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

Abstract

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.

Keywords: DLL4 (delta like ligand 4); IRES (internal ribosome entry site); PERK (PKR-Like endoplasmic reticulum kinase); UPR (unfolded protein response); angiogenesis; endoplasmic reticulum stress; hypoxia.

Figure 1

DLL4 5′-UTR contains an internal ribosome entry site. (A) Schematic representation of bicistronic constructs. IRESs cloned within the inter-cistronic region were either viral (EMCV) or cellular (VEGF-A IRESA, VEGF-A IRESB, FGF-1, DLL4), (B–D) Analysis of the DLL4 IRES activity in transiently transfected (B) HeLa, (C) HUVEC or (D) NIH3T3 cells. 48h after transfection IRES activity was determined by calculating the LucF/LucR ratio. DLL4 IRES activity was compared to that of the cellular IRES-A of VEGF-A, the viral EMCV IRES or the FGF-1A IRES, known to be highly tissue and cell line specific. (E) HeLa Tet off cells were transfected with TET sensitive bicistronic constructs containing the full 5′-untranslated region of DLL4. At 2 h prior to transfection, cells were treated with 0.5 nM, 5 nM or 50 nM doxycycline (Dox). Forty-eight hours after transfection, luciferase activities were measured as described. As positive controls, the VEGF-A full 5′-UTR (containing a cryptic promoter) was introduced in the intercistronic region and only the VEGF-A IRES A or B as negative controls (sequences without cryptic promoter). Data are means ± SEM from 3 independent experiments in duplicates, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

The DLL4 IRES is activated by hypoxia. HeLa (A,B) or HUVEC endothelial cells (C,D) cells were transfected by bicistronic constructs containing either the EMCV or the DLL4 IRES and submitted to 24 h hypoxia. (A,C) Hypoxia was confirmed through verifying both expression induction of HIF-1α and 4E-BP1 dephosphorylation (lower band visible at 24 h of hypoxia) by western blotting. β-ACTIN was used as a loading control. (B,D) Relative luciferase activities LucR, LucF or LucF/LucFR ratio (fold increase) under normoxia (black bars) and hypoxia (white bars) in HeLa (B) or HUVEC (D) cells. Results represent the means of three independent experiments (±SEM), * p < 0.05, *** p < 0.001.

Figure 3

The DLL4 IRES is activated by ER stress. HeLa cells were transfected by bicistronic constructs containing either the EMCV or the DLL4 IRES and treated or not for 6 h with increasing concentrations of DTT. (A) ER stress induction was verified by monitoring both cytoplasmic XBP1 splicing by RT-PCR and eIF2 phosphorylation by western blotting (B) Relative luciferase activities (LucR, LucF or LucF/LucR ratio) after ER stress induction by treatment with increasing amounts of DTT for 6 h. IRES activities were determined by calculating the LucF/LucR ratios and are expressed as fold change versus untreated cells. The results represent the means of three independent experiments (±SEM), ** p < 0.01, *** p < 0.001.

Figure 4

Role of three UPR sensors in DLL4 IRES translation activation by ER Stress. (A) HeLa cells were transfected with PERK, ATF6, IRE1 or control (Scr) siRNA. The knockdown efficiency of the targeted transcripts was determined by western blotting with PERK, IRE1 and β-ACTIN antibodies, and by semi-quantitative RT-PCR against ATF6 and GAPDH as a control. (B) Western blot analysis of phosphorylated eIF2α and total eIF2α and RT-PCR analysis of XBP1 splicing performed after transfection of HeLa cells either with siRNA specific for PERK, ATF6, or IRE1 or with control siRNA (Scr), after treatment with DTT. (C) Relative IRES activities in HeLa cells treated with DTT/control after co-transfection with siRNA specific for PERK, ATF6 or IRE1, or with control siRNA (scr) and DLL4 or EMCV bicistronic vectors. (D) Western blot analysis of phosphorylated eIF2α and total eIF2α and RT-PCR analysis of XBP1 splicing performed after transfection of HeLa cells with DLL4 or EMCV bicistronic vectors, after treatment with DTT and increasing concentration of the PERK inhibitor GSK2606414. (E) Western blot analysis of phosphorylated eIF2α and total eIF2α and RT-PCR analysis of XBP1 splicing performed after transfection of HeLa cells with DLL4 or EMCV bicistronic vectors, after treatment with increasing concentration of the PERK activator CCT020312. (F) Relative IRES activities in HeLa cells treated with DTT/control after transfection with DLL4 or EMCV bicistronic vectors and treatment with PERK activator (CCT020312) or inhibitor (GSK2606414). The results represent the means of three independent experiments (±SEM), * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

PERK kinase activity stimulates DLL4 IRES-mediated translation in a phosphorylated eIF2α–dependent manner. (A) Linear schematic representation of PERK and PERK-LZ showing the locations of the major functional domains (SP = Signal Peptide, L zip = Leucine Zipper, HA = HA Tag). Numbers indicate amino acid positions. (B) Western blot analysis of doxycycline-induced-PERK-LZ (HA) expression and eIF2α phosphorylation after 48 h treatment with increasing amounts of doxycycline. (C) Relative IRES activities in PERK-LZ expressing HeLa cells treated with 1 µg/mL doxycycline /control after transfection of EMCV or DLL4 bicistronic vectors. Means ± SEM are shown, *** p < 0.001. (D) Western blot analysis of ATF4, total and phosphorylated eIF2α, as well as total PERK in HeLa cells, after transfection with ATF4 or scramble (Scr) siRNA and treatment with DTT. β-ACTIN was used as a loading control. (E) Relative IRES activities in HeLa cells treated with DTT/control, after cotransfection with the EMCV or DLL4 bicistronic vectors and with either siRNA specific for ATF4 or control siRNA (Scr). Results represent the means of three independent experiments ± SEM. (F) Wild-type (WT) and eIF2αS51A MEFs transfected with the bicistronic LucR-IRES-LucF vectors and treated with increasing concentrations of DTT. ER stress induction was verified by monitoring eIF2α phosphorylation by western blot and cytoplasmic XBP1 splicing by RT-PCR. (G) Relative IRES activities were determined as previously described in WT (left) and eIF2αS51A (right) MEFs. Results represent the means of three independent experiments ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

hnRNPA1 modulates DLL4 IRES activity during ER stress. (A) Western blot analysis of hnRNPA1 in HeLa nuclear, cytoplasmic or total extract after treatment with increasing concentrations of DTT. β-ACTIN was used as a loading control for cytoplasmic and total extracts and LAMIN-B for nuclear extract. (B) Western blot analysis of hnRNPA1, total and phosphorylated eIF2 in HeLa cells, after transfection with hnRNPA1 or scramble (Scr) siRNA and treatment with DTT. β-ACTIN was used as a loading control. (C) Relative IRES activities in HeLa cells treated with DTT/control, after cotransfection with the EMCV or DLL4 bicistronic vectors and with either siRNA specific for hnRNPA1 or control siRNA (Scr). Results represent the means of three independent experiments ± SEM. * p < 0.05, *** p < 0.001.

Figure 7

Schematic model of the network of gene expression co-regulation by stress during tumoral progression. During tumor progression, the stress zone encompasses the growing tumor, but also its microenvironment. The neo vessels and more particularly the Tip cells, present at the extremity and which guide the neo vessels towards the tumor, are located in this unfavorable microenvironment. Hypoxia, nutrient starvation and acidosis will irremediably induce the accumulation of unfolded protein in the reticulum of cells located in this area, leading to ER stress and UPR activation. Thus, in addition to transcriptional regulations, the activation of the PERK pathway will induce the co-regulation of an UPR dependent gene network containing IRES elements, revealing a translational regulon in which the synthesis of a cohort of angiogenic related genes is activated in response to ER stress. The fine-tuning of gene expression allows an efficient control of angiogenesis, which is a highly regulated process.