Berberine induces anti-atopic dermatitis effects through the downregulation of cutaneous EIF3F and MALT1 in NC/Nga mice with atopy-like dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with severe pruritus. Berberine, a naturally occurring isoquinoline alkaloid, has anti-inflammatory effects. This study investigated the effects and molecular mechanisms of berberine on AD-like symptoms in mice. In this study, NC/Nga mice with atopy-like dermatitis (dermatitis mice), fibroblast and mast cells were used. In dermatitis mice, intermittent oral administrations of berberine 3 times a week for 12 days inhibited skin symptom, itching, cutaneous infiltration of eosinophils and mast cells, and the expression of cutaneous eotaxin, macrophage migration inhibitory factor (MIF) and IL-4. Berberine also attenuated IL-4/MIF-induced eotaxin in fibroblasts and allergen-induced MIF and IL-4 in mast cells. In mast cells, the GeneChip® microarray showed that antigen increased the expression of EIF3F and MALT1, inhibited by berberine. The siRNAs for them inhibited the expression of MIF and IL-4 in antigen-stimulated mast cells. These results suggest that berberine improves AD-like symptoms through the inhibition of the eotaxin and pro-inflammatory cytokine expression and the related inflammatory cell recruitment. It is also suggested that the downregulation of EIF3F and MALT1 by berberine is involved in suppressing the cytokine expression. Taken together, berberine or berberine-containing crude drugs are expected to contribute to the improvement of AD symptoms.

Effects of EIF3B gene downregulation on apoptosis and proliferation of human ovarian cancer SKOV3 and HO-8910 cells.

In order to investigate the role of EIF3B (eukaryotic translation initiation factor 3B, EIF3B) gene in the proliferation and apoptosis of ovarian cancer cells, a lentiviral vector system and shEIF3B lentiviral vector were constructed to transfect human ovarian cancer cells. SKOV3 and HO-8910 cells were used in this experiment. The cell growth was detected by Celigo cell counting assay, the apoptosis rate was measured by flow cytometry and the cell proliferation ability of lentivirus transfected cells was tested by MTT assay and clone formation assay. Results showed that the specific shRNA had a significant inhibitory effect on the expression of EIF3B gene. Compared with the negative control, the expression of EIF3B mRNA and protein in SKOV3 and HO-8910 cells were significantly inhibited in the knockdown group. Then the proliferation rate of each group was tested, we found that SKOV3 and HO-8910 cells in siRNA lentivirus infected group was significantly decreased. Same result was obtained from the clonogenic assay of which the colony formation of transfected cells was significantly inhibited compared with the control group. Further study showed that the proliferation inhibitory effect was associated with as increased apoptosis rate of SKOV3 and HO-8910 cells in EIF3B knockdown groups. All in all, inhibition of EIF3B gene expression significantly inhibit the proliferation of ovarian cancer cells and increase the apoptosis of ovarian cancer cells. These results provide a new basis for the study of the molecular mechanism of ovarian cancer development and provide new target gene for ovarian cancer treatment.

[Rutaecarpine protects against bleomycin-induced pulmonary fibrosis through inhibiting Notch1/eIF3a signaling pathway in rats].

To investigate whether the protection of rutaecarpine against bleomycin-induced pulmonary fibrosis is mediated by inhibiting Notch1/eukaryotic initiation factor 3a (eIF3a) signaling pathway, and whether these effects are related to the synthesis and release of calcitonin gene-related peptide (CGRP) and inhibition of epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, male Sprague-Dawley rats were randomly divided into five groups (n=12), respectively, Control group, bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group and capsaicin plus rutaecarpine (300 mg·kg⁻¹) group. Bleomycin (5 mg·kg⁻¹) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg·kg⁻¹) by subcutaneous injections 1 days before and 7, 14, 21 days after induce pulmonary fibrosis rat model to deplete endogenous CGRP. At the end of experiments, blood was collected from carotid artery to determinate the plasma levels of CGRP by ELISA. Pulmonary tissue change was observed by HE staining. Masson's trichrome stain was used to demonstration collagen deposition. The collagen I expression in pulmonary tissue was measured by immunohistochemisty. The expression of CGRP, Notch1, eIF3a, collagen I, vimentin, alpha-smooth muscle actin (α-SMA), E-cadherin and zonula occludens-1 (ZO-1) was detected by qPCR or Western blot. Compared with the control group, the pulmonary tissue of the bleomycin group showed significant fibrosis, including significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, and concomitantly with the decrease in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was decreased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was increased in bleomycin group (P<0.05 or P<0.01). Compared with the bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group significantly reduced bleomycin-induced pulmonary injury concomitantly with the increase in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was increased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was decreased by rutaecarpine treatment (P<0.05 or P<0.01). All these effects of rutaecarpine were abolished by capsaicin.These results suggest that rutaecarpine protects against bleomycin-induced pulmonary fibrosis by inhibiting Notch1/eIF3a signaling pathway, alleviating EMT process, which is related to the increased synthesis and release of CGRP.

Hrp48 and eIF3d contribute to msl-2 mRNA translational repression.

Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. Here we identify Hrp48 as a SXL co-factor. Hrp48 binds to the 3' UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5' UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. Our results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.

Ser-486/491 phosphorylation and inhibition of AMPKα activity is positively associated with Gleason score, metastasis, and castration-resistance in prostate cancer: A retrospective clinical study.

BACKGROUND: We previously demonstrated that adenosine monophosphate-activated protein kinase (AMPKα) activity is significantly inhibited by Ser-486/491 phosphorylation in cell culture and in vivo models of metastatic and castration-resistant prostate cancer, and hypothesized these findings may translate to clinical specimens. METHODS: In this retrospective, single-institution pilot study, 45 metastatic prostate cancer cases were identified within the University Hospitals Cleveland Medical Center Pathology Archive with both metastasis and matched primary prostate tumor specimens in formalin-fixed, paraffin-embedded blocks, and complete electronic medical records. Thirty non-metastatic, hormone-dependent prostate cancer controls, who were progression-free as defined by undetectable prostate specific antigen for at least 79.6 months (range 79.6-136.0 months), and matched metastatic cases based on age, race, and year of diagnosis. All specimens were collected from 1991 to 2014; primary tumor specimens were obtained via diagnostic biopsy or prostatectomy, and metastasis specimens obtained via surgery or perimortem. 5-µ sequential slides were processed for phospho-Ser-486/491 AMPKα1 /α2 , phospho-Thr-172 AMPKα, AMPKα1 /α2 , phospho-Ser-792 Raptor, phospho-Ser-79 acetyl-CoA carboxylase, and phospho-Ser-872, 3-hydroxy-3-methylglutaryl-CoA reductase immunohistochemistry to determine expression, phosphorylation pattern, and activity of AMPKα. RESULTS: Increased inhibitory Ser-486/491 AMPKα1 /α2 phosphorylation, increased AMPKα protein expression, decreased AMPKα activity, and loss of nuclear AMPKα and p-AMPKα are associated with prostate cancer progression to metastasis. Increased p-Ser-486/491 AMPKα1 /α2 was also positively correlated with higher Gleason grade and progression to castration-resistance. CONCLUSIONS: p-Ser-486/491 AMPKα1 /α2 is a novel marker of prostate cancer metastasis and castration-resistance. Ser-486/491 phosphokinases should be pursued as targets for metastatic and castration-resistant prostate cancer chemotherapy.

Knockdown of elF3a inhibits TGF­ß1­induced extracellular matrix protein expression in keloid fibroblasts.

Keloid formation is characterized by hyperproliferation of secretory and responsive keloid fibroblasts (KFs) and overproduction of extracellular matrix (ECM). Eukaryotic translation initiation factor 3 subunit A (eIF3a) one of the core subunits of the translation initiation complex, eIF3, has previously been reported to possess an anti­fibrogenic effect. However, the role of eIF3a in keloid formation has not yet been investigated. Therefore, the present study examined the effect of eIF3a on transforming growth factor­ß1 (TGF­ß1)­mediated ECM expression in KFs. The expression levels of eIF3a in human keloid tissues was evaluated using reverse transcription­quantitative polymerase chain reaction and western blotting. KFs were incubated with siRNA­eIF3a or siRNA­mock for 48 h. The cells were then treated with TGF­ß1 (10 ng/ml) for 72 h. Cell proliferation was evaluated using the CCK­8 assay. The expression levels of α­SMA, collagen type I, TGF­ß receptor I (RI), TGF­ß RII, phosphorylated (p)­mothers against decapentaplegic homolog (Smad2), Smad2, p­Smad3 and Smad3 were detected western blotting. The present study identified significant upregulation of eIF3a mRNA and protein and in human keloid tissues compared with in normal tissues. Knockdown of eIF3a inhibited KF proliferation induced by TGF­ß1. In addition, eIF3a silencing significantly suppressed the TGF­ß1­induced expression of α­smooth muscle actin, collagen I, TGF­ß RI and TGF­ß RII in KFs. Furthermore, eIF3a silencing inhibited the phosphorylation levels of Smad2 and Smad3 in TGF­ß1­induced KFs. To the best of our knowledge, the current study is the first to demonstrate that siRNA­eIF3a inhibits the expression ECM proteins via the TGF­ß1/Smad signaling pathway in KFs. Therefore, eIF3a may be a potential, novel target for treatment of keloids.

eIF3a: A new anticancer drug target in the eIF family.

eIF3a is the largest subunit of eIF3, which is a key player in all steps of translation initiation. During the past years, eIF3a is recognized as a proto-oncogene, which is an important discovery in this field. It is widely reported to be correlated with cancer occurrence, metastasis, prognosis, and therapeutic response. Recently, the mechanisms of eIF3a action in the carcinogenesis are unveiled gradually. A number of cellular, physiological, and pathological processes involving eIF3a are identified. Most importantly, it is emerging as a new potential drug target in the eIF family, and some small molecule inhibitors are being developed. Thus, we perform a critical review of recent advances in understanding eIF3a physiological and pathological functions, with specific focus on its role in cancer and anticancer drug targets.

The eIF3 complex of Trypanosoma brucei: composition conservation does not imply the conservation of structural assembly and subunits function.

The multisubunit eukaryotic initiation factor 3 (eIF3) plays multiple roles in translation but is poorly understood in trypanosomes. The putative subunits eIF3a and eIF3f of Trypanosoma brucei (TbIF3a and TbIF3f) were overexpressed and purified, and 11 subunits were identified, TbIF3a through l minus j, which form a tight complex. Both TbIF3a and TbIF3f are essential for the viability of T. brucei RNAi knockdown of either of them severely reduced total translation and the ratio of the polysome/80S peak area. TbIF3f and TbIF3a RNAi cell lines were modified to express tagged-TbIF3a and -TbIF3f, respectively. RNAi in combination with affinity purification assays indicated that both subunits are variably required for TbIF3 stability and integrity. The relative abundance of other subunits in the TbIF3f-tag complex changed little upon TbIF3a depletion; while only subunits TbIF3b, i, and e copurified comparably with TbIF3a-tag upon TbIF3f depletion. A genome-wide UV-crosslinking assay showed that several TbIF3 subunits have direct RNA-binding activity, with TbIF3c showing the strongest signal. In addition, CrPV IRES, but neither EMCV IRES nor HCV IRES, was found to mediate translation in T. brucei These results together imply that the structure of TbIF3 and the subunits function have trypanosome-specific features, although the composition is evolutionarily conserved.

Using Histone Deacetylase Inhibitors to Analyze the Relevance of HDACs for Translation.

Gene expression is regulated in part through the reversible acetylation of histones, by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). HAT activity results in the addition of acetyl groups on the lysine residues of histone tails leading to decondensation of the chromatin, and increased gene transcription in general, whereas HDACs remove these acetyl groups, thus leading to an overall suppression of gene transcription. Recent evidence has elucidated that histones are not the only components of the proteome that are targeted by HATs and HDACs. A large number of nonhistone proteins undergo posttranslational acetylation. They include proteins involved in mRNA stability, protein localization and degradation, as well as protein-protein and protein-DNA interactions. In recent years, numerous studies have discovered increased HDAC expression and/or activity in numerous disease states, including cancer, where the upregulation of HDAC family members leads to dysregulation of genes and proteins involved in cell proliferation, cell cycle regulation, and apoptosis. These observations have pushed HDAC inhibitors (HDACi) to the forefront of therapeutic development of oncological conditions. HDACi, such as Vorinostat (Suberoylanilide hydroxamic acid (SAHA)), affect cancer cells in part by suppressing the translation of key proteins linked to tumorigenesis, such as cyclin D1 and hypoxia inducible factor 1 alpha (HIF-1α). Herein we describe methodologies to analyze the impact of the HDACi Vorinostat on HIF-1α translational regulation and downstream effectors.

Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes Cell Proliferation and Motility in Pancreatic Cancer.

Identifying a target molecule that is crucially involved in pancreatic tumor growth and metastasis is necessary in developing an effective treatment. The study aimed to investigate the role of the eukaryotic translation initiation factor 3a (eIF3a) in the cell proliferation and motility in pancreatic cancer. Our data showed that the expression of eIF3a was upregulated in pancreatic ductal adenocarcinoma as compared with its expression in normal pancreatic tissues. Knockdown of eIF3a by a specific shRNA caused significant decreases in cell proliferation and clonogenic abilities in pancreatic cancer SW1990 and Capan-1 cells. Consistently, the pancreatic cancer cell growth rates were also impaired in xenotransplanted mice. Moreover, wound-healing assay showed that depletion of eIF3a significantly slowed down the wound recovery processes in SW1990 and Capan-1 cells. Transwell migration and invasion assays further showed that cell migration and invasion abilities were significantly inhibited by knockdown of eIF3a in SW1990 and Capan-1 cells. Statistical analysis of eIF3a expression in 140 cases of pancreatic ductal adenocarcinoma samples revealed that eIF3a expression was significantly associated with tumor metastasis and TNM staging. These analyses suggest that eIF3a contributes to cell proliferation and motility in pancreatic ductal adenocarcinoma.

Fluorofenidone attenuates bleomycin-induced pulmonary fibrosis by inhibiting eukaryotic translation initiation factor 3a (eIF3a) in rats.

Fluorofenidone is a novel derivative of l-mimosine. It has remarkable anti-fibrotic properties. In this study, we established that fluorofenidone ameliorates pulmonary fibrosis (PF) both in vivo and in vitro by specifically inhibiting the expression of eukaryotic translation initiation factor 3a (eIF3a). eIF3a plays an important role in the development and progression of PF. An animal model of PF was induced by intratracheal instillation of bleomycin (5mg/kg) in rats. Rats were orally administered with fluorofenidone (250, 500 mg/kg/d·[i.g.]) and pirfenidone (500 mg/kg/d·[i.g.]) for 28 days. Primary pulmonary fibroblasts were cultured to determine the effect of fluorofenidone on TGF-ß1-induced (5 ng/ml) proliferation and differentiation of fibroblasts. The expression/level of eIF3a, TGF-ß1, α-SMA, collagen I, and collagen III were analyzed by ELISA, real-time PCR, and western blot. The cell proliferation rate was determined by MTS assay. The results indicate that fluorofenidone significantly improves the pathological changes in lung tissues and reduces the deposition of collagen by inhibiting eIF3a in rats with bleomycin-induced PF. Moreover, in a culture of pulmonary fibroblasts, fluorofenidone decreased the up-regulation of TGF-ß1-induced eIF3a by inhibiting the proliferation of cells and reducing the expression of α-SMA, collagen I, and collagen III. These findings suggest that eIF3a is a new and special target of fluorofenidone, which could be potentially used in the development of a drug that treats PF.

Eukaryotic initiation factor 3C silencing inhibits cell proliferation and promotes apoptosis in human glioma.

Eukaryotic initiation factor 3, subunit c (eIF3c), an oncogene overexpressed in human cancers, plays an important role in cell tumorigenesis and proliferation. However, studies assessing its function in gliomas are scarce. The present study evaluated for the first time, the role of eIF3c in gliomas. Immunohistochemistry was carried out to assess eIF3c expression in 95 human glioma samples and normal brain tissues. Then, the eIF3c mRNA levels were detected in tumor and normal brain specimens by quantitative RT-PCR. In addition, eIF3c mRNA levels were assessed in four glioma cell lines (U87, U251, A172 and U373) by semi-quantitative RT-PCR. The RNA interference (RNAi) technology was employed to knock down the eIF3c gene in the U251 cells. Western blot analysis, BrdU assay and flow cytometry were used to measure eIF3c protein levels, cell proliferation, cell apoptosis and cell cycle, respectively. The eIF3c protein was overexpressed in the human glioma specimens. In agreement, the eIF3c mRNA expression levels were significantly higher in the human glioma tissues compared with the normal brain samples (P<0.0001). In addition, eIF3c mRNA was detected in all the glioma cell lines. Silencing the eIF3c gene in the U251 cells by RNAi significantly suppressed cell proliferation (P<0.01) and increased apoptosis (P<0.01). Finally, a stark decrease was observed in the G1 phase cell number (P<0.01), while the S and G2 phase cells were significantly increased (P<0.01) after eIF3c knockdown. These findings suggest that eIF3c is overexpressed in human gliomas and essential for their proliferation and survival. Therefore, inhibiting eIF3c expression may constitute an effective therapy for human glioma.

The translation initiation factor eIF3i up-regulates vascular endothelial growth factor A, accelerates cell proliferation, and promotes angiogenesis in embryonic development and tumorigenesis.

Vascular endothelial growth factor A (VEGFA) is a critical proangiogenic factor that is activated by hypoxia at both the transcriptional and post-transcriptional levels. In hypoxia conditions, stabilized hypoxia-inducible factor 1α (HIF1A) is the key regulator for transcriptional activation of VEGFA. However, the post-transcriptional control of VEGFA expression remains poorly understood. Here, we report that the eukaryotic translation initiation factor 3i (eIF3i) is required for VEGFA protein expression in both normal embryonic and tumorigenic angiogenesis. eIF3i is dynamically expressed in the early stages of zebrafish embryogenesis and in human hepatocellular carcinoma tissues. eIF3i homozygous mutant zebrafish embryos show severe angiogenesis defects and human hepatocellular cancer cells with depletion of eIF3i to induce less angiogenesis in tumor models. Under hypoxia, the HIF1A protein can interact with its binding sequence in the eIF3i promoter and activate eIF3i transcription. The expression of VEGFA, which should rise in hypoxia, is significantly inhibited by eIF3i siRNA treatment. Moreover, eIF3i knockdown did not cause a general translation repression but specifically reduced the translation efficiency of the VEGFA mRNAs. Taken together, our results suggest that eIF3i is induced by HIF1A under hypoxia and controls normal and tumorigenic angiogenesis through regulating VEGFA protein translation.

N-myc downstream regulated 1 (NDRG1) is regulated by eukaryotic initiation factor 3a (eIF3a) during cellular stress caused by iron depletion.

Iron is critical for cellular proliferation and its depletion leads to a suppression of both DNA synthesis and global translation. These observations suggest that iron depletion may trigger a cellular "stress response". A canonical response of cells to stress is the formation of stress granules, which are dynamic cytoplasmic aggregates containing stalled pre-initiation complexes that function as mRNA triage centers. By differentially prioritizing mRNA translation, stress granules allow for the continued and selective translation of stress response proteins. Although the multi-subunit eukaryotic initiation factor 3 (eIF3) is required for translation initiation, its largest subunit, eIF3a, may not be essential for this activity. Instead, eIF3a is a vital constituent of stress granules and appears to act, in part, by differentially regulating specific mRNAs during iron depletion. Considering this, we investigated eIF3a's role in modulating iron-regulated genes/proteins that are critically involved in proliferation and metastasis. In this study, eIF3a was down-regulated and recruited into stress granules by iron depletion as well as by the classical stress-inducers, hypoxia and tunicamycin. Iron depletion also increased expression of the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), and a known downstream repressed target of eIF3a, namely the cyclin-dependent kinase inhibitor, p27(kip1). To determine if eIF3a regulates NDRG1 expression, eIF3a was inducibly over-expressed or ablated. Importantly, eIF3a positively regulated NDRG1 expression and negatively regulated p27(kip1) expression during iron depletion. This activity of eIF3a could be due to its recruitment to stress granules and/or its ability to differentially regulate mRNA translation during cellular stress. Additionally, eIF3a positively regulated proliferation, but negatively regulated cell motility and invasion, which may be due to the eIF3a-dependent changes in expression of NDRG1 and p27(kip1) observed under these conditions.

Inhibition of translation initiation factors might be the potential therapeutic targets for HCV patients with hepatic iron overload.

Standard therapy, interferon-alpha (IFN-α) and ribavirin, remains the only available option for treatment of patients with hepatitis C virus (HCV) infection. However, iron overload, a common finding among HCV patients, have a poor response to treatment with current therapy. These data suggest that both host and viral factors are involved in the determination of the outcome of the therapy. Currently, novel antiviral compounds focus on the development of indirect antiviral drugs. The process of the viral translation is considered as the potential therapeutic targets. Coincidentally, study has found that hepatic iron load enhances the levels of eukaryotic initiation factor 3 (eIF3), which is essential for HCV translation. Reversely, iron chelation could reduce eIF3 p170 translation. Our hypothesis is that iron overload may specifically enhance cellular eIFs. As a result, the cellular mechanisms, in patients with iron overload, are utilized for translating viral mRNA into protein. Thus, treatment strategies that target eIFs should be an exceptionally good candidate therapeutic method for HCV patients with hepatic iron overload.

Role of eIF3a in regulating cell cycle progression.

Translational control is an essential process in regulation of gene expression, which occurs at the initiation step performed by a number of translation initiation factor complexes. eIF3a (eIF3 p170) is the largest subunit of the eIF3 complex. eIF3a has been suggested to play roles in regulating translation of a subset of mRNAs and in regulating cell cycle progression and cell proliferation. In this study, we examined the expression profile of eIF3a in cell cycle and its role in cell cycle progression. We found that eIF3a expression oscillated with cell cycle and peaked in S phase. Reducing eIF3a expression also reduced cell proliferation rate by elongating cell cycle but did not change the cell cycle distribution. However, eIF3a appears to play an important role in cellular responses to external cell cycle modulators likely by affecting synthesis of target proteins of these modulators.

Novel characteristics of the function and induction of murine p56 family proteins.

The interferon-stimulated gene 56 (ISG56) family is induced strongly in response to virus infection, interferons (IFNs) and double-stranded RNA (dsRNA). In the mouse, this family comprises three members, ISG56, ISG54, and ISG49, which are clustered on chromosome 19 and encode the corresponding proteins p56, p54, and p49. Here, we report differential properties of these proteins and their distinct induction patterns in different cell types. All three murine proteins bound to the c-subunit of the translation initiation factor eIF3, but unlike the other members, p49 did not inhibit protein synthesis. Using a newly raised antibody, we demonstrated that both in vitro and in vivo, p49 expression was strongly induced by IFN, dsRNA, and Sendai virus. However, in kidney mesangial cells, as opposed to podocytes, encephalomyocarditis virus, vesicular stomatitis virus, or extracellular dsRNA did not induce any of the p56 family proteins, although they were robustly expressed after Sendai virus infection or dsRNA transfection. Furthermore, protein-specific differences in the regulation of p56 family members became evident in various leukocyte types: all three proteins were induced by IFN in T cells, but in B cells p56 and ISG56 mRNA could not be detected. Similarly, p56 was selectively uninducible in plasmacytoid dendritic cells, whereas in myeloid dendritic cells, all three family members were expressed. These results revealed novel cell type-, inducer-, and gene-specific regulation of the ISG56 family of genes.

An oncogenic role for the phosphorylated h-subunit of human translation initiation factor eIF3.

Dysregulation of protein synthesis has been implicated in oncogenesis through a mechanism whereby "weak" mRNAs encoding proteins involved in cell proliferation are strongly translated when the protein synthesis apparatus is activated. Previous work has determined that many cancer cells contain high levels of eIF3h, a protein subunit of translation initiation factor eIF3, and overexpression of eIF3h malignantly transforms immortal NIH-3T3 cells. This is a general feature of eIF3h, as high levels also affect translation, proliferation, and a number of malignant phenotypes of CHO-K1 and HeLa cells and, most significantly, of a primary prostate cell line. Furthermore, overexpressed eIF3h inhibits Myc-dependent induction of apoptosis of primary prostate cells. eIF3h appears to function through translation, as the initial appearance of overexpressed eIF3h in rapidly induced NIH-3T3 cells correlates tightly with the stimulation of protein synthesis and the generation of malignant phenotypes. This oncogenic potential of eIF3h is enhanced by phosphorylation at Ser(183). Finally, reduction of eIF3h levels in breast and prostate cancer cell lines by short interfering RNA methods reduces their rates of proliferation and anchorage-independent growth in soft agar. The results provide compelling evidence that high eIF3h levels directly stimulate protein synthesis, resulting in the establishment and maintenance of the malignant state in cells.

eIF3-f function in skeletal muscles: to stand at the crossroads of atrophy and hypertrophy.

The control of muscle cell size is a physiological process balanced by a fine tuning between protein synthesis and protein degradation. MAFbx/Atrogin-1 is a muscle specific E3 ubiquitin ligase upregulated during disuse, immobilization and fasting or systemic diseases such as diabetes, cancer, AIDS and renal failure. This response is necessary to induce a rapid and functional atrophy. To date, the targets of MAFbx/Atrogin-1 in skeletal muscle remain to be identified. We have recently presented evidence that eIF3-f, a regulatory subunit of the eukaryotic translation factor eIF3 is a key target that accounts for MAFbx/Atrogin-1 function in muscle atrophy. More importantly, we showed that eIF3-f acts as a "translational enhancer" that increases the efficiency of the structural muscle proteins synthesis leading to both in vitro and in vivo muscle hypertrophy. We propose that eIF3-f subunit, a mTOR/S6K1 scaffolding protein in the IGF-1/Akt/mTOR dependent control of protein translation, is a positive actor essential to the translation of specific mRNAs probably implicated in muscle hypertrophy. The central role of eIF3-f in both the atrophic and hypertrophic pathways will be discussed in the light of its promising potential in muscle wasting therapy.

Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development.

Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.