c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis

Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5' untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5' untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.     

HER2 signaling enhances 5'UTR-mediated translation of c-Myc mRNA

The increased levels of c-Myc protein observed previously in an ovarian carcinoma cell line stably transfected to express HER2 has suggested a role for the HER2 pathway in c-Myc expression. Analysis of HER2-transfected cells stimulated with heregulin beta1 (HRG) revealed increased c-Myc protein levels but not a corresponding increase in c-Myc mRNA expression or any change in c-Myc protein half-life. Transfection of HER2-overexpressing cells with a construct containing the 5' untranslated region (5'UTR) of c-Myc mRNA originated from the P2 promoter and placed upstream of the Renilla luciferase gene, enhanced reporter expression upon stimulation with HRG. The HRG-mediated increase in reporter activity correlated with the HRG-mediated induction observed for c-Myc protein, identifying the P2-derived leader (P2L) of c-Myc mRNA as the cis-element involved in c-Myc translational induction. Both the increase in c-Myc protein levels and P2L-enhanced translational activity were inhibited by the PI3K inhibitor wortmannin. Together, these results demonstrate that HRG stimulation of HER2 overexpressing cells leads to enhanced c-Myc protein synthesis through activation of the PI3K/Akt/mTOR pathway and that the P2L of c-Myc mRNA is the element responsible for induction of c-Myc translation.     

Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by AKT activity and enhanced by rapamycin through a p38 MAPK- and ERK-dependent pathway

The macrolide antibiotic rapamycin inhibits the mammalian target of rapamycin protein (mTOR) kinase resulting in the global inhibition of cap-dependent protein synthesis, a blockade in ribosome component biosynthesis, and G1 cell cycle arrest. G1 arrest may occur by inhibiting the protein synthesis of critical factors required for cell cycle progression. Hypersensitivity to mTOR inhibitors has been demonstrated in cells having elevated levels of AKT kinase activity, whereas cells containing quiescent AKT activity are relatively resistant. Our previous data suggest that low AKT activity induces resistance by allowing continued cap-independent protein synthesis of cyclin D1 and c-Myc proteins. In support of this notion, the current study demonstrates that the human cyclin D1 mRNA 5' untranslated region contains an internal ribosome entry site (IRES) and that both this IRES and the c-myc IRES are negatively regulated by AKT activity. Furthermore, we show that cyclin D1 and c-myc IRES function is enhanced following exposure to rapamycin and requires both p38 MAPK and RAF/MEK/ERK signaling, as specific inhibitors of these pathways reduce IRES-mediated translation and protein levels under conditions of quiescent AKT activity. Thus, continued IRES-mediated translation initiation may permit cell cycle progression upon mTOR inactivation in cells in which AKT kinase activity is relatively low.     

Dipeptidyl Peptidase 2 is an essential survival factor in the regulation of cell quiescence

Most cells in the body are in a resting state and undergo cell cycle progression only upon growth factor stimulation or activation. While much research on proliferation and activation has been performed, very little about signals that maintain quiescent cells in G₀ is known, preventing cell cycle entry or apoptosis. In this study, the pathways of apoptosis induction in quiescent peripheral blood cells and fibroblasts mediated by inhibition or down-regulation of Dipeptidyl Peptidase 2 (DPP2) have been explored. A decrease in DPP2 activity was found to cause resting cells to exit from G0, accompanied by a decrease in p130, p27^Kip1 and p21^Cip1 protein levels. In addition, DPP2-inhibited or down-regulated cells exhibit an increase in early G1/S progressors, with increases in the levels of retinoblastoma (pRb), p107 and cyclin D proteins. Furthermore, decrease of DPP2 activity leads to an increase in c-Myc and a decrease in Bcl-2, two events that have been associated with apoptosis induction. This apoptosis by DPP2 down-regulation is prevented in p53-/- cells or by ectopic expression of proteins that suppress p53 or c-Myc activity. Thus, DPP2 is essential for maintaining lymphocytes and fibroblasts in G₀, and its inhibition results in apoptosis mediated by induction of c-Myc and p53.     

Phosphorylation of initiation factor-2 alpha is required for activation of internal translation initiation during cell differentiation.

The long uORF-burdened 5'UTRs of many genes encoding regulatory proteins involved in cell growth and differentiation contain internal ribosomal entry site (IRES) elements. In a previous study we showed that utilization of the weak IRES of platelet-derived growth factor (PDGF2) is activated during megakaryocytic differentiation. The establishment of permissive conditions for IRES-mediated translation during differentiation has been confirmed by our demonstration of the enhanced activity of vascular endothelial growth factor, c-Myc and encephalomyocarditis virus IRES elements under these conditions, although their mRNAs are not naturally expressed in differentiated K562 cells. In contrast with the enhancement of IRES-mediated protein synthesis during differentiation, global protein synthesis is reduced, as judged by polysomal profiles and radiolabelled amino acid incorporation rate. The reduction in protein synthesis rate correlates with increased phosphorylation of the translation initiation factor eIF2 alpha. Furthermore, IRES use is decreased by over-expression of the dominant-negative form of the eIF2 alpha kinase, PKR, the vaccinia virus K3L gene, or the eIF2 alpha-S51A variant which result in decreased eIF2 alpha phosphorylation. These data demonstrate a connection between eIF2 alpha phosphorylation and activation of cellular IRES elements. It suggests that phosphorylation of eIF2 alpha, known to be important for cap-dependent translational control, serves to fine-tune the translation efficiency of different mRNA subsets during the course of differentiation and has the potential to regulate expression of IRES-containing mRNAs under a range of physiological circumstances.     

IRES-mediated translation of utrophin A is enhanced by glucocorticoid treatment in skeletal muscle cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6alpha-methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5'UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5'UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5'UTR. Additional experiments identified a distinct region within the utrophin A 5'UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.     

Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5'-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival.     

Translation mediated by the internal ribosome entry site of the cat-1 mRNA is regulated by glucose availability in a PERK kinase-dependent manner.

The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene is known to be regulated by amino acid availability. It is shown here that cat-1 gene expression is also induced by Glc limitation, which causes a 7-fold increase in cat-1 mRNA, a 30-fold induction of Cat-1 protein levels, and a 4-fold stimulation of arginine uptake. Glc limitation is known to induce the unfolded protein response (UPR) by altering protein glycosylation in the endoplasmic reticulum (ER). The studies here demonstrate that synthesis of Cat-1 occurs during the UPR when global protein synthesis is inhibited. The 5'-UTR of the cat-1 mRNA contains an internal ribosomal entry site (IRES) that is activated by amino acid starvation by a mechanism that involves phosphorylation of the translation initiation factor, eukaryotic initiation factor 2alpha, by the GCN2 kinase. It is shown here that translation from the cat-1/IRES is also induced by Glc deprivation in a manner dependent upon phosphorylation of eukaryotic initiation factor 2alpha by the transmembrane ER kinase, PERK. Because PERK is a key constituent of the UPR, it is concluded that induction of cat-1 gene expression is part of the adaptive response of cells to ER stress. These results also demonstrate that regulation of IRES activity in cellular mRNAs is part of the mechanism by which the UPR protects cells from unfolded proteins in the ER.     

Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis.

c-Myc plays a central role in the regulation of cell cycle progression, differentiation, and apoptosis. However, the proteins which mediate c-Myc function(s) remain to be determined. Enforced c-myc expression rapidly induces apoptosis in interleukin-3 (IL-3)-dependent 32D.3 murine myeloid cells following IL-3 withdrawal, and this is associated with the constitutive, growth factor-independent expression of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis. Here we have examined the role of ODC in c-Myc-induced apoptosis. Enforced expression of ODC, like c-myc, is sufficient to induce accelerated death following IL-3 withdrawal. ODC induced cell death in a dose-dependent fashion, and alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzyme activity, effectively blocked ODC-induced cell death. ODC-induced cell death was due to the induction of apoptosis. We also demonstrate that ODC is a mediator of c-Myc-induced apoptosis. 32D.3-derived c-myc clones have augmented levels of ODC enzyme activity, and their rates of death were also a function of their ODC enzyme levels. Importantly, the rates of death of c-myc clones were inhibited by treatment with DFMO. These findings demonstrate that ODC is an important mediator of c-Myc-induced apoptosis and suggest that ODC mediates other c-Myc functions.     

Coupling of the Cell Cycle and Apoptotic Machineries in Developing T Cells

Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4⁺CD8⁺ thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4⁺CD8⁺ thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4⁺CD8⁺ thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4⁺CD8⁺ thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis.     

Functional characterization of the X-linked inhibitor of apoptosis (XIAP) internal ribosome entry site element: role of La autoantigen in XIAP translation

X-linked inhibitor of apoptosis protein (XIAP) is a key regulator of programmed cell death triggered by various apoptotic triggers. Translation of XIAP is controlled by a 162-nucleotide (nt) internal ribosome entry site (IRES) element located in the 5' untranslated region of XIAP mRNA. XIAP IRES mediates efficient translation of XIAP under physiological stress and enhances cell protection against serum deprivation and radiation-induced apoptosis. In the present report we describe the assembly of a sequence-specific RNA-protein complex consisting of at least four cytosolic proteins on the XIAP IRES element. We determine that the core binding sequence is approximately 28 nt long and is located 34 nt upstream of the initiation site. Moreover, we identify the La autoantigen as a protein that specifically binds XIAP IRES in vivo and in vitro. The biological relevance of this interaction is further demonstrated by the inhibition of XIAP IRES-mediated translation in the absence of functional La protein. The results suggest an important role for the La protein in the regulation of XIAP expression, possibly by facilitating ribosome recruitment to the XIAP IRES.     

BiP internal ribosomal entry site activity is controlled by heat-induced interaction of NSAP1

TheBiP protein, a stress response protein, plays an important role in the proper folding and assembly of nascent protein and in the scavenging of misfolded proteins in the endoplasmic reticulum lumen. Translation of BiP is directed by an internal ribosomal entry site (IRES) in the 5' nontranslated region of the BiP mRNA. BiP IRES activity increases when cells are heat stressed. Here we report that NSAP1 specifically enhances the IRES activity of BiP mRNA by interacting with the IRES element. Overexpression of NSAP1 in 293T cells increased the IRES activity of BiP mRNA, whereas knockdown of NSAP1 by small interfering RNA (siRNA) reduced the IRES activity of BiP mRNA. The amount of NSAP1 bound to the BiP IRES increased under heat stress conditions, and the IRES activity of BiP mRNA was increased. Moreover, the increase in BiP IRES activity with heat treatment was not observed in cells lacking NSAP1 after siRNA treatment. BiP mRNAs were redistributed from the heavy polysome to the light polysome in NSAP1 knockdown cells. Together, these data indicate that NSAP1 modulates IRES-dependent translation of BiP mRNA through an RNA-protein interaction under heat stress conditions.     

Hepatocyte growth factor induces pro-apoptotic genes in HepG2 hepatoma but not in B16-F1 melanoma cells

Hepatocyte growth factor (HGF) exerts a cytostatic effect on HepG2 and B16-F1 cell lines. To evaluate the possible involvement of the apoptotic process in this effect, we performed studies at cellular and molecular levels. HGF induced apoptosis only in HepG2 hepatoma cells at day 3 in about 20% of the cells undergoing growth inhibition, while hallmarks of apoptosis did not occur in B16-F1 melanoma cells. During the first 24 h after HGF treatment, enhanced expression of the pro-apoptotic genes bax and c-Myc was observed at level of mRNA and protein. Concomitant induction of antizyme (AZ) might lower ornithine decarboxylase (ODC) protein level though a huge increase in ODC mRNA level took place. This was suggested as a signal for apoptosis decisional phase. The levels of the proteins examined except that of AZ fell down thereafter when HepG2 cells underwent apoptosis. In B16-F1 cells, only ODC and AZ protein levels were elevated probably in relation to the initial elevated growth rate and the absence of apoptosis involvement in the following cytostatic effect of HGF in melanoma cells. Consistent with this hypothesis, bax mRNA and protein levels were unchanged or even lower relative to control values.