Expression of angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 is highly responsive to ambient glutamine availability: role of nuclear factor-kappaB and activating protein-1.

Vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8) are prominent pro-angiogenic and pro-metastatic proteins that represent negative prognostic factors in many types of cancer. Hypoxia is thought to be the primary environmental cause of VEGF and IL-8 expression in solid tumors. We hypothesized that a lack of nutrients other than oxygen could stimulate the expression of these factors and previously demonstrated that expression of VEGF and IL-8 is responsive to amino acid deprivation. In the present study, we examined the effect of glutamine availability on the expression of these factors as well as the role of transcription factors NFkappaB and activating protein-1 (AP-1) in the response of TSE human breast carcinoma cells to glutamine deprivation. VEGF and IL-8 secretion and mRNA levels were dramatically induced by glutamine deprivation. mRNA stabilization contributed to this response. Glutamine deprivation increased NFkappaB (p65/p50) and AP-1 (Fra-1/c-Jun+JunD) DNA-binding activities. Blocking NFkappaB and AP-1 activation with curcumin as well as expression of dominant inhibitors, inhibitor of nuclear factor-kappaB (IkappaB) super repressor (IkappaBM), and a mutant form of c-Fos (A-Fos) demonstrated that the activation of NFkappaB and AP-1 transcription factors was necessary for the induction of IL-8 expression but dispensable for the induction of VEGF expression. A macro-array containing 111 NFkappaB target genes identified a total of 17 that were up-regulated 2-fold or more in response to glutamine deprivation. These included growth regulated oncogene alpha (GROalpha/GRO1/CXCL1), another neutrophil chemoattractant implicated in tumor angiogenesis and metastasis.

Aberrant accumulation of fibulin-3 in the endoplasmic reticulum leads to activation of the unfolded protein response and VEGF expression.

PURPOSE: The inherited early-onset macular degenerative disease known as malattia leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) have been linked to a missense mutation leading to production of a mutant fibulin-3 protein (R345W). R345W is poorly secreted and accumulates in the RPE of ML/DHRD retinas. Accumulation of misfolded proteins within the endoplasmic reticulum (ER) causes activation of unfolded protein response (UPR) signaling and expression of ER stress-responsive genes, including vascular endothelial growth factor (VEGF). Therefore, we hypothesized that the expression of R345W activates the UPR, leading to VEGF expression. METHODS: Adenoviral vectors were used to overexpress fibulin-3 wild-type (Wt) and R345W mutant proteins in ARPE-19 cells. Secretion and intracellular accumulation of Wt and R345W were compared by Western blot analysis and immunocytochemistry. Activation of the UPR was evaluated by measuring the expression of glucose-regulated protein 78 (GRP78 [BiP]) and editing of the X-box binding protein (XBP-1) mRNA. VEGF expression and transcriptional activation of the VEGF promoter were determined by Northern blot analysis, Western blot analysis, and use of a novel VEGF promoter-reporter construct containing 8.2 kb of the human VEGF gene. RESULTS: R345W was poorly secreted by ARPE-19 cells and accumulated in the ER, leading to UPR activation and increased VEGF expression. Compared with Wt mutant proteins, the expression of R345W was more effective at causing UPR activation, increasing VEGF expression, and stimulating transcription from the VEGF promoter. CONCLUSIONS: These findings demonstrated that the expression of mutated fibulin-3 caused UPR activation and increased VEGF expression. Expression of mutant fibulin proteins may contribute to macular degeneration and choroidal neovascularization by causing ER stress leading to RPE dysfunction and increased VEGF expression.

Expression of the pro-angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 by human breast carcinomas is responsive to nutrient deprivation and endoplasmic reticulum stress.

BACKGROUND: The expression of pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8), plays an important role in tumor growth and metastasis. Low oxygen tension within poorly-vascularized tumors is thought to be the prime stimulus causing the secretion of VEGF. The expression of IL-8 by solid tumors is thought to be primarily due to intrinsic influences, such as constitutive activation of nuclear factor kappa B (NF-kappaB). However, VEGF expression is responsive to glucose deprivation, suggesting that low concentrations of nutrients other than oxygen may play a role in triggering the pro-angiogenic phenotype. Glucose deprivation causes endoplasmic reticulum (ER) stress and alters gene expression through the unfolded protein response (UPR) signaling pathway. A branch of the UPR, known as the ER overload response (EOR), can cause NF-kappaB activation. Thus, we hypothesized that treatments that cause ER stress and deprivation of other nutrients, such as amino acids, would trigger the expression of angiogenic cytokines by breast cancer cell lines. RESULTS: We found that glutamine deprivation and treatment with a chemical inducer of ER stress (tunicamycin) caused a marked induction of the secretion of both VEGF and IL-8 protein by a human breast adenocarcinoma cell line (TSE cells). Glutamine deprivation, glucose deprivation and several chemical inducers of ER stress increased VEGF and IL-8 mRNA expression in TSE and other breast cancer cell lines cultured under both normoxic and hypoxic conditions, though hypoxia generally diminished the effects of glucose deprivation. Of all amino acids tested, ambient glutamine availability had the largest effect on VEGF and IL-8 mRNA expression. The induction of VEGF mRNA expression, but not IL-8, was sustained and closely corresponded with the upregulated expression of the ER stress-responsive genes glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153). CONCLUSION: These results suggest that nutrient deprivation within the solid tumor microenvironment might contribute to the activation of a pro-angiogenic phenotype. The angiogenic switch may act to increase blood supply in response to nutrient deprivation as well as hypoxia.

Response of VEGF expression to amino acid deprivation and inducers of endoplasmic reticulum stress.

PURPOSE: Vascular endothelial growth factor (VEGF) plays an important role in initiation of the angiogenesis that leads to proliferative retinopathy. Several environmental conditions and chemical agents that influence the expression of VEGF can also cause endoplasmic reticulum (ER) stress. The hypothesis for the current study was that expression of VEGF is responsive to conditions that cause ER stress, including amino acid deprivation. METHODS: Confluent cultures of a human retinal pigmented epithelial cell line (ARPE-19) were deprived of amino acids or treated with chemical inducers of ER stress. Treatment with cobalt was used to mimic hypoxia-induced expression of VEGF. Northern blot analysis was used to measure intracellular VEGF mRNA, and ELISA was used to measure secreted VEGF protein. Glucose-regulated protein 78 (GRP78) mRNA levels were compared with those of VEGF. Glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA was used as a control. RESULTS: Conditions and chemical agents known to activate ER stress response (ERSR) pathways also induced the expression of VEGF. Deprivation of amino acids in the culture medium increased VEGF mRNA expression by 1.3- to 6-fold. Glucose deprivation or treatment of ARPE-19 cells with tunicamycin, brefeldin A, the calcium ionophore A23187, or thapsigargin increased the expression of VEGF mRNA in these cells by 8- to 10-fold. Expression of GRP78 mRNA was well correlated with that of VEGF mRNA under all conditions. These treatments also increased the secretion of VEGF protein by up to twofold. The increase in VEGF mRNA level in response to glutamine deprivation was rapid (greater than 10-fold) and was observed in a physiologically relevant range of glutamine concentrations. The half-life of VEGF mRNA was increased 2.5-fold by glutamine starvation. CONCLUSIONS: These results indicate that VEGF is an ER stress-responsive gene and suggest that cells can respond to nutrient deprivation by increasing VEGF expression through both transcriptional and posttranscriptional mechanisms.

Effect of IL-1beta on survival and energy metabolism of R28 and RGC-5 retinal neurons.

PURPOSE: Interleukin-(IL)1beta expression is increased in the retina during a variety of diseases involving the death of retinal neurons and contributes to neurodegenerative processes through an unknown mechanism. This study was conducted to examine the effects of IL-1beta on the metabolism and viability of RGC-5 and R28 retinal neuronal cells. METHODS: Cellular reductive capacity was evaluated using WST-1 tetrazolium salt. Mitochondrial transmembrane potential was determined by JC-1 fluorescence. Cellular ATP levels were measured with a luciferase assay. Caspase-3/7 activation was detected with a DEVDase activity assay. Cell death and lysis was evaluated by measuring release of lactate dehydrogenase (LDH). Glycolysis was assessed by measuring glucose disappearance and lactate appearance in cell culture medium. Cellular respiration was followed polarographically. RESULTS: IL-1beta treatment caused a pronounced decrease in cellular reductive potential. IL-1beta caused depletion of intracellular ATP, loss of mitochondrial transmembrane potential, caspase-3/7 activation, and LDH release. IL-1beta treatment increased rates of glucose utilization and lactate production. The cells were partially protected from IL-1beta toxicity by ample ambient glucose. However, glucose did not block the ability of IL-1beta to cause a decline in mitochondrial transmembrane potential or ATP depletion. IL-1beta decreased oxygen consumption of the R28 cells by nearly half, but did not lower cytochrome c oxidase activity. CONCLUSIONS: The present results suggest that IL-1beta inhibits mitochondrial energy metabolism of these retinal neuronlike cells.

Activation of NFkappaB is inhibited by curcumin and related enones.

The transcription factor NFkappaB (NFkappaB) is up-regulated in many cancer cells where it contributes to development of the pro-survival, anti-apoptotic state. The natural product curcumin is a known inhibitor of activation of NFkappaB. Enone analogues of curcumin were compared with curcumin for their abilities to inhibit the TNFalpha-induced activation of NFkappaB, using the Panomics' NFkappaB Reporter Stable Cell Line. The enones tested included curcumin analogues that retained the 7-carbon spacer between the aromatic rings, analogues with a 5-carbon spacer, and analogues with a 3-carbon spacer. Inhibitors of NFkappaB activation were identified in all three series, a number of which were more active than curcumin. Enone analogues in the series with the 5-carbon spacer were especially active, including members that contained heterocyclic rings. 1,5-Bis(3-pyridyl)-1,4-pentadien-3-one was the most active analogue, IC50 = 3.4 +/- 0.2 microM. The most active analogues retain the enone functionality, although some analogues devoid of the enone functionality exhibited activity. The activity of the analogues as inhibitors of the activation of NFkappaB did not correlate with their anti-oxidant activity. The data suggest that the abilities of curcumin and analogues to prevent the stress-induced activation of NFkappaB result from the inhibition of specific targets rather than from activity as anti-oxidants.

The oxidative stressor arsenite activates vascular endothelial growth factor mRNA transcription by an ATF4-dependent mechanism.

Aberrant retinal expression of vascular endothelial growth factor (VEGF) leading to neovascularization is a central feature of age-related macular degeneration and diabetic retinopathy, two leading causes of vision loss. Oxidative stress is suggested to occur in retinal tissue during age-related macular degeneration and diabetic retinopathy and is suspected in the mechanism of VEGF expression in these diseases. Arsenite, a thiol-reactive oxidative stressor, induces VEGF expression by a HIF-1alpha-independent mechanism. Previously, we demonstrated that homocysteine, an endoplasmic reticulum stressor, increases VEGF transcription by a mechanism dependent upon activating transcription factor ATF4. Because ATF4 is expressed in response to oxidative stress, we hypothesized that ATF4 was also responsible for increased VEGF transcription in response to arsenite. We now show that arsenite increased steady state levels of VEGF mRNA and activated transcription from a VEGF promoter construct. Arsenite induced eIF2alpha phosphorylation, resulting in increased ATF4 protein levels. Inactivation or loss of ATF4 greatly diminished the VEGF response to arsenite treatment. Overexpression of ATF4 was sufficient to activate the VEGF promoter, and arsenite cooperated with exogenous ATF4 to further activate the promoter. A complex containing ATF4 binds a DNA element at +1767 bp relative to the VEGF transcription start site, and DNA binding activity is increased by arsenite treatment. In addition, the ability of a thiol antioxidant, N-acetylcysteine, to inhibit the effect of arsenite on VEGF expression coincided with its ability to inhibit phosphorylation of eIF2alpha and ATF4 protein expression. Thus, arsenite-induced up-regulation of VEGF gene transcription occurs by an ATF4-dependent mechanism.

Anti-oxidant activities of curcumin and related enones.

The natural product curcumin (diferuloylmethane, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), obtained from the spice turmeric, exhibits numerous biological activities including anti-cancer, anti-inflammatory, and anti-angiogenesis activities. Some of these biological activities may derive from its anti-oxidant properties. There are conflicting reports concerning the structural/electronic basis of the anti-oxidant activity of curcumin. Curcumin is a symmetrical diphenolic dienone. A series of enone analogues of curcumin were synthesized that included: (1) curcumin analogues that retained the 7-carbon spacer between the aryl rings; (2) curcumin analogues with a 5-carbon spacer; and (3) curcumin analogues with a 3-carbon spacer (chalcones). These series included members that retained or were devoid of phenolic groups. Anti-oxidant activities were determined by the TRAP assay and the FRAP assay. Most of the analogues with anti-oxidant activity retained the phenolic ring substituents similar to curcumin. However, a number of analogues devoid of phenolic substituents were also active; these non-phenolic analogues are capable of forming stable tertiary carbon-centered radicals.

Induction of Grp78/BiP by translational block: activation of the Grp78 promoter by ATF4 through and upstream ATF/CRE site independent of the endoplasmic reticulum stress elements.

Mammalian cells respond to endoplasmic reticulum (ER) stress by attenuation of protein translation mediated through the PERK-eIF2alpha pathway and transcriptional activation of genes such as Grp78/BiP encoding ER chaperone proteins. The disruption of PERK function or the blocking of eIF2alpha Ser51 phosphorylation fails to attenuate translation after ER stress and also results in substantial impairment of Grp78/BiP induction by ER stress. While the activation of the Grp78 promoter by the ATF6 pathway through the endoplasmic reticulum stress elements (ERSEs) is well documented, the molecular mechanism linking PERK activation to Grp78 stress induction is unknown. We report here that ATF4, a transcription factor whose translation is up-regulated by the PERK-eIF2alpha pathway, can activate the Grp78 promoter independent of the ERSE. The ATF4-activating site is localized to an ATF/CRE sequence upstream of the ERSEs and is distinct from the C/EBP-ATF composite site previously identified as the ATF4 binding site in the ER stress-inducible chop promoter. In vitro translated ATF4 binding to the ATF/CRE site requires other nuclear co-factors from non-stressed cells, forming a complex that exhibits identical electrophoretic mobility as a thapsigargin-stress induced complex. Here we have identified the closely related ATF1 and CREB1 as nuclear co-factors that form in vivo complexes with endogenous ATF4. ER stress induces CREB1 phosphorylation and ATF1/CREB1 binding to the Grp78 promoter. Through the use of adenoviral vector expression systems, we provide evidence that when ATF4 function is suppressed and its binding partners are not able to compensate for its function, Grp78 induction by Tg and Tu is partially inhibited. Our studies resolve a mechanism responsible for inhibition of Grp78 mRNA induction by ER stress in cells that are functionally null for PERK or devoid of eIF2alpha phosphorylation.

Homocysteine increases the expression of vascular endothelial growth factor by a mechanism involving endoplasmic reticulum stress and transcription factor ATF4.

Vascular endothelial growth factor (VEGF) plays a key role in the development and progression of diabetic retinopathy. We previously demonstrated that amino acid deprivation and other inducers of endoplasmic reticulum-stress (ER stress) up-regulate the expression of VEGF in the retinal-pigmented epithelial cell line ARPE-19. Because homocysteine causes ER stress, we hypothesized that VEGF expression is increased by ambient homocysteine. dl-Homocysteine-induced VEGF expression was investigated in confluent ARPE-19 cultures. Northern analysis showed that homocysteine increased steady state VEGF mRNA levels 4.4-fold. Other thiol-containing compounds, including l-homocysteine thiolactone and DTT, induced VEGF expression 7.9- and 8.8-fold. Transcriptional run-on assays and mRNA decay studies demonstrated that the increase in VEGF mRNA levels was caused by increased transcription rather than mRNA stabilization. VEGF mRNA induction paralleled that of the ER-stress gene GRP78. Homocysteine treatment caused transient phosphorylation of eIF2alpha and an increase in ATF4 protein level. Overexpression of a dominant-negative ATF4 abolished the VEGF response to homocysteine treatment and to amino acid deprivation. VEGF mRNA expression by ATF4-/- MEF did not respond to homocysteine treatment and the response was restored with expression of wild-type ATF4. These studies indicate that expression of the pro-angiogenic factor VEGF is increased by homocysteine and other thiol-containing reductive compounds via ATF4-dependent activation of VEGF transcription.

Molecular and functional analysis of glutamine uptake in human hepatoma and liver-derived cells.

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter B(0) (ATB(0)), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by ATB(0). The 2.9-kb ATB(0) mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. ATB(0) mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of ATB(0)-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels.