eIF4B controls survival and proliferation and is regulated by proto-oncogenic signaling pathways

Messenger RNA translation or protein synthesis, is a fundamental biological process affecting cell growth, survival and proliferation. Initiation is the rate limiting and hence the most regulated step of translation. In eukaryotes, translation initiation is facilitated by multiple protein factors collectively called eIFs (for eukaryotic translation initiation factors). The complex consisting of the eIF4 group factors including the mRNA cap-binding eIF4E protein, large scaffolding protein eIF4G and RNA helicase eIF4A is assisted by the eIF4B co-factor to unwind local secondary structures and create a ribosome landing pad on mRNA. Recruitment of the ribosome and augmentation in the mRNA scanning process culminates in the positioning of the ribosome over the start codon. Deregulated translational control is believed to play an important role in oncogenic transformation. Indeed, many eIFs are bona fide proto-oncogenes. In many types of human cancers, eIFs are either overexpressed or ectopically activated by Ras-MAPK and PI3K-mTOR signaling cascades, resulting in increased survival and accelerated proliferation. In this review we will analyze the bulk of data describing eIF4B and its role in cell survival and proliferation. Recent studies have shown that eIF4B is phosphorylated and activated by Ras-MAPK and PI3K-mTOR signaling cascades. In addition, eIF4B regulates translation of proliferative and pro-survival mRNAs. Moreover, eIF4B depletion in cancer cells attenuates proliferation, sensitizes them to genotoxic stress-driven apoptosis. Taken together, these findings identify eIF4B as a potential target for development of anti-cancer therapies.Key words: eIF4B, translation, signaling, structured 5′UTR, helicase activity, survival, proliferation, apoptosis     

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2-L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.     

Xenopus galectin-VIa shows highly specific expression in cement glands and is regulated by canonical Wnt signaling

Anterior-posterior neural patterning of Xenopus embryo is determined during gastrulation and then followed by differentiation of neural structures including brain and eye. The cement gland is a mucus-secreting neural organ located in the anterior end of the neural plate. This study analyzed expression patterns of Xenopus galectin-VIa (Xgalectin-VIa) by whole-mount in situ hybridization, and found highly restricted expression of this gene in the cement gland region. These patterns were similar to those of XAG-1 and XCG, known cement gland-specific genes. In addition, Xgalectin-VIa was expressed in the dorsal edge of eye vesicles, the otic vesicle, and in part of the hatching gland at the tadpole stage. Although the spatial expression pattern was similar, the temporal expression of Xgalectin-VIa differed from that of XAG-1 and XCG. RT-PCR analysis showed only weak Xgalectin-VIa expression in early neurula embryos, whereas both XAG-1 and CGS were strongly expressed at that stage. We also showed that Xgalectin-VIa expression is repressed by enhancement of Wnt signaling and increased by its inhibition. Furthermore, Xgalectin-VIa expression was activated by neural-gene inducer Xotx2, as is the case for XAG-1 and CGS. Together, these results indicated that Xgalectin-VIa possesses different features from other cement gland genes and is a novel and useful marker of the cement gland in developing embryos.     

The Wnt antagonist Dickkopf-1 is regulated by Bmp signaling and c-Jun and modulates programmed cell death

Dickkopf-1 (Dkk-1) has been shown to be a potent inhibitor of Wnt/beta-catenin signaling in a variety of assays and organisms. In this study, we show that expression of Dkk-1 overlaps significantly with the sites of programmed cell death in normal as well as mutant vertebrate limb development, and identify several of its upstream regulators, one of which is Bmp-4. Interestingly, Bmp-4 only activates Dkk-1 when it concomitantly induces apoptosis. Moreover, Dkk-1 is heavily up-regulated by UV irradiation and several other genotoxic stimuli. We further show that normal expression of Dkk-1 is dependent on the Ap-1 family member c-Jun and that overexpression of Dkk-1 enhances Bmp-triggered apoptosis in the vertebrate limb. Taken together, our results provide evidence for an important role of Dkk-1-mediated inhibition of Wnt/beta-catenin signaling in response to different stress signals that all converge on the activation of c-Jun in vivo.     

HRV signaling in airway epithelial cells is regulated by ITAM-mediated recruitment and activation of Syk

Human rhinovirus (HRV), cause of the common cold, is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease (COPD). Binding of HRV to ICAM (intercellular adhesion molecule)-1, its major receptor, induces a profound inflammatory response from airway epithelial cells. My laboratory has identified Syk tyrosine kinase to be an early regulator of HRV-ICAM-1 signalling: Syk mediates replication-independent p38 mitogen-activated protein (MAP) kinase and phosphatidyl-inositol 3 (PI3)-kinase activation, interleukin (IL)-8 expression, as well as HRV internalization via clathrin-mediated endocytosis. Syk activation is accompanied by formation of a protein complex consisting of ICAM-1, ezrin and Syk at the plasma membrane. However, the molecular mechanisms that regulate this process are not understood. In this report, we investigated the role of the Syk-SH2 domains and the ezrin ITAM (immuno-tyrosine activation motif)-like motif in HRV-induced cell activation using the human BEAS-2B airway epithelial cells. Our observations suggest that the ezrin-ITAM plays a role in Syk recruitment and activation by binding to the Syk tandem SH2 domains, as originally described in the canonical ITAM-mediating signal transduction pathway in hematopoietic cells. This report is the first to demonstrate ITAM-mediated signaling in non-hematopoietic cells, suggesting that this signaling paradigm may be more ubiquitous than previously recognized.     

Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation

Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1^−/− mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1^−/− esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1^−/− foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.     

Murine folliculogenesis in vitro is stage-specifically regulated by insulin via the Akt signaling pathway

Folliculogenesis is a critical process during mammalian development, but little is known about the mechanisms underlying primordial follicular formation, growth, and activation. The objective of this study was to assess the effect of continuous insulin exposure on folliculogenesis in 16.5 dpc and 3 dpp mouse ovaries in vitro. The results demonstrated that 5 μg/ml insulin promotes the phosphorylation of Akt and the primordial follicular assembly and activation process via the InsR/PI3K/Akt signaling pathway in the 16.5 dpc fetal mouse ovaries cultured in vitro. However, the presence of 5 μg/ml insulin represses the phosphorylation of Akt and the follicular assembly and activation process in the 3 dpp mouse ovaries cultured in vitro. Furthermore, in vitro cultures of 3 dpp mouse ovaries with insulin can repress the expression of Pten. In conclusion, folliculogenesis was stage-specifically regulated by insulin via the Akt signaling pathway in vitro.     

Regulation of hair follicle development by the TNF signal ectodysplasin and its receptor Edar

X-linked and autosomal forms of anhidrotic ectodermal dysplasia syndromes (HED) are characterized by deficient development of several ectodermal organs, including hair, teeth and exocrine glands. The recent cloning of the genes that underlie these syndromes, ectodysplasin (ED1) and the ectodysplasin A receptor (EDAR), and their identification as a novel TNF ligand-receptor pair suggested a role for TNF signaling in embryonic morphogenesis. In the mouse, the genes of the spontaneous mutations Tabby (Ta) and downless (dl) were identified as homologs of ED1 and EDAR, respectively. To gain insight into the function of this signaling pathway in development of skin and hair follicles, we analyzed the expression and regulation of Eda and Edar in wild type as well as Tabby and Lef1 mutant mouse embryos. We show that Eda and Edar expression is confined to the ectoderm and occurs in a pattern that suggests a role of ectodysplasin/Edar signaling in the interactions between the ectodermal compartments and the formation and function of hair placodes. By using skin explant cultures, we further show that this signaling pathway is intimately associated with interactions between the epithelial and mesenchymal tissues. We also find that Ta mutants lack completely the placodes of the first developing tylotrich hairs, and that they do not show patterned expression of placodal genes, including Bmp4, Lef1, Shh, Ptch and Edar, and the genes for beta-catenin and activin A. Finally, we identified activin as a mesenchymal signal that stimulates Edar expression and WNT as a signal that induces Eda expression, suggesting a hierarchy of distinct signaling pathways in the development of skin and hair follicles. In conclusion, we suggest that Eda and Edar are associated with the onset of ectodermal patterning and that ectodysplasin/edar signaling also regulates the morphogenesis of hair follicles.     

eIF4B controls survival and proliferation and is regulated by proto-oncogenic signaling pathways

Messenger RNA translation, or protein synthesis, is a fundamental biological process affecting cell growth, survival and proliferation. Initiation is the rate limiting and hence the most regulated step of translation. In eukaryotes, translation initiation is facilitated by multiple protein factors collectively called eIFs (for eukaryotic translation initiation factors). The complex consisting of the eIF4 group factors including the mRNA cap-binding eIF4E protein, large scaffolding protein eIF4G and RNA helicase eIF4A is assisted by the eIF4B co-factor to unwind local secondary structures and create a ribosome landing pad on mRNA. Recruitment of the ribosome and augmentation in the mRNA scanning process culminates in the positioning of the ribosome over the start codon. Deregulated translational control is believed to play an important role in oncogenic transformation. Indeed, many eIFs are bona fide proto-oncogenes. In many types of human cancers, eIFs are either overexpressed or ectopically activated by Ras-MAPK and PI3K-mTOR signaling cascades, resulting in increased survival and accelerated proliferation. In this review we will analyze the bulk of data describing eIF4B and its role in cell survival and proliferation. Recent studies have shown that eIF4B is phosphorylated and activated by Ras-MAPK and PI3K-mTOR signaling cascades. In addition, eIF4B regulates translation of proliferative and pro-survival mRNAs. Moreover, eIF4B depletion in cancer cells attenuates proliferation, sensitizes them to genotoxic stress driven apoptosis. Taken together, these findings identify eIF4B as a potential target for development of anti-cancer therapies.     

Corneal epithelial cell fate is maintained during repair by Notch1 signaling via the regulation of vitamin A metabolism

Integrity and preservation of a transparent cornea are essential for good vision. The corneal epithelium is stratified and nonkeratinized and is maintained and repaired by corneal stem cells. Here we demonstrate that Notch1 signaling is essential for cell fate maintenance of corneal epithelium during repair. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1-deficient corneal progenitor cells differentiate into a hyperplastic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell nonautonomous processes, characterized by secretion of fibroblast growth factor-2 (FGF-2) through Notch1(-/-) epithelium followed by vascularization and remodeling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, we found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of cellular retinol binding protein 1 (CRBP1), required to generate a pool of intracellular retinol.     

Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements

During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.     

Wnt signaling and the regulation of stem cell function

Canonical Wnt signaling plays a crucial role in controlling cell expansion in many types of stem cells. Recent studies, however, demonstrated that Wnt is not only a general stem cell growth factor but can also influence cell lineage decisions in certain stem cell types by promoting specific fates at the expense of others. Thus, Wnt signaling elicits multiple functions in stem cells. Wnt activity appears to depend on cell-intrinsic properties that might change with time during development, thereby altering the cellular response to Wnt. Moreover, the spatial context of a stem cell also determines how the cell interprets Wnt signal activity, in that synergistic or antagonistic signaling pathways can modulate Wnt signaling. How a stem cell integrates Wnt and other signals and how such signaling networks regulate stem cell function on the molecular level remains to be elucidated.     

Wnt signaling regulates the function of MyoD and myogenin

The myogenic regulatory factors (MRFs), MyoD and myogenin, can induce myogenesis in a variety of cell lines but not efficiently in monolayer cultures of P19 embryonal carcinoma stem cells. Aggregation of cells expressing MRFs, termed P19[MRF] cells, results in an approximately 30-fold enhancement of myogenesis. Here we examine molecular events occurring during P19 cell aggregation to identify potential mechanisms regulating MRF activity. Although myogenin protein was continually present in the nuclei of >90% of P19[myogenin] cells, only a fraction of these cells differentiated. Consequently, it appears that post-translational regulation controls myogenin activity in a cell lineage-specific manner. A correlation was obtained between the expression of factors involved in somite patterning, including Wnt3a, Wnt5b, BMP-2/4, and Pax3, and the induction of myogenesis. Co-culturing P19[Wnt3a] cells with P19[MRF] cells in monolayer resulted in a 5- to 8-fold increase in myogenesis. Neither BMP-4 nor Pax3 was efficient in enhancing MRF activity in unaggregated P19 cultures. Furthermore, BMP-4 abrogated the enhanced myogenesis induced by Wnt signaling. Consequently, signaling events resulting from Wnt3a expression but not BMP-4 signaling or Pax3 expression, regulate MRF function. Therefore, the P19 cell culture system can be used to study the link between somite patterning events and myogenesis.     

SMAD4-mediated WNT signaling controls the fate of cranial neural crest cells during tooth morphogenesis

TGFβ/BMP signaling regulates the fate of multipotential cranial neural crest (CNC) cells during tooth and jawbone formation as these cells differentiate into odontoblasts and osteoblasts, respectively. The functional significance of SMAD4, the common mediator of TGFβ/BMP signaling, in regulating the fate of CNC cells remains unclear. In this study, we investigated the mechanism of SMAD4 in regulating the fate of CNC-derived dental mesenchymal cells through tissue-specific inactivation of Smad4. Ablation of Smad4 results in defects in odontoblast differentiation and dentin formation. Moreover, ectopic bone-like structures replaced normal dentin in the teeth of Osr2-IresCre;Smad4(fl/fl) mice. Despite the lack of dentin, enamel formation appeared unaffected in Osr2-IresCre;Smad4(fl/fl) mice, challenging the paradigm that the initiation of enamel development depends on normal dentin formation. At the molecular level, loss of Smad4 results in downregulation of the WNT pathway inhibitors Dkk1 and Sfrp1 and in the upregulation of canonical WNT signaling, including increased β-catenin activity. More importantly, inhibition of the upregulated canonical WNT pathway in Osr2-IresCre;Smad4(fl/fl) dental mesenchyme in vitro partially rescued the CNC cell fate change. Taken together, our study demonstrates that SMAD4 plays a crucial role in regulating the interplay between TGFβ/BMP and WNT signaling to ensure the proper CNC cell fate decision during organogenesis.     

Neural stem cells in the adult ciliary epithelium express GFAP and are regulated by Wnt signaling

The identification of neural stem cells with retinal potential in the ciliary epithelium (CE) of the adult mammals is of considerable interest because of their potential for replacing or rescuing degenerating retinal neurons in disease or injury. The evaluation of such a potential requires characterization of these cells with regard to their phenotypic properties, potential, and regulatory mechanisms. Here, we demonstrate that rat CE stem cells/progenitors in neurosphere culture display astrocytic nature in terms of expressing glial intermediate neurofilament protein, GFAP. The GFAP-expressing CE stem cells/progenitors form neurospheres in proliferating conditions and generate neurons when shifted to differentiating conditions. These cells express components of the canonical Wnt pathway and its activation promotes their proliferation. Furthermore, we demonstrate that the activation of the canonical Wnt pathway influences neuronal differentiation of CE stem cells/progenitors in a context dependent manner. Our observations suggest that CE stem cells/progenitors share phenotypic properties and regulatory mechanism(s) with neural stem cells elsewhere in the adult CNS.     

miR-199b-5p enhances the proliferation of medullary thymic epithelial cells via regulating Wnt signaling by targeting Fzd6

Thymic epithelial cells (TECs) are essential regulators of T-cell development and selection. miRNAs play critical roles in regulating TEC proliferation during the process of thymic aging. Our previous studies revealed that miR-199b-5p was upregulated in TECs from 1- to 3-month-old mice. But its function and potential mechanism are not clear. We hypothesized that miR-199b-5p may play an important role in age-related thymus involution via targeting some genes. To confirm it, the murine thymic epithelial cell line 1 (MTEC1) cells were used. Our results showed that overexpression of miR-199b-5p can enhance MTEC1 cell proliferation. On the contrary, repression of miR-199b-5p can inhibit MTEC1 cell proliferation. Meanwhile, it was confirmed that frizzled receptor 6 (Fzd6) is the direct target gene of miR-199b-5p. Furthermore, overexpression of miR-199b-5p can upregulate the expressions of β-catenin, Tcf7, Wnt4, and C-myc to activate Wnt signaling and cell cycle signaling. Silence of Fzd6 and co-transfection with siFzd6 and miR-199b-5p mimic/inhibitor confirmed that the biological function of miR-199b-5p is indeed by targeting Fzd6 in medullary TECs. Overall, miR-199b-5p is an important regulator in medullary TEC proliferation through targeting Fzd6 to activate Wnt signaling and cell cycle signaling. Our data indicate that miR-199b-5p may block the process of thymic aging and be a potential therapeutic target for thymus involution.