Vascular Endothelial Growth Factor mRNA Translation Research Articles

MCP-1 facilitates VEGF production by removing miR-374b-5p blocking of VEGF mRNA translation

Monocyte chemotactic protein-1 (MCP-1) is known to be able to facilitate vascular endothelial growth factor (VEGF) gene expression, hence promoting vascular hyperpermeability and neovascularization. We show here that a microRNA molecule, miR-374b-5p can target the 3′-untranslated region of the VEGF mRNA, thus preventing VEGF production. Additionally, MCP-1 promotes the acetylation of transcription factor stat3 at Lys685, which facilitates the formation of an ac-stat3-DNA methyltransferase-histone methyltransferase complex (ac-stat3/DNMT1/EZH2) that binds to the promoter of the miR-374b-5p gene. This results in diminished miR-374b-5p expression and enhanced VEGF production. Moreover, treatment of appropriate animal models either with a miR-374b-5p mimicry or with inhibitors of either stat3 acetylation, DNMT1, or EZH2, leads to marked inhibition of MCP-1-promoted neovascularization and tumor growth. These findings indicate that MCP-1 facilitated inhibition of miR-374b-5p gene expression leads to the removal of a block of VEGF mRNA translation by miR-374b-5p. This mechanism could be of importance in the modulation of inflammatory conditions.

Regulated in Development and DNA Damage 1 Is Necessary for Hyperglycemia-induced Vascular Endothelial Growth Factor Expression in the Retina of Diabetic Rodents

Vascular endothelial growth factor (VEGF) is considered a major role player in the pathogenesis of diabetic retinopathy, yet the mechanisms regulating its expression are not fully understood. Our laboratory previously demonstrated that diabetes-induced VEGF expression in the retina was dependent on the repressor of mRNA translation 4E-BP1. Interaction of 4E-BP1 with the cap-binding protein eIF4E regulates protein expression by controlling the selection of mRNAs for translation. The process is regulated by the master kinase mTOR in complex 1 (mTORC1), which phosphorylates 4E-BP1, thus promoting its disassociation from eIF4E. In the present study, we investigated the role of the Akt/mTORC1 repressor REDD1 (regulated in development and DNA damage) in diabetes-induced VEGF expression. REDD1 expression was induced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in Müller cells concomitant with increased VEGF expression. In Müller cells, hyperglycemic conditions attenuated global rates of protein synthesis and cap-dependent mRNA translation concomitant with up-regulated cap-independent VEGF mRNA translation, as assessed by a bicistronic luciferase reporter assay. Hyperglycemic conditions also attenuated mTORC1 signaling and enhanced 4E-BP1 binding to eIF4E. Furthermore, ectopic expression of REDD1 in Müller cells was sufficient to promote both increased 4E-BP1 binding to eIF4E and VEGF expression. Whereas the retina of wild-type mice exhibited increased expression of VEGF and tumor necrosis factor alpha (TNF-α) 4 weeks after streptozotocin administration, the retina of REDD1 knock-out mice failed to do so. Overall, the results demonstrate that REDD1 contributes to the pathogenesis of diabetes in the retina by mediating the pathogenic effects of hyperglycemia.

The ShcA adaptor activates AKT signaling to potentiate breast tumor angiogenesis by stimulating VEGF mRNA translation in a 4E-BP-dependent manner.

The ShcA adaptor protein is engaged by numerous receptor tyrosine kinases (RTKs) in breast cancer cells. Once activated, RTKs phosphorylate three key tyrosine phosphorylation sites (Y239, Y240 and Y317) within ShcA that creates a docking site for Grb2/SOS and Grb2/Gab-containing complexes to activate the MAPK and AKT signaling pathways, respectively. We previously demonstrated that a tyrosine to phenylalanine substitution of the ShcA tyrosine phosphorylation sites (Shc3F-Y239/240/313F) significantly impairs breast tumor growth and angiogenesis in transgenic mouse models, in part, through the regulation of vascular endothelial growth factor (VEGF) production. Despite this fact, the underlying molecular mechanisms by which ShcA transduces pro-tumorigenic signals in breast cancer cells remain poorly defined. In this study, we demonstrate that ShcA-dependent activation of AKT, but not the RAS/MAPK pathway, induces VEGF production by bolstering VEGF mRNA translation. Accordingly, ShcA drives breast tumor growth and angiogenesis in vivo in a 4E-BP-dependent manner. These findings establish ShcA as a biological bridge that links AKT activation downstream of RTKs to cap-dependent VEGF mRNA translation in order to promote mammary tumorigenesis.

AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1)

The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine-glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3′UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 Variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.

AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1)

The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine-glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3’UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.

MDM2 Regulates Vascular Endothelial Growth Factor mRNA Stabilization in Hypoxia

Expression of vascular endothelial growth factor (VEGF) increases in cancer cells during hypoxia. Herein, we report that the MDM2 oncoprotein plays a role in hypoxia-mediated VEGF upregulation. In studying the characteristics of MDM2 and VEGF expression in neuroblastoma cells, we found that hypoxia induced significantly higher upregulation of both VEGF mRNA and protein in MDM2-positive cells than in the MDM2-negative cells, even in cells without wild-type (wt) p53. We found that hypoxia induced translocation of MDM2 from the nucleus to the cytoplasm, which was associated with increased VEGF expression. Enforcing overexpression of cytoplasmic MDM2 by transfection of the mutant MDM2/166A enhanced expression of VEGF mRNA and protein production, even without hypoxia. The results of mechanistic studies demonstrated that the C-terminal RING domain of the MDM2 protein bound to the AU-rich sequence within the 3' untranslated region (3'UTR) of VEGF mRNA; this binding increased VEGF mRNA stability and translation. In addition, knockdown of MDM2 by small interfering RNA (siRNA) in MDM2-overexpressing cancer cells resulted in inhibition of VEGF protein production, cancer cell survival, and angiogenesis. Our results suggest that MDM2 plays a p53-independent role in the regulation of VEGF, which may promote tumor growth and metastasis.

RNA Binding Protein CUGBP2/CELF2 Mediates Curcumin-Induced Mitotic Catastrophe of Pancreatic Cancer Cells

BackgroundCurcumin inhibits the growth of pancreatic cancer tumor xenografts in nude mice; however, the mechanism of action is not well understood. It is becoming increasingly clear that RNA binding proteins regulate posttranscriptional gene expression and play a critical role in RNA stability and translation. Here, we have determined that curcumin modulates the expression of RNA binding protein CUGBP2 to inhibit pancreatic cancer growth.Methodology/Principal FindingsIn this study, we show that curcumin treated tumor xenografts have a significant reduction in tumor volume and angiogenesis. Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells. This was further confirmed by increased phosphorylation of checkpoint kinase 2 (Chk2) protein coupled with higher levels of nuclear cyclin B1 and Cdc-2. Curcumin increased the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA, but protein levels were lower. Furthermore, curcumin increased the expression of RNA binding proteins CUGBP2/CELF2 and TIA-1. CUGBP2 binding to COX-2 and VEGF mRNA was also enhanced, thereby increasing mRNA stability, the half-life changing from 30 min to 8 h. On the other hand, silencer-mediated knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF even with curcumin treatment. COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.Conclusion/SignificanceCurcumin inhibits pancreatic tumor growth through mitotic catastrophe by increasing the expression of RNA binding protein CUGBP2, thereby inhibiting the translation of COX-2 and VEGF mRNA. These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers.

Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. Acute hyperglycemia increased synthesis of intrarenal Ang I and Ang II and resulted in activation of both Ang II receptors, AT1 and AT2, in the kidney. Losartan (specific AT1 antagonist) or PD123319 (specific AT2 antagonist) did not affect hyperglycemia but prevented activation of renal AT1 and AT2, respectively. In murine renal cortex, acute hyperglycemia increased VEGF protein but not mRNA content after 24 h, which suggested translational regulation. Blockade of AT2, but not AT1, prevented increase in VEGF synthesis by inhibiting translation of VEGF mRNA in renal cortex. Acute hyperglycemia increased VEGF expression in wild type but not in AT2 knockout mice. Binding of heterogeneous nuclear ribonucleoprotein K to VEGF mRNA, which stimulates its translation, was prevented by blockade of AT2, but not AT1. The Akt-mTOR-p70 S6K signaling pathway, involved in the activation of mRNA translation, was activated in hyperglycemic kidneys and was blocked by the AT2 antagonist. Elongation phase is an important step of mRNA translation that is controlled by elongation factor 1A (eEF1A) and 2 (eEF2). Expression of eEF1A and activity of eEF2 was higher in kidney cortex from hyperglycemic mice and only the AT2 antagonist prevented these changes. To assess selectivity of translational control of VEGF expression, we measured expression of fibronectin (FN) and laminin β1 (lamβ1): acute hyperglycemia increased FN expression at both protein and mRNA levels, indicating transcriptional control, and did not affect the expression of lamβ1. To confirm results obtained with PD123319, we induced hyperglycemia in AT2 knockout mice and found that in the absence of AT2, translational control of VEGF expression by hyperglycemia was abolished. Our data show that acute hyperglycemia stimulates Ang II synthesis in murine kidney cortex, this leads to AT2 activation and stimulation of VEGF mRNA translation, via the Akt-mTOR-p70 S6K signaling pathway. Our data show that exclusive translational control of protein expression in the kidney by acute hyperglycemia is not a general phenomenon, but do not prove that it is restricted to VEGF.

Biphasic Regulation of HMG-CoA Reductase Expression and Activity during Wound Healing and Its Functional Role in the Control of Keratinocyte Angiogenic and Proliferative Responses

In this study, we determined the regulation and potential function of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) during skin repair in mice. Upon skin injury, healthy mice exhibited a biphasic increase in HMGR expression and activity with elevated levels at days 3 and 13 post-wounding. In situ hybridization revealed wound margin keratinocytes as a cellular source of HMGR expression. In vitro experiments using cultured HaCaT keratinocytes uncovered epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, and insulin as potent co-inducers of HMGR activity and vascular endothelial growth factor (VEGF) in the cells. Insulin-, but not EGF-mediated VEGF protein expression was functionally connected to co-induced HMGR activity, as simvastatin restrictively interfered only with insulin-induced translation of VEGF mRNA by inhibition of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation. Functional ablation of insulin-induced sterol regulatory element-binding protein (SREBP)-2 by siRNA abolished HMGR expression and insulin-triggered VEGF protein release from keratinocytes. Simvastatin also blocked proliferation of cultured keratinocytes. The observed inhibitory effects of simvastatin on keratinocyte VEGF expression and proliferation could be reversed by mevalonate, the product of HMGR enzymatic activity. In accordance, simvastatin-mediated inhibition of HMGR activity in acutely regenerating tissue of wounded mice was paralleled by a marked loss of VEGF protein expression and disturbances of normal proliferation processes in wound margin keratinocytes during skin repair.

Angiotensin II stimulation of VEGF mRNA translation requires production of reactive oxygen species

ANG II, a mediator of renal injury in diabetic renal disease, promotes vascular endothelial growth factor (VEGF) mRNA translation in proximal tubular epithelial (MCT) cells (Feliers D, Duraisamy S, Barnes JL, Ghosh-Choudhury G, and Kasimath BS. Am J Physiol Renal Physiol 288: F521-F529, 2005). The mechanism by which ANG II elicits this effect is not known. ANG II is known to induce oxidative stress and the rapidity of the effect suggested a role for reactive oxygen species (ROS). The aim of this study is to test the hypothesis that ANG II regulates VEGF mRNA translation in MCT cells through ROS production. In MCT cells exposed to 1 nM ANG II, ROS production was increased in a time-dependent manner. Inhibition of ROS production by N-acetylcysteine (NAC), a precursor of glutathione, and diphenyleneiodonium (DPI), an inhibitor of flavoproteins that include NAD(P)H oxidase, prevented ANG II-stimulated VEGF protein expression. NAC and DPI also inhibited phosphorylation of 4E-BP1 on Thr46 and association of eIF4E with eIF4G, steps that are important in the initiation phase of mRNA translation. NAC and DPI also blocked Akt activation which is required for 4E-BP1 phosphorylation. LY-294002, a selective phosphatidylinositol (PI 3-kinase) inhibitor, did not prevent ROS accumulation in response to ANG II, whereas DPI blocked ANG II activation of PI 3-kinase, demonstrating that ROS production is upstream of the PI 3-kinase signaling pathway. Preincubation with catalase abolished ANG II stimulation of VEGF expression and mRNA translation, suggesting involvement of hydrogen peroxide (H(2)O(2)). H(2)O(2) reproduced the effects of ANG II on VEGF expression and aforementioned parameters of mRNA translation. Finally, neither preincubation of MCT cells with specific inhibitors of the mitochondrial respiratory chain nor inactivation of the mitochondrial respiratory chain in MCT cells prevented ANG II stimulation of VEGF expression. Inhibition of nitric oxide synthase by l-NAME had no effect on ANG II stimulation of VEGF expression. These data show that ROS, generated probably through activation of an NAD(P)H oxidase, mediate ANG II stimulation of VEGF mRNA translation.

Non-Angiogenic Functions of VEGF in Breast Cancer

This review advances the hypothesis that the function of vascular endothelial growth factor (VEGF) in breast cancer is not limited to angiogenesis, and that VEGF signaling in breast carcinoma cells is important for the ability of these cells to evade apoptosis and progress towards invasive and metastatic disease. In other terms, VEGF signaling provides a selective advantage for the survival and dissemination of breast carcinoma cells that may be independent of angiogenesis. The key component of this hypothesis is that breast carcinoma cells express specific VEGF receptors and that these receptors respond to autocrine VEGF, resulting in the activation of signaling pathways that impede apoptosis and promote cell migration. A related hypothesis, which is developed in this review, is that the alpha6beta4 integrin, which has been implicated in the survival and motility of breast cancer cells, can stimulate the translation of VEGF mRNA and, consequently, autocrine VEGF signaling. These findings imply that VEGF and VEGF receptor-based therapeutics, in addition to targeting angiogenesis, may also target tumor cells directly.

Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation

Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

Translational regulation of vascular endothelial growth factor expression in renal epithelial cells by angiotensin II

ANG II regulates growth factor expression in the kidney. We investigated whether ANG II regulated vascular endothelial growth factor (VEGF) synthesis in proximal tubular epithelial (MCT) cells. ANG II (1 nM) increased VEGF protein expression within 5 min, the effect lasting for 30 min. There was no change in VEGF mRNA levels or mRNA stability, and transcription inhibitors did not affect ANG II-induced VEGF expression. Regulation of VEGF translation was investigated. Polyribosomal analysis revealed selective enrichment of heavy ribosomes (polysomes) with VEGF mRNA transcripts compared with light ribosomes in ANG II-treated cells, although distribution of GAPDH was unaltered. In vitro translation of total RNA from polysomal fractions showed selective increase in VEGF protein synthesis in ANG II-treated cells. Preincubation with LY-294002, a PI 3-kinase inhibitor, or expression of dominant-negative Akt prevented ANG II-stimulated increase in VEGF translation. ANG II increased phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1, critical events that regulate the initiation phase of protein translation. ANG II failed to increase VEGF mRNA translation in cells stably expressing the phosphorylation mutant of 4E-BP1. Our data illustrate that a rapid increase in VEGF protein expression by ANG II is regulated at the initiation phase of translation of VEGF mRNA in renal epithelial cells. Regulation of VEGF translation by ANG II represents a novel pathway of renal response to injury.

Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia

Vascular endothelial growth factor (VEGF) promotes neovascularization, microvascular permeability, and endothelial proliferation. We described previously VEGF mRNA and protein induction by estradiol (E2) in human endometrial fibroblasts. We report here E2 induction of VEGF expression in human venous muscle cells [smooth muscle cells (SMC) from human saphenous veins; HSVSMC] expressing both ER-alpha and ER-beta estrogen receptors. E2 at 10(-9) to 10(-8) M increases VEGF mRNA in HSVSMC in a time-dependent manner (3-fold at 24 h), as analyzed by semiquantitative RT-PCR. This level of induction is comparable with E2 endometrial induction of VEGF mRNA. Tamoxifen and hypoxia also increase HSVSMC VEGF mRNA expression over control values. Immunocytochemistry of saphenous veins and isolated SMC confirms translation of VEGF mRNA into protein. Immunoblot analysis of HSVSMC-conditioned medium detects three bands of 18, 23, and 28 kDa, corresponding to VEGF isoforms of 121, 165, and 189 amino acids. Radioreceptor assay of the conditioned medium produced by E2-stimulated HSVSMC reveals an increased VEGF secretion. Our data indicate that VEGF is E2, tamoxifen, and hypoxia inducible in cultured HSVSMC and E2 inducible in aortic SMC, suggesting E2 modulation of VEGF effects in angiogenesis, vascular permeability, and integrity.

Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA.

The mRNA of vascular endothelial growth factor (VEGF), the major angiogenic growth factor, contains an unusually long (1,038 nucleotides) and structured 5' untranslated region (UTR). According to the classical translation initiation model of ribosome scanning, such a 5' UTR is expected to be a strong translation inhibitor. In vitro and bicistronic strategies were used to show that the VEGF mRNA translation was cap independent and occurred by an internal ribosome entry process. For the first time, we demonstrate that two independent internal ribosome entry sites (IRESs) are present in this 5' UTR. IRES A is located within the 300 nucleotides upstream from the AUG start codon. RNA secondary structure prediction and site-directed mutagenesis allowed the identification of a 49-nucleotide structural domain (D4) essential to IRES A activity. UV cross-linking experiments revealed that IRES A activity was correlated with binding of a 100-kDa protein to the D4 domain. IRES B is located in the first half of the 5' UTR. An element between nucleotides 379 and 483 is required for its activity. Immunoprecipitation experiments demonstrated that a main IRES B-bound protein was the polypyrimidine tract binding protein (PTB), a well-known regulator of picornavirus IRESs. However, we showed that binding of the PTB on IRES B does not seem to be correlated with its activity. Evidence is provided of an original cumulative effect of two IRESs, probably controlled by different factors, to promote an efficient initiation of translation at the same AUG codon.