Sylvie Mader

The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E‐binding protein (BP) 1] that binds to the mRNA 5′ cap binding protein eukaryotic initiation factor (eIF)‐4E and inhibits cap‐dependent translation. Phosphorylation of the repressor decreases its affinity for eIF‐4E, and thus relieves translational inhibition. eIF‐4E forms a complex with two other polypeptides, eIF‐4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E‐BP1 inhibits translation. We show that 4E‐BP1 inhibits 48S pre‐initiation complex formation. Furthermore, we demonstrate that 4E‐BP1 competes with p220 for binding to eIF‐4E. Mutants of 4E‐BP1 that are deficient in their binding to eIF‐4E do not inhibit the interaction between p220 and eIF‐4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E‐BP1 and p220 for eIF‐4E.

Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis

The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin β1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin β1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells.

Glutathione depletion overcomes resistance to arsenic trioxide in arsenic-resistant cell lines

Arsenic trioxide (As2O3) is an effective treatment for acute promyelocytic leukemia (APL), but is less effective against other leukemias. Although the response of APL cells to As2O3 has been linked to degradation of the PML/RARα fusion oncoprotein, there is evidence that PML/RARα expression is not the only mediator of arsenic sensitivity. Indeed, we found that exogenous expression of PML/RARα did not sensitize a non-APL leukemic line to As2O3. To evaluate possible other determinants of sensitivity of leukemic cells to As2O3, we derived two arsenic-resistant NB4 subclones. Despite being approximately 10-fold more resistant to arsenic than their parental cell line, PML/RARα protein was still degraded by As2O3 in these cells, providing further evidence that loss of expression of the oncoprotein does not confer arsenic sensitivity. Both arsenic-resistant clones contained high glutathione (GSH) levels, however, and we found that GSH depletion coupled with As2O3 treatment dramatically inhibited their growth. Annexin V-staining and TUNEL analysis confirmed a synergistic induction of apoptosis. In addition, these cells failed to accumulate ROS in response to arsenic treatment, in contrast to their arsenic-sensitive parental cells, unless cotreated with buthionine sulfoximine. While other malignant cells did not show a good correlation between arsenic sensitivity and GSH content, GSH depletion nevertheless sensitized all cell lines examined, regardless of their initial response to arsenic alone. These findings suggest that PML/RARα expression is not a determinant of arsenic sensitivity, and further support the coupling of GSH depletion and arsenic treatment as a novel treatment for human malignancies that are unresponsive to arsenic alone.

Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells

Estrogen receptors (ERs), which mediate the proliferative action of estrogens in breast cancer cells, are ligand-dependent transcription factors that regulate expression of their primary target genes through several mechanisms. In addition to direct binding to cognate DNA sequences, ERs can be recruited to DNA through other transcription factors (tethering), or affect gene transcription through modulation of signaling cascades by non-genomic mechanisms of action. To better characterize the mechanisms of gene regulation by estrogens, we have identified more than 700 putative primary and about 1300 putative secondary target genes of estradiol in MCF-7 cells through microarray analysis performed in the presence or absence of the translation inhibitor cycloheximide. Although siRNA-mediated inhibition of ERα expression antagonized the effects of estradiol on up- and down-regulated primary target genes, estrogen response elements (EREs) were enriched only in the vicinity of up-regulated genes. Binding sites for several other transcription factors, including proteins known to tether ERα, were enriched in up- and/or down-regulated primary targets. Secondary estrogen targets were particularly enriched in sites for E2F family members, several of which were transcriptionally regulated by estradiol, consistent with a major role of these factors in mediating the effects of estrogens on gene expression and cellular growth.

Phosphorylation of eIF-4E on Serine 209 by Protein Kinase C Is Inhibited by the Translational Repressors, 4E-binding Proteins

Translation initiation in eukaryotes is facilitated by the mRNA 5′ cap structure (m7GpppX, where X is any nucleotide) that binds the multisubunit initiation factor eIF4F through one of its subunits, eIF4E. eIF4E is a phosphoprotein whose phosphorylation state positively correlates with cell growth. Protein kinase C phosphorylates eIF4E in vitro, and possibly in vivo. Using recombinant eIF4E incubated in vitro with purified protein kinase C and analyzed by solid-phase phosphopeptide sequencing in combination with high performance liquid chromatography coupled to mass spectrometry, we demonstrated that the third amino acid of the peptide SGSTTK (Ser) is the major site of phosphorylation. This finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E (Joshi, B., Cai, A. L., Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J., Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E.(1995) J. Biol. Chem. 270, 14597-14603). A S209A mutation resulted in dramatically reduced phosphorylation, both in vitro and in vivo. Furthermore, the mutant protein was phosphorylated on threonine (most probably threonine 210) in vivo. Here we show that in the presence of the recently characterized translational repressors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase C is strongly reduced. This suggests a two-step model for the phosphorylation (and activation) of eIF4E by growth factors and hormones: first, dissociation of eIF4E from 4E-BPs, followed by eIF4E phosphorylation.

Different Classes of Coactivators Recognize Distinct but Overlapping Binding Sites on the Estrogen Receptor Ligand Binding Domain

We have analyzed interaction of coactivators with the wild-type estrogen receptor α (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells.

Corepressor Recruitment by Agonist-Bound Nuclear Receptors

Members of the nuclear receptor superfamily are ligand-regulated transcription factors that are composed of a series of conserved domains. These receptors are targets of a wide range of lipophilic signaling molecules that modulate many aspects of physiology and metabolism. Binding of cognate ligands to receptors induces a conformational change in the ligand binding domain (LBD) that creates a pocket for recruitment of coregulatory proteins, which are essential for ligand-dependent regulation of transcription. Several coregulatory proteins that interact with hormone-bound receptors contain characteristic helical LXXLL motifs, known as nuclear receptor (NR) boxes. Generally, ligand binding to receptors is associated with activation of transcription, and most of the NR box-containing proteins characterized to date are coactivators. However, a full understanding of the function of hormone-bound receptors must also incorporate their recruitment of corepressors. The recent identification of ligand-dependent corepressor (LCoR) is a case in point. LCoR contains a single NR box that mediates its hormone-dependent interaction with several nuclear receptors. It functions as a molecular scaffold that recruits several proteins that function in transcriptional repression. Remarkably, although the two proteins share only very limited homology, LCoR and another NR box-containing corepressor RIP140 recruit similar cofactors implicated in transcriptional repression, suggesting many parallels in their mechanisms of action. Corepressors such as LCoR and RIP140 may function in negative feedback loops to attenuate hormone-induced transactivation, act more transiently as part of a cycle of cofactors recruited to target promoters by ligand-bound receptors, or function in hormone-induced target gene repression.

Growth-stimulatory and transcriptional activation properties of raloxifene in human endometrial Ishikawa cells.

Raloxifene (Ral) has estrogenic activity in bone and cardiovascular tissues, but is antiestrogenic in breast and has limited uterotrophic activity in mice. Here we report that Ral stimulates the growth of human endometrial Ishikawa tumors implanted in the mammary fat pad of nude ovariectomized mice. In cultured Ishikawa cells, Ral has agonist effects on transcription mediated by the progesterone receptor, an endogenous estrogen target gene, and on expression of reporter genes containing estrogen response elements (EREs). Both Ral and tamoxifen (Tam), but not estradiol, stimulated transcription mediated by the activator protein 1 at micromolar concentrations. However, this effect correlated with induction of cellular death at high concentrations of Ral or Tam and was not observed at lower concentrations. Our results suggest that Ral has stimulatory effects in Ishikawa cells on both cellular growth and gene transcription, and that EREs can mediate some of these effects.