Translation control of UCP2 synthesis by the upstream open reading frame

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane. In vivo low expression of UCP2 contrasts with a high UCP2 mRNA level, and induction of UCP2 expression occurs without change in mRNA level, demonstrating a translational control. The UCP2 mRNA is characterized by a long 5′ untranslated region (5′UTR), in which an upstream open reading frame (uORF) codes for a 36-amino-acid sequence. The 5′UTR and uORF have an inhibitory role in the translation of UCP2. The present study demonstrates that the 3′ region of the uORF is a major determinant for this inhibitory role. In this 3′ region, a single-base substitution that kept the codon sense unchanged significantly modified UCP2 translation, whereas some important amino acid changes had no effect. We discuss our results within the framework of the existing models explaining initiation of translation downstream of a uORF. Received 22 March 2006; received after revision 19 May 2006; accepted 8 June 2006 C. Hurtaud and C. Gelly contributed equally to this work.     

Opposite actions of testosterone and progesterone on UCP1 mRNA expression in cultured brown adipocytes

The brown adipose tissue (BAT) thermogenic response to diet-induced obesity and cold has been found to be gender dependent. In the present work, we aimed to investigate the effects of the main physiological male and female sex hormones, i.e. testosterone, progesterone and 17-β-estradiol, on the expression of uncoupling protein 1 (UCP1) – the main mediator of BAT thermogenesis – and on UCP2 and lipid accumulation in rodent brown adipocytes differentiated in culture. Testosterone-treated cells showed fewer and smaller lipid droplets than control cells and a dose-dependent inhibition of UCP1 mRNA expression, under adrenergic stimulation by norepinephrine (NE). These effects were reverted by the androgen receptor antagonist flutamide, suggesting they are dependent, at least in part, on the androgen receptor. Progesterone- and 17-β-estradiol-treated cells showed more and larger lipid droplets and progesterone stimulated NE-induced UCP1 mRNA expression at the lower concentration tested, but not at higher concentrations, suggesting that for brown adipocytes, this hormone is dose dependent. 17-β-Estradiol did not have any remarkable effect either on UCP1 or UCP2 mRNA expression. Interestingly, the specific progesterone receptor antagonist RU486 induced UCP1 and UCP2 mRNAs, including UCP1 mRNA expression in non-NE-treated brown adipocytes, suggesting a profound effect of this anti-progestagen on brown adipocyte thermogenic capacity. Thus, are conclude that testosterone, 17-β-estradiol, progesterone and RU486 have distinct actions on brown adipocytes, thus modulating UCP1 and UCP2 mRNA expression and/or lipid accumulation, and that sex hormones are factors that may explain in part the gender-dependent BAT thermogenic response. Received 24 June 2002; received after revision 20 August 2002; accepted 26 August 2002 RID="*" ID="*"Corresponding author.     

Leptin, but not a β3-adrenergic agonist, upregulates muscle uncoupling protein-3 messenger RNA expression: short-term thermogenic interactions

The short-term effects of leptin and a β₃-adrenoceptor agonist on thermogenesis and expression of uncoupling proteins (UCPs) in brown adipose tissue (BAT) and muscle and their possible interactions were assessed. One hour after administration of the β₃-adrenoceptor agonist Trecadrine, a statistically significant increase in UCP1 messenger RNA (mRNA) expression in BAT was observed, whereas UCP2 and UCP3 in both BAT and gastrocnemius muscle were unaffected. Leptin induced an upregulation of UCP3 mRNA in muscle, with no changes in BAT UCP1 mRNA. A statistical interaction was found between leptin and Trecadrine in rectal temperature. The present study provides evidence, for the first time, of the induction of UCP3 mRNA expression in skeletal muscle by leptin in nongenetically obese animals. Received 5 March 1999; received after revision 19 April 1999; accepted 21 April 1999     

KIF5C: A new binding partner for protein kinase CK2 with a preference for the CK2α’ subunit

Protein kinase CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in eukaryotic cells. CK2 is a constitutively active tetrameric enzyme composed of two catalytic α and/or α’-subunits and two regulatory β-subunits. There is increasing evidence that the individual subunits may have independent functions and that they are asymmetrically distributed inside the cell. To gain a better understanding of the functions of the individual subunits, we employed a yeast-two-hybrid screen with CK2α and CK2α’. We identified the motor neuron protein KIF5C as a new binding partner for CK2. The interaction found in the yeast-two-hybrid screen was confirmed by co-sedimentation analysis on a sucrose density gradient and by co-immunoprecipitation analysis. Pull-down experiments and surface plasmon resonance spectrometry revealed a direct binding of KIF5C to CK2α’. Co-localization studies with neuroblastoma cells, bone marrow and with primary neurons confirmed the biochemical analysis that KIF5C preferentially bound to CK2α’. Received 8 August 2008; received after revision 3 November 2008; accepted 4 November 2008     

‘Heated’ Debates in Apoptosis

Hippocrates’ assertion that ‘what the lance does not heal, fire will’ underscores the fact that for thousands of years heat has been used to treat a variety of diseases, including cancer. Indeed, spontaneous tumor remission has been observed in patients following feverish infection [1], and expression of activated oncogenes, such as Ras, can render tumor cells sensitive to heat compared with normal cells [2, 3]. In the past, a primary drawback to the use of heat as a clinical therapy was the inability to selectively focus heat to tumors in situ. Of late, however, several approaches have been devised to deliver heat more precisely, including the use of heated nanoparticles, making hyperthermia a more clinically tractable treatment option [4, 5]. Despite these practical advances, the mechanisms responsible for heat shock-induced cell death remain controversial and ill-defined. In this Visions and Reflections we discuss recent findings surrounding the initiation of heat shock-induced apoptosis, and propose future areas of research. Received 17 March 2007; received after revision 25 April 2007; accepted 22 May 2007     

Differential expression of genes for uncoupling proteins 1, 2 and 3 in brown and white adipose tissue depots during rat development

The different expression patterns of genes for uncoupling proteins (UCPs) 1, 2 and 3 (ucp1, ucp2 and ucp3) were studied in interscapular brown adipose tissue (BAT) and in four white adipose tissue (WAT) depots (epididymal, inguinal, mesenteric and retroperitoneal) in male rats of different ages (18 days-12 months). UCP mRNA expression levels were determined by Northern blotting. In BAT, there were high levels of expression of UCP1 and UCP3 mRNA, but no detectable levels of UCP2 mRNA. Both ucp1 and ucp3 followed a similar expression pattern with age, with high levels in suckling rats which decreased to 50% or less in rats just under 2 months old, declining thereafter until 5 months and then recovering with age. However, an additional peak of expression was observed for ucp3 at the age of 3 months. In WAT, ucp1 expression was rare: occasional expression was found for UCP1 mRNA in the retroperitoneal depot in suckling rats and in the epididymal and inguinal depots in suckling and mature adult rats. ucp2 and ucp3 had different developmental expression patterns, but these were similar for each gene in the different depots studied. UCP3 mRNA was highly expressed in rats soon after birth, it decreased until 3 months, and increased thereafter, except for the mesenteric WAT where ucp3 expression decreased until 7 months before recovering. The fact that changes with age of both ucp1 and ucp3 expression have a similar profile in BAT, which is also similar to the ucp3 and also ucp1 profiles in some WAT depots, might reflect a common regulatory pattern for the expression of these genes, and also a common function. In contrast to ucp1 and ucp3, ucp2 had a peak of expression at about 2 months, and lower expression at 3 months, suggesting different regulation and probably a different role for this UCP.     

The functional RNA domain 5BSL3.2 within the NS5B coding sequence influences hepatitis C virus IRES-mediated translation

Hepatitis C virus (HCV) translation is mediated by an internal ribosome entry site (IRES) located at the 5′ end of the genomic RNA. The 3′ untranslatable region (3′UTR) stimulates translation by the recruitment of protein factors that simultaneously bind to the 5′ end of the viral genome. This leads to the formation of a macromolecular complex with a closed loop conformation, similar to that described for the cap-translated mRNAs. We previously demonstrated the existence of a long-range RNA–RNA interaction involving subdomain IIId of the IRES region and the stem–loop 5BSL3.2 of the CRE element at the 3′ end of the viral genome. The present study provides evidence that the enhancement of HCV IRES-dependent translation mediated by the 3′UTR is negatively controlled by the CRE region in the human hepatoma cell lines Huh-7 and Hep-G2 in a time-dependent manner. Domain 5BSL3.2 is the major partner in this process. Mutations in this motif lead to an increase in IRES activity by up to eightfold. These data support the existence of a functional high order structure in the HCV genome that involves two evolutionarily conserved RNA elements, domain IIId in the IRES and stem–loop 5BSL3.2 in the CRE region. This interaction could have a role in the circularisation of the viral genome.     

Anti-adipogenesis by 6-thioinosine is mediated by downregulation of PPAR γ through JNK-dependent upregulation of iNOS

Adipocyte dysfunction is associated with the development of obesity. This study shows that 6-thioinosine inhibits adipocyte differentiation. The mRNA levels of PPAR γ and C/EBPα, but not C/EBPβ and δ, were reduced by 6-thioinosine. Moreover, the mRNA levels of PPAR γ target genes (LPL, CD36, aP2, and LXRα) were down-regulated by 6-thioinosine. We also demonstrated that 6-thioinosine inhibits the transactivation activity and the mRNA level of PPAR γ. Additionally, attempts to elucidate a possible mechanism underlying the 6-thioinosine-mediated effects revealed that 6-thioinosine induced iNOS gene expression without impacting eNOS expression, and that this was mediated through activation of AP-1, especially, JNK. In addition, 6-thioinosine was found to operate upstream of MEKK-1 in JNK activation signaling. Taken together, these findings suggest that the inhibition of adipocyte differentiation by 6-thioinosine occurs primarily through the reduced expression of PPAR γ, which is mediated by upregulation of iNOS via the activation of JNK.     

Protein kinase CK2 and new binding partners during spermatogenesis

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α′-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α′, TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.     

MicroRNA-128 downregulates Bax and induces apoptosis in human embryonic kidney cells

MicroRNAs (miRNAs) are short ~21-nt non-coding RNA molecules that have been shown to regulate a number of biological processes. Previous reports have shown that overexpression of miR-128 in glioma cells inhibited cell proliferation. Literature also suggests that miR-128 negatively regulates prostate cancer cell invasion. Here, we show that overexpression of hsa-miR-128, a brain-enriched microRNA, induces apoptosis in HEK293T cells as elucidated by apoptosis assay, cell cycle changes, loss of mitochondrial membrane potential and multicaspase assay. By in silico analysis, we identified a putative target site within the 3′ untranslated region (UTR) of Bax, a proapoptotic member of the apoptosis pathway. We found that ectopic expression of hsa-miR-128 suppressed a luciferase reporter containing the Bax-3′ UTR and reduced the levels of Bax in HEK293T cells. Taken together, our study demonstrates that overexpression of hsa-miR-128 not only induces apoptosis in HEK293T cells but also is an endogenous regulator of Bax protein.     

Impaired apoptosis in lymphoblasts from Alzheimer’s disease patients: Cross-talk of Ca<Superscript>2+</Superscript>/calmodulin and ERK1/2 signaling pathways

We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer’s disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca²⁺/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca²⁺/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawal. F. Bartolomé, N. de las Cuevas: These authors contributed equally to this work. Received 14 February 2007; received after revision 16 April 2007; accepted 23 April 2007     

The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen

The life of aerobes is dependent on iron and oxygen for efficient bioenergetics. Due to potential risks associated with iron/oxygen chemistry, iron acquisition, concentration, storage, utilization, and efflux are tightly regulated in the cell. A central role in regulating iron/oxygen chemistry in animals is played by mRNA translation or turnover via the iron responsive element (IRE)/iron regulatory protein (IRP) system. The IRE family is composed of three-dimensional RNA structures located in 3′ or 5′ untranslated regions of mRNA. To date, there are 11 different IRE mRNAs in the family, regulated through translation initiation or mRNA stability. Iron or oxidant stimuli induce a set of graded responses related to mRNA-specific IRE substructures, indicated by differential responses to iron in vivo and binding IRPs in vitro. Molecular effects of phosphorylation, iron and oxygen remain to be added to the structural information of the IRE-RNA and IRP repressor in the regulatory complex. Received 21 April 2007; received after revision 13 July 2007; accepted 2 August 2007     

Polyphenolic phytochemicals – just antioxidants or much more?

Polyphenolic phytochemicals are ubiquitous in plants, in which they function in various protective roles. A ‘recommended’ human diet contains significant quantities of polyphenolics, as they have long been assumed to be ‘antioxidants’ that scavenge excessive, damaging, free radicals arising from normal metabolic processes. There is recent evidence that polyphenolics also have ‘indirect’ antioxidant effects through induction of endogenous protective enzymes. There is also increasing evidence for many potential benefits through polyphenolic-mediated regulation of cellular processes such as inflammation. Inductive or signalling effects may occur at concentrations much lower than required for effective radical scavenging. Over the last 2 – 3 years, there have been many exciting new developments in the elucidation of the in vivo mechanisms of the health benefits of polyphenolics. We summarise the current knowledge of the intake, bio-availability and metabolism of polyphenolics, their antioxidant effects, regulatory effects on signalling pathways, neuro-protective effects and regulatory effects on energy metabolism and gut health. Received 14 May 2007; received after revision 27 June 2007; accepted 24 July 2007     

靶向CD133的RNA干扰对人肝癌SMMC-7721细胞的生长抑制作用

目的利用RNA干扰技术下调SMMC-7721肝癌细胞中CD133的表达,观察其在肝癌细胞增殖和侵袭方面的作用.方法构建CD133特异性的siRNA真核表达载体,转染SMMC-7721肝癌细胞并筛选出稳定表达siRNA的转染克隆;应用RT-PCR和Western blot检测CD133 mRNA和蛋白的表达;流式细胞仪检...     

Hormonal control of mammalian oocyte meiosis at diplotene stage

Mammalian oocytes grow and undergo meiosis within ovarian follicles. Fully grown oocytes are arrested at the first meiotic prophase by a mural granulosa origin “arrester” until a surge of luteinizing hormone (LH) from the pituitary at the mid-cycle stimulates the immature oocyte to resume meiosis. Recent evidence indicates that natriuretic peptide precursor type C (NPPC) produced by mural granulosa cells stimulates the generation of cyclic guanosine 3′,5′-monophosphate (cGMP) by cumulus cell natriuretic peptide receptor 2 (NPR2), which diffuses into oocyte via gap junctions and inhibits oocyte phosphodiesterase 3A (PDE3A) activity and cyclic adenosine 3′,5′-monophosphate (cAMP) hydrolysis and maintains meiotic arrest with a high intraoocyte cAMP level. This cAMP is generated through the activity of the Gs G-protein by the G-protein-coupled receptor, GPR3 and GPR12, and adenylyl cyclases (ADCY) endogenous to the oocyte. Further studies suggest that endocrine hormones, such as follicle-stimulating hormone (FSH), LH, 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), participate in oocyte meiosis possibly by the regulation of NPPC and/or NPR2. A detailed investigation of NPPC and NPR2 expression in follicle cells will elucidate the precise molecular mechanisms of gonadotropins, and control the arrest as well as resumption of meiosis.     

Synphilin-1 isoforms in Parkinson’s disease: regulation by phosphorylation and ubiquitylation

Parkinson’s disease (PD) is characterized by the death of dopaminergic neurons and the presence of Lewy bodies in the substantia nigra pars compacta. The mechanisms involved in the death of neurons as well as the role of Lewy bodies in the pathogenesis of the disease are still unclear. Lewy bodies are made of aggregated proteins, in which α-synuclein represents their major component. α-Synuclein interacts with synphilin-1, a protein that is also present in Lewy bodies. When expressed in cells, synphilin-1 forms inclusions together with α-synuclein that resemble Lewy bodies. Synphilin-1 is ubiquitylated by various E3 ubiquitin-ligases, such as SIAH, parkin and dorfin. Ubiquitylation of synphilin-1 by SIAH is essential for its aggregation into inclusions. We recently identified a new synphilin-1 isoform, synphilin-1A, that is toxic to neurons, aggregation-prone and accumulates in detergent-insoluble fractions of brains from α-synucleinopathy patients. Synphilin-1A inclusions recruit both α-synuclein and synphilin-1. Aggregation of synphilin-1 and synphilin-1A seems to be protective to cells. We now discuss several aspects of the neurobiology and pathology of synphilin-1 isoforms, focusing on possible implications for PD. Received 26 July 2007; received after revision 19 September 2007; accepted 15 October 2007     

Do cells think?

A microorganism has to adapt to changing environmental conditions in order to survive. Cells could follow one of two basic strategies to address such environmental fluctuations. On the one hand, cells could anticipate a fluctuating environment by spontaneously generating a phenotypically diverse population of cells, with each subpopulation exhibiting different capacities to flourish in the different conditions. Alternatively, cells could sense changes in the surrounding conditions – such as temperature, nutritional availability or the presence of other individuals – and modify their behavior to provide an appropriate response to that information. As we describe, examples of both strategies abound among different microorganisms. Moreover, successful application of either strategy requires a level of memory and information processing that has not been normally associated with single cells, suggesting that such organisms do in fact have the capacity to ‘think’. Received 3 January 2007; accepted 4 April 2007