Alterations to DNA methylation and expression of CXCL14 are associated with suboptimal birth outcomes.

CXCL14 is a chemokine that has previously been implicated in insulin resistance in mice. In humans, the role of CXCL14 in metabolic processes is not well established, and we sought to determine whether CXCL14 is a risk susceptibility gene important in fetal programming of metabolic disease. For this purpose, we investigated whether CXCL14 is differentially regulated in human umbilical cords of infants with varying birth weights. We found an elevated expression of CXCL14 in human low birth weight (LBW) cords, as well as in cords from nutritionally restricted Macaca fascicularis macaques. To further analyze the regulatory mechanisms underlying the expression of CXCL14, we examined CXCL14 in umbilical cord-derived mesenchymal stem cells (MSCs) that provide an in vitro cell-based system amenable to experimental manipulation. Using both whole frozen cords and MSCs, we determined that site-specific CpG methylation in the CXCL14 promoter is associated with altered expression, and that changes in methylation are evident in LBW infant-derived umbilical cords that may indicate future metabolic compromise through CXCL14.

Molecular pathways reflecting poor intrauterine growth are found in Wharton's jelly-derived mesenchymal stem cells.

STUDY QUESTION: Are molecular pathways reflecting the biology of small for gestational age (SGA) neonates preserved in umbilical cord-derived mesenchymal stem cells (MSCs)? SUMMARY ANSWER: MSCs from SGA newborns were found to express an altered EGR-1-dependent gene network involved in the regulation of cell proliferation and oxidative stress. WHAT IS KNOWN ALREADY: Individuals with suboptimal intrauterine development are at greater risk of metabolic diseases such as type II diabetes, obesity and cardiovascular disease. STUDY DESIGN, SIZE, DURATION: Umbilical cords (n = 283) from the GUSTO (growing up in Singapore towards healthy outcomes) birth cohort study, and primary MSC isolates established from SGA and matched control cases (n = 6 per group), were subjected to gene expression analysis and candidate genes were studied for functional validation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Umbilical cord specimens were derived from babies born at the National University Hospital (NUH) in Singapore. Local ethical approval was obtained. MSC isolates were established in Wharton's jelly and molecular analysis was conducted by gene expression microarrays and RT-PCR. Cells from SGA and control groups were compared in the presence and absence of insulin and candidate gene function was studied via siRNA-mediated gene knockdown and over-expression experiments in MSCs. MAIN RESULTS AND THE ROLE OF CHANCE: Using repeated measure ANOVAs, proliferation rates of MSCs isolated from SGA neonates were found to be significantly increased (P < 0.01). In the absence of insulin, EGR-1 levels were found to be significantly reduced in the group of SGA-derived MSCs, whereas EGR-1 expression was found to be up-regulated in the same group in the presence of insulin (P < 0.01). EGR-1 was found to induce expression of COX-2 in the SGA group (P < 0.01) and both, EGR-1 and COX-2 stimulated glucose uptake in MSCs (P < 0.01). EGR-1 and COX-2 levels were associated in whole umbilical cords (n = 283, P < 0.01) and EGR-1 positively correlated with abdominal circumference and birthweight (n = 91, P < 0.01 and n = 91, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: Cell models may not entirely reflect the physiology of the host and patient follow-up studies will be necessary for further clinical validation. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that Wharton's jelly-derived MSCs are useful in identifying pathways specific for fetal growth restriction. STUDY FUNDING/COMPETING INTERESTS: This work is supported by the Translational Clinical Research (TCR) Flagship Program on Developmental Pathways to Metabolic Disease funded by the National Research Foundation (NRF) and administered by the National Medical Research Council (NMRC), Singapore- NMRC/TCR/004-NUS/2008'. SICS Investigators are supported through the Agency for Science Technology and Research (A*STAR) funding. No potential conflicts of interest relevant to this article were reported.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

ACSL1 Is Associated With Fetal Programming of Insulin Sensitivity and Cellular Lipid Content.

Individuals who are born small for gestational age (SGA) have a risk to develop various metabolic diseases during their life course. The biological memory of the prenatal state of growth restricted individuals may be reflected in epigenetic alterations in stem cell populations. Mesenchymal stem cells (MSCs) from the Wharton's jelly of umbilical cord tissue are multipotent, and we generated primary umbilical cord MSC isolates from SGA and normal neonates, which were subsequently differentiated into adipocytes. We established chromatin state maps for histone marks H3K27 acetylation and H3K27 trimethylation and tested whether enrichment of these marks was associated with gene expression changes. After validating gene expression levels for 10 significant chromatin immunoprecipitation sequencing candidate genes, we selected acyl-coenzyme A synthetase 1 (ACSL1) for further investigations due to its key roles in lipid metabolism. The ACSL1 gene was found to be highly associated with histone acetylation in adipocytes differentiated from MSCs with SGA background. In SGA-derived adipocytes, the ACSL1 expression level was also found to be associated with increased lipid loading as well as higher insulin sensitivity. ACSL1 depletion led to changes in expression of candidate genes such as proinflammatory chemokines and down-regulated both, the amount of cellular lipids and glucose uptake. Increased ACSL1, as well as modulated downstream candidate gene expression, may reflect the obese state, as detected in mice fed a high-fat diet. In summary, we believe that ACSL1 is a programmable mediator of insulin sensitivity and cellular lipid content and adipocytes differentiated from Wharton's jelly MSCs recapitulate important physiological characteristics of SGA individuals.

The antidepressant sertraline inhibits translation initiation by curtailing mammalian target of rapamycin signaling.

Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner.

Silibinin inhibits translation initiation: implications for anticancer therapy.

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E.

The signal transduction pathway wherein mTOR regulates cellular growth and proliferation is an active target for drug discovery. The search for new mTOR inhibitors has recently yielded a handful of promising compounds that hold therapeutic potential. This search has been limited by the lack of a high-throughput assay to monitor the phosphorylation of a direct rapamycin-sensitive mTOR substrate in cells. Here we describe a novel cell-based chemical genetic screen useful for efficiently monitoring mTOR signaling to 4E-BPs in response to stimuli. The screen is based on the nuclear accumulation of eIF4E, which occurs in a 4E-BP-dependent manner specifically upon inhibition of mTOR signaling. Using this assay in a small-scale screen, we have identified several compounds not previously known to inhibit mTOR signaling, demonstrating that this method can be adapted to larger screens.

Antitumor activity and mechanism of action of the cyclopenta[b]benzofuran, silvestrol.

BACKGROUND: Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. METHODOLOGY/PRINCIPAL FINDINGS: Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. CONCLUSIONS/SIGNIFICANCE: Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors.

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.

Bradykinin induced a positive chronotropic effect via stimulation of T- and L-type calcium currents in heart cells.

Using Fluo-3 calcium dye confocal microscopy and spontaneously contracting embryonic chick heart cells, bradykinin (10(-10) M) was found to induce positive chronotropic effects by increasing the frequency of the transient increase of cytosolic and nuclear free Ca2+. Pretreatment of the cells with either B1 or B2 receptor antagonists (R126 and R817, respectively) completely prevented bradykinin (BK) induced positive chronotropic effects on spontaneously contracting single heart cells. Using the whole-cell voltage clamp technique and ionic substitution to separate the different ionic current species, our results showed that BK (10(-6) M) had no effect on fast Na+ inward current and delayed outward potassium current. However, both L- and T-type Ca2+ currents were found to be increased by BK in a dose-dependent manner (10(-10)-10(-7) M). The effects of BK on T- and L-type Ca2+ currents were partially blocked by the B1 receptor antagonist [Leu8]des-Arg9-BK (R592) (10(-7) M) and completely reversed by the B2 receptor antagonist D-Arg[Hyp3,D-Phe7,Leu8]BK (R-588) (10(-7) M) or pretreatment with pertussis toxin (PTX). These results demonstrate that BK induced a positive chronotropic effect via stimulation of T- and L-type Ca2+ currents in heart cells mainly via stimulation of B2 receptor coupled to PTX-sensitive G-proteins. The increase of both types of Ca2+ current by BK in heart cells may explain the positive inotropic and chronotropic effects of this hormone.