem cells whilst AKT1 becomes preferentially expressed as cells differentiate into granulocytes or monocytes. AKT2 expression remains unchanged. In AML, AKT3 expression varies widely among patient samples and is counterintuitively high in mature/monocytic leukemia. Furthermore, a low level of AKT3 expression is strongly correlated to genetic alterations associated with a better outcome (NPM1 mutations and RUNX1-RUNX1T1 translocation), while a high level is correlated to alterations associated to a bad outcome (RUNX1 mutations; and SRSF2, U2AF1, SF3B1, ASXL1 and BCOR mutations occurring frequently in MDS and MPN). Consistently, a high AKT3 expression level appears as a very strong predictor of poor survival. Curiously, although modestly varying among AML samples, a high AKT1 expression shows in contrast as a strong predictor of a better patient outcome. These data suggest that AKT3 and AKT1 expressions have strong, yet opposite, prognostic values.

A synonymous polymorphism of the Tristetraprolin (TTP) gene, an AU-rich mRNA-binding protein, affects translation efficiency and response to Herceptin treatment in breast cancer patients.

Post-transcriptional regulation plays a central role in cell differentiation and proliferation. Among the regulatory factors involved in this mechanism, Tristetraprolin (ZFP36 or TTP) is the prototype of a family of RNA-binding proteins that bind to adenylate and uridylate (AU)-rich sequences in the 3'UTR of mRNAs, which promotes their physiological decay. Here, we investigated whether TTP correlates with tumor aggressiveness in breast cancer and is a novel prognostic factor for this neoplasia. By immunoblot analysis, we determined the amount of TTP protein in different breast cancer cell lines and found an inverse correlation between aggressiveness and metastatic potential. TTP mRNA levels were very variable among cells lines and did not correlate with protein levels. Interestingly, by sequencing the entire TTP coding region in Hs578T cells that do not express the TTP protein, we identified a synonymous polymorphism (rs3746083) that showed a statistically significant association with a lack of response to Herceptin/Trastuzumab in HER2-positive-breast cancer patients. Even though this genetic change did not modify the corresponding amino acid, we performed functional studies and showed an effect on protein translation associated with the variant allele with respect to the wild-type. These data underline the importance of synonymous variants on gene expression and the potential role of TTP genetic polymorphisms as a prognostic marker for breast cancer.

Pivotal role of translokin/CEP57 in the unconventional secretion versus nuclear translocation of FGF2.

Intracellular trafficking of fibroblast growth factor 2 (FGF2) exhibits two unusual features: (i) it is secreted despite the lack of signal peptide and (ii) it can translocate to the nucleus after interaction with high- and low-affinity receptors on the cell surface, although it does not possess any classical nuclear localization signal. This nuclear translocation constitutes an important part of the response to the growth factor. Previously, we identified Translokin/CEP57, an FGF2 binding partner, as an intracellular mediator of FGF2 trafficking, which is essential for the nuclear translocation of the growth factor. Here, we report the identification of four Translokin partners: sorting nexin 6, Ran-binding protein M and the kinesins KIF3A and KIF3B. These proteins, through their interaction with Translokin, are involved in two exclusive complexes allowing the bidirectional trafficking of FGF2. Thus, Translokin plays a pivotal role in this original mechanism. In addition, we show that FGF2 secretion is regulated by a negative loop, retro-controlled by FGF receptor and involving FGF2 itself.

The VEGF IRESes are differentially susceptible to translation inhibition by miR-16.

Experiments with EMCV (Encephalomyocarditis virus) internal ribosome entry sites (IRESes) have shown that microRNAs (miRs) are unable to inhibit IRES driven translation. However, it is accepted that miRs can inhibit translation through multiple mechanisms, only some of which require interaction with the 5' cap structure. In this report, we first validate the targeting of miR-16 to a predicted binding site in the VEGF 3'UTR. We developed a series of experiments to ascertain whether or not miR-16 can inhibit translation of transcripts driven by either of the VEGF IRESes. Our results indicate that cellular IRESes can be classified as both sensitive and insensitive to miR control. While VEGF IRES-A activity was not altered by miR-16 targeting to the 3'UTR, IRES-B was susceptible to miR-16 inhibition. Taken together with previous results that show that IRES-B selectively translates the CUG initiated VEGF-121 isoform, we can conclude that the existence of two differentially susceptible IRESes in the VEGF 5'UTR leads to even more complex regulatory control of VEGF isoform production. This study demonstrates for the first time the inhibition of cellular IRES driven translation by a miR.

Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production.

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.

A novel leukemic route of mutant NPM1 through nuclear import of the overexpressed long noncoding RNA LONA.

The most frequent genetic alteration in acute myeloid leukemia (AML) is the mutation of nucleophosmin 1 (NPM1). Yet, its downstream oncogenic routes are not fully understood. Here, we report the identification of one long noncoding RNA (lncRNA) overexpressed in NPM1-mutated AML patients (named LONA) whose intracellular localization inversely reflects that of NPM1. While NPM1 is nuclear and LONA cytoplasmic in wild-type NPM1 AML cells, LONA becomes nuclear as mutant NPM1 moves toward the cytoplasm. Gain or loss of function combined with a genome-wide RNA-seq search identified a set of LONA mRNA targets encoding proteins involved in myeloid cell differentiation (including THSB1, MAFB, and ASB2) and interaction with its microenvironment. Consistently, LONA overexpression in mutant NPM1 established cell lines and primary AML cells exerts an anti-myeloid differentiation effect, whilst it exerts an opposite pro-myeloid differentiation effect in a wild type NPM1 setting. In vivo, LONA overexpression acts as an oncogenic lncRNA reducing the survival of mice transplanted with AML cells and rendering AML tumors more resistant to AraC chemotherapy.These data indicate that mutation-dependent nuclear export of NPM1 leads to nuclear retention and consequent oncogenic functions of the overexpressed lncRNA LONA, thus uncovering a novel NPM1 mutation-dependent pathway in AML pathogenesis.

ER Stress and Unfolded Protein Response in Leukemia: Friend, Foe, or Both?

The unfolded protein response (UPR) is an evolutionarily conserved adaptive signaling pathway triggered by a stress of the endoplasmic reticulum (ER) lumen compartment, which is initiated by the accumulation of unfolded proteins. This response, mediated by three sensors-Inositol Requiring Enzyme 1 (IRE1), Activating Transcription Factor 6 (ATF6), and Protein Kinase RNA-Like Endoplasmic Reticulum Kinase (PERK)-allows restoring protein homeostasis and maintaining cell survival. UPR represents a major cytoprotective signaling network for cancer cells, which frequently experience disturbed proteostasis owing to their rapid proliferation in an usually unfavorable microenvironment. Increased basal UPR also participates in the resistance of tumor cells against chemotherapy. UPR activation also occurs during hematopoiesis, and growing evidence supports the critical cytoprotective role played by ER stress in the emergence and proliferation of leukemic cells. In case of severe or prolonged stress, pro-survival UPR may however evolve into a cell death program called terminal UPR. Interestingly, a large number of studies have revealed that the induction of proapoptotic UPR can also strongly contribute to the sensitization of leukemic cells to chemotherapy. Here, we review the current knowledge on the consequences of the deregulation of UPR signaling in leukemias and their implications for the treatment of these diseases.

Adenosine triphosphatase pontin is overexpressed in hepatocellular carcinoma and coregulated with reptin through a new posttranslational mechanism.

UNLABELLED: Reptin and Pontin are related ATPases associated with stoichiometric amounts in several complexes involved in chromatin remodeling, transcriptional regulation, and telomerase activity. We found that Reptin was up-regulated in hepatocellular carcinoma (HCC) and that down-regulation of Reptin led to growth arrest. We show here that Pontin messenger RNA (mRNA) is also up-regulated in human HCC 3.9-fold as compared to nontumor liver (P = 0.0004). Pontin expression was a strong independent factor of poor prognosis in a multivariate analysis. As for Reptin, depletion of Pontin in HuH7 cells with small interfering RNAs (siRNAs) led to growth arrest. Remarkably, Pontin depletion led to down-regulation of Reptin as shown with western blot, and vice versa. Whereas siRNAs induced a decrease of their cognate mRNA targets, they did not affect the transcripts of the partner protein. Translation of Pontin or Reptin was not altered when the partner protein was silenced. However, pulse-chase experiments demonstrated that newly synthesized Pontin or Reptin stability was reduced in Reptin- or Pontin-depleted cells, respectively. This phenomenon was reversed upon inhibition of proteasome or ubiquitin-activating enzyme (E1). In addition, proteasome inhibition could partly restore Pontin steady-state levels in Reptin-depleted cells, as shown by western blot. This restoration was not observed when cells were also treated with cycloheximide, thus confirming that proteasomal degradation in this setting was restricted to newly synthesized Pontin. CONCLUSION: Reptin and Pontin protein levels are strictly controlled by a posttranslational mechanism involving proteasomal degradation of newly synthesized proteins. These data demonstrate a tight regulatory and reciprocal interaction between Reptin and Pontin, which may in turn lead to the maintenance of their 1:1 stoichiometry.

An upstream open reading frame within an IRES controls expression of a specific VEGF-A isoform.

Vascular endothelial growth factor A (VEGF-A) is a potent secreted mitogen critical for physiological and pathological angiogenesis. Regulation of VEGF-A occurs at multiple levels, including transcription, mRNA stabilization, splicing, translation and differential cellular localization of various isoforms. Recent advances in our understanding of the posttranscriptional regulation of VEGF-A are comprised of the identification of stabilizing mRNA-binding proteins and the discovery of two internal ribosomal entry sites (IRES) as well as two alternative initiation codons in the 5'UTR of the VEGF-A mRNA. We have previously reported that VEGF-A translation initiation at both the AUG and CUG codons is dependent on the exon content of the coding region. In this report, we show that the expression of different VEGF-A isoforms is regulated by a small upstream open reading frame (uORF) located within an internal ribosome entry site, which is translated through a cap-independent mechanism. This uORF acts as a cis-regulatory element that regulates negatively the expression of the VEGF 121 isoform. Our data provide a framework for understanding how VEGF-A mRNAs are translated, and how the production of the VEGF 121 isoform is secured under non-hypoxic environmental conditions.

Translational Regulations in Response to Endoplasmic Reticulum Stress in Cancers.

During carcinogenesis, almost all the biological processes are modified in one way or another. Among these biological processes affected, anomalies in protein synthesis are common in cancers. Indeed, cancer cells are subjected to a wide range of stresses, which include physical injuries, hypoxia, nutrient starvation, as well as mitotic, oxidative or genotoxic stresses. All of these stresses will cause the accumulation of unfolded proteins in the Endoplasmic Reticulum (ER), which is a major organelle that is involved in protein synthesis, preservation of cellular homeostasis, and adaptation to unfavourable environment. The accumulation of unfolded proteins in the endoplasmic reticulum causes stress triggering an unfolded protein response in order to promote cell survival or to induce apoptosis in case of chronic stress. Transcription and also translational reprogramming are tightly controlled during the unfolded protein response to ensure selective gene expression. The majority of stresses, including ER stress, induce firstly a decrease in global protein synthesis accompanied by the induction of alternative mechanisms for initiating the translation of mRNA, later followed by a translational recovery. After a presentation of ER stress and the UPR response, we will briefly present the different modes of translation initiation, then address the specific translational regulatory mechanisms acting during reticulum stress in cancers and highlight the importance of translational control by ER stress in tumours.

The StkP/PhpP signaling couple in Streptococcus pneumoniae: cellular organization and physiological characterization.

In Streptococcus pneumoniae, stkP and phpP, encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called "soluble" protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone "competence stimulating peptide." We show that PhpP activity is essential in a stkP(+) genetic background, suggesting tight control of StkP activity by PhpP. Proteins PhpP and StkP colocalized to the cell membrane subcellular fraction and likely belong to the same complex, as revealed by coimmunoprecipitation in cellular extracts. Specific coimmunoprecipitation of the N-kinase domain of StkP and PhpP recombinant proteins by PhpP-specific antibodies demonstrates direct interaction between these proteins. Consistently, flow cytometry analysis allowed the determination of the cytoplasmic localization of PhpP and of the N-terminal kinase domain of StkP, in contrast to the periplasmic localization of the StkP C-terminal PASTA (penicillin-binding protein and serine-threonine kinase associated) domain. A signaling route involving interplay between serine, threonine, and histidine phosphorylation is thus described for the first time in this human pathogen.

Translational induction of VEGF internal ribosome entry site elements during the early response to ischemic stress.

Vascular endothelial growth factor-A (VEGF), a powerful factor involved in vasculogenesis and angiogenesis, is translationally regulated through 2 independent internal ribosome entry sites (IRESs A and B). IRESs enable an mRNA to be translated under conditions in which 5'-cap-dependent translation is inhibited, such as low oxygen stress. In the VEGF mRNA, IRES A influences translation at the canonical AUG codon, whereas the 5' IRES B element regulates initiation at an upstream, in frame CUG. In this study, we have developed transgenic mice expressing reporter genes under the control of these 2 IRESs. We reveal that although these IRESs display low activity in embryos and adult tissues, they permit efficient translation at early time points in ischemic muscle, a stress under which cap-dependent translation is inhibited. These results demonstrate the in vivo efficacy of the VEGF IRESs in response to a local environmental stress such as hypoxia.

The PERK Branch of the Unfolded Protein Response Promotes DLL4 Expression by Activating an Alternative Translation Mechanism.

Delta-like 4 (DLL4) is a pivotal endothelium specific Notch ligand that has been shown to function as a regulating factor during physiological and pathological angiogenesis. DLL4 functions as a negative regulator of angiogenic branching and sprouting. Interestingly, Dll4 is with Vegf-a one of the few examples of haplo-insufficiency, resulting in obvious vascular abnormalities and in embryonic lethality. These striking phenotypes are a proof of concept of the crucial role played by the bioavailability of VEGF and DLL4 during vessel patterning and that there must be a very fine-tuning of DLL4 expression level. However, to date the expression regulation of this factor was poorly studied. In this study, we showed that the DLL4 5'-UTR harbors an Internal Ribosomal Entry Site (IRES) that, in contrast to cap-dependent translation, was efficiently utilized in cells subjected to several stresses including hypoxia and endoplasmic reticulum stress (ER stress). We identified PERK, a kinase activated by ER stress, as the driver of DLL4 IRES-mediated translation, and hnRNP-A1 as an IRES-Trans-Acting Factor (ITAF) participating in the IRES-dependent translation of DLL4 during endoplasmic reticulum stress. The presence of a stress responsive internal ribosome entry site in the DLL4 msRNA suggests that the process of alternative translation initiation, by controlling the expression of this factor, could have a crucial role in the control of endothelial tip cell function.