Hepatitis C virus-mediated Aurora B kinase inhibition modulates inflammatory pathway and viral infectivity.

BACKGROUND & AIMS: Chronic hepatitis C is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. DNA methylation and histone covalent modifications constitute crucial mechanisms of genomic instability in human disease, including liver fibrosis and hepatocellular carcinoma. The present work studies the consequences of HCV-induced histone modifications in early stages of infection. METHODS: Human primary hepatocytes and HuH7.5 cells were transiently transfected with the core protein of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a. Infectious genotype 2a HCV in culture was also used. RESULTS: We show that HCV and core protein inhibit the phosphorylation of Serine 10 in histone 3. The inhibition is due to the direct interaction between HCV core and Aurora B kinase (AURKB) that results in a decrease of AURKB activity. HCV and core significantly downregulate NF-κB and COX-2 transcription, two proteins with anti-apoptotic and proliferative effects implicated in the control of the inflammatory response. AURKB depletion reduced HCV and core repression of NF-κB and COX-2 gene transcription and AURKB overexpression reversed the viral effect. AURKB abrogation increased HCV specific infectivity which was decreased when AURKB was overexpressed. CONCLUSIONS: The core-mediated decrease of AURKB activity may play a role in the inflammatory pathway during the initial steps of viral infection, while ensuring HCV infectivity.

Hepatitis C virus fitness can influence the extent of infection-mediated epigenetic modifications in the host cells.

Introduction: Cellular epigenetic modifications occur in the course of viral infections. We previously documented that hepatitis C virus (HCV) infection of human hepatoma Huh-7.5 cells results in a core protein-mediated decrease of Aurora kinase B (AURKB) activity and phosphorylation of Serine 10 in histone H3 (H3Ser10ph) levels, with an affectation of inflammatory pathways. The possible role of HCV fitness in infection-derived cellular epigenetic modifications is not known. Methods: Here we approach this question using HCV populations that display a 2.3-fold increase in general fitness (infectious progeny production), and up to 45-fold increase of the exponential phase of intracellular viral growth rate, relative to the parental HCV population. Results: We show that infection resulted in a HCV fitness-dependent, average decrease of the levels of H3Ser10ph, AURKB, and histone H4 tri-methylated at Lysine 20 (H4K20m3) in the infected cell population. Remarkably, the decrease of H4K20m3, which is a hallmark of cellular transformation, was significant upon infection with high fitness HCV but not upon infection with basal fitness virus. Discussion: Here we propose two mechanisms ─which are not mutually exclusive─ to explain the effect of high viral fitness: an early advance in the number of infected cells, or larger number of replicating RNA molecules per cell. The implications of introducing HCV fitness as an influence in virus-host interactions, and for the course of liver disease, are warranted. Emphasis is made in the possibility that HCV-mediated hepatocellular carcinoma may be favoured by prolonged HCV infection of a human liver, a situation in which viral fitness is likely to increase.

Function of partially duplicated human α77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1ß, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.

Randomized controlled trial of early arachidonic acid and docosahexaenoic acid enteral supplementation in very preterm infants.

Objective: To evaluate changes in blood long-chain polyunsaturated fatty acid (LCPUFA) and oxylipin concentrations in very preterm infants from birth to 36 weeks' postmenstrual age (WPA) after providing an emulsified arachidonic acid (ARA):docosahexaenoic acid (DHA) supplement at two different concentrations. Study design: This prospective, randomized trial assigned infants to receive a supplement (1) 80:40 group (80 mg/kg/day ARA and 40 mg/kg/day DHA, n = 9) or (2) 120:60 group (120 mg/kg/day ARA and 60 mg/kg/day DHA, n = 9). Infants received supplement daily from birth until 36 WPA. At baseline, 21 days of life and 36 WPA, the LCPUFAs were measured in plasma by gas chromatography/mass spectrophotometry. Additionally, LCPUFAs and oxylipins were analyzed in whole blood by ultra-high-performance liquid chromatography-tandem mass spectrometry. Furthermore, a sample of oral mucosa was obtained to analyze single-nucleotide polymorphism located in the FADS1 gene by PCR. Results: Gestational age was similar between groups (80:40 = 28+6 [27+3; 30+3] completed weeks+days ; 120:60 = 29+6 [27+3; 30+5] completed weeks+days , p = 0.83). At 36 WPA, the change in plasma ARA was significantly different between groups (80:40 group = 0.15 [-0.67; 0.69] %nmol, 120:60 = 1.68 [1.38; 3.16] %nmol, p = 0.031). In whole blood, the levels of ARA-derived oxylipins (5-, 8-, 9-, 11-, 15-HETE and 8,9-EET) and EPA-derived oxylipins (18-HEPE) significantly increase from baseline to 36 WPA in the 120:60 group than the 80:40 group. Conclusion: Supplementation at high doses (120:60 mg/kg/day) increased levels of ARA, and EPA- and ARA-derived oxylipins compared to low doses (80:40 mg/kg/day). Differences were detected in EPA metabolites without a significant increase in plasma DHA.

Thyroid hormone-mediated activation of the ERK/dual specificity phosphatase 1 pathway augments the apoptosis of GH4C1 cells by down-regulating nuclear factor-kappaB activity.

Thyroid hormone (T3) plays a crucial role in processes such as cell proliferation and differentiation, whereas its implication on cellular apoptosis has not been well documented. Here we examined the effect of T3 on the apoptosis of GH4C1 pituitary cells and the mechanisms underlying this effect. We show that T3 produced a significant increase in apoptosis in serum-depleted conditions. This effect was accompanied by a decrease in nuclear factor-kappaB (NF-kappaB)-dependent transcription, IkappaBalpha phosphorylation, translocation of p65/NF-kappaB to the nucleus, phosphorylation, and transactivation. Moreover, these effects were correlated with a T3-induced decrease in the expression of antiapoptotic gene products, such as members of the inhibitor of apoptosis protein and Bcl-2 families. On the other hand, ERK but not c-Jun N-terminal kinase or MAPK p38, was activated upon exposure to T3, and inhibition of ERK alone abrogated T3-mediated apoptosis. In addition, T3 increased the expression of the MAPK phosphatase, dual specificity phosphatase 1 (DUSP1), in an ERK-dependent manner. Interestingly, the suppression of DUSP1 expression abrogated T3-induced inhibition of NF-kappaB-dependent transcription and p65/NF-kappaB translocation to the nucleus, as well as T3-mediated apoptosis. Overall, our results indicate that T3 induces apoptosis in rat pituitary tumor cells by down-regulating NF-kappaB activity through a mechanism dependent on the ERK/DUSP1 pathway.

Histone deacetylase inhibitors regulate retinoic acid receptor beta expression in neuroblastoma cells by both transcriptional and posttranscriptional mechanisms.

The retinoic acid receptor beta (RARbeta) is a retinoic acid (RA)-inducible tumor suppressor, which plays an important role in the arrest of neuroblastoma cell growth. Using human neuroblastoma SH-SY5Y cells, we have examined the regulation of RARbeta expression by histone deacetylase inhibitors (HDACi), considered to be promising agents in anticancer therapy. Our results show that HDACi cooperated with RA to increase RARbeta mRNA levels and to activate the RARbeta2 promoter in transient transfection assays. Chromatin immunoprecipitation assays showed that the basal RARbeta2 promoter that contains the RA response element was refractory to acetylation by both HDACi and RA. In addition, HDACi caused a transient increase in acetylation of a downstream RARbeta2 region, even though global histones remain hyperacetylated after a prolonged treatment with the inhibitors. RA potentiated this response and maintained acetylation for a longer period. Despite the cooperation of RA with HDACi to increase transcription of the RARbeta gene, these inhibitors caused a paradoxical reduction of the cellular levels of the RARbeta protein in cells treated with the retinoid. This reduction is secondary to a change in the protein half-life that is decreased by the HDACi due to increased ubiquitin-independent proteasomal degradation. These results show that HDACi regulate expression of the tumor suppressor gene RARbeta by both transcriptional and posttranscriptional mechanisms and might then modulate sensitivity to the retinoid in neuroblastoma cells.

The use of an active learning approach to teach metabolism to students of nutrition and dietetics.

This article describes the transition from a traditional instructor-centered course, based on lectures, to a student-centered course based on active learning methodologies as part of the reform of the Spanish higher education system within the European Higher Education Area (EHEA). Specifically, we describe the use of active learning methodologies to teach metabolism to students of nutrition and dietetics during the first year of their professional training in a 4-year undergraduate degree (Bachelor of Human Nutrition and Dietetics). In the new course design, the number of didactic lectures was largely reduced and complemented with a series of activities (problems/case studies, discussion workshops, self-assessment quizzes) aimed to get students actively engaged, to encourage self-learning, and to promote sustained work throughout the length of the course. The article presents quantitative data demonstrating a clear and significant improvement in students' performance when an active approach was implemented. Importantly, the improved performance was achieved without work overload. Finally, students' responses to this new teaching methodology have been very positive and overall satisfaction high. In summary, our results strongly argue in favor of the teaching model described herein.

Aurora kinase B activity is modulated by thyroid hormone during transcriptional activation of pituitary genes.

Covalent histone modifications clearly play an essential role in ligand-dependent transcriptional regulation by nuclear receptors. One of the predominant mechanisms used by nuclear receptors to activate or repress target-gene transcription is the recruitment of coregulatory factors capable of covalently modify the amino terminal ends of histones. Here we show that the thyroid hormone (T3) produces a rapid increase in histone H3Ser10 phosphorylation (H3Ser10ph) concomitant to the rapid displacement of the heterochromatin protein 1ß (HP1ß) to the nuclear periphery. Moreover, we found that T3-mediated pituitary gene transcription is associated with an increase in H3Ser10ph. Interestingly, the Aurora kinase B inhibitor ZM443979 abolishes the effect of T3 on H3Ser10ph, blocks HP1ß delocalization, and significantly reduces ligand-dependent transactivation. Similar effects were shown when Aurora kinase B expression was abrogated in small interfering RNA assays. In an effort to understand the underlying mechanism by which T3 increases H3Ser10ph, we demonstrate that liganded thyroid hormone receptor directly interacts with Aurora kinase B, increasing its kinase activity. Moreover, using chromatin immunoprecipitation assays, we have shown that Aurora kinase B participates of a mechanism that displaces HP1ß from promoter region, thus preparing the chromatin for the transcriptional activation of T3 regulated genes. Our findings reveal a novel role for Aurora kinase B during transcriptional initiation in GO/G1, apart from its well-known mitotic activity.

Residues K128, 132, and 134 in the thyroid hormone receptor-alpha are essential for receptor acetylation and activity.

The thyroid hormone receptor (TR)-alpha is a nuclear receptor that mediates both transrepression and ligand-dependent transactivation. Here we show that TRalpha is posttranslationally modified by acetylation in response to its own ligand (T(3)). Acetylation increases binding to DNA. Using mutagenesis, we identified three conserved lysine residues in the carboxi-terminal extension (CTE) of the DNA binding domain that are targets of the cAMP-response element-binding protein acetyltransferase. Substitution of these lysines by arginines in TRalpha decreased ligand binding affinity and precluded ligand-dependent release of corepressors and recruitment of coactivators. The acetylation TRalpha mutant lost the ability to transactivate even at high T(3) concentrations and acts as a dominant-negative inhibitor of wild-type TR activity. In addition, whereas native TRalpha interferes with AP-1 function, the mutant is unable to mediate transrepression. Finally, TRalpha suppresses NIH-3T3 fibroblast transformation by the Ras oncogene both in a ligand-dependent and -independent manner, but the CTE mutant is unable to mediate ligand-dependent repression of transformation. These results reveal a key role for the CTE region on acetylation, ligand affinity, transactivation, transrepression, and antitransforming properties of TRalpha.

Activation of the human immunodeficiency virus type I long terminal repeat by 1 alpha,25-dihydroxyvitamin D3.

The genetic predisposition of the host and the virus is the most important determinant for prediction of the course of human immunodeficiency virus type I (HIV-1) viral infection and acquired immune deficiency syndrome (AIDS) progression. Transcription from the HIV-1 long terminal repeat (LTR) is a crucial step for viral replication. Here, we describe a stimulatory role of the vitamin D receptor (VDR) on HIV-1 LTR transactivation. Transient transfections reveal that VDR activates the LTR in HeLa, U937, and Cos-1 cells in a ligand-dependent manner. 1alpha,25-Dihydroxyvitamin D3 (vitD3) promotes activation of a minimal LTR construct (from nucleotides -35 to +89), lacking a previously described hormone response element that binds several nuclear receptors. NF-kappaB (nuclear factor-kappa B) and Sp1-binding sites, which are responsible for most basal LTR activity in HeLa cells, are also dispensable for vitD3-dependent HIV-1 transcription. Although the tat response element element is not required for VDR-mediated HIV-1 gene expression, the viral protein Tat acts in a synergistic manner with the receptor to stimulate LTR activity. Furthermore, our data also show cooperation of the receptor with various cellular coactivators for HIV-1 transactivation by vitD3. Paradoxically, mutations in the VDR ligand-dependent transcriptional activation function-2 that abrogate vitD3-dependent stimulation through classical vitamin D response elements, do not reduce vitD3-mediated LTR transactivation. Furthermore, point mutations in the DNA-binding domain that abolish receptor binding to consensus DNA sequences do not affect ligand-dependent HIV-1 stimulation. These results show that VDR activates the HIV-1 LTR through different mechanisms, including non-classical nuclear receptor transcriptional actions that may ensure viral transcription under different physiological scenarios.

Binding of the thyroid hormone receptor to a negative element in the basal growth hormone promoter is associated with histone acetylation.

Nuclear thyroid hormone receptors (TRs) act as ligand-dependent activators, but paradoxically unliganded TRs can increase transcription of promoters containing negative response elements (nTRE), and hormone binding represses this activation. The rat growth hormone (GH) promoter contains a positive TRE and a nTRE. Ligand-dependent negative regulation mediated by the nTRE could play an important physiological role in restricting GH gene expression in non-pituitary cells that express TRs. With chromatin immunoprecipitation assays, we show here that the nTRE is responsible for binding of TR to the promoter in non-pituitary HeLa cells and that this element also governs transactivation by the unoccupied receptor and repression by triiodothyronine. Occupancy of the promoter by TR is concomitant with appearance of acetylated histone H3, and triiodothyronine causes release of the receptor as well as disappearance of the acetylated histone from the promoter. Although the nTRE overlaps the TATA box, the receptor does not exclude binding of TATA-binding protein, but could rather facilitate formation of the preinitiation complex. Furthermore, the proximal GH promoter is synergistically stimulated by unliganded TR and TATA-binding protein, whereas the ligand represses this cooperation. Constitutive receptor activity and synergism with TATA-binding protein require binding of corepressors. Furthermore, inhibitors of histone deacetylases enhance promoter activation by the unliganded receptor and reduce triiodothyronine-dependent repression, whereas expression of HDAC1 reverses promoter stimulation. This suggests that partitioning of histone acetylases and deacetylases between the receptors and basal transcription factors could be involved in regulation of the basal GH promoter by TRs.

The thyroid hormone receptor antagonizes CREB-mediated transcription.

Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein-protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)-containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein-protein interactions. In turn, expression of CREB reduces TR-dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand-dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross-talk between CREB and TR signalling pathways, which can have important functional consequences.