Claudia Banchio

Transcriptional regulation of phosphatidylcholine biosynthesis.

Phosphatidylcholine biosynthesis in animal cells is primarily regulated by the rapid translocation of CTP:phosphocholine cytidylyltransferase alpha between a soluble form that is inactive and a membrane-associated form that is activated. Until less than 10 years ago there was no information on the transcriptional regulation of phosphatidylcholine biosynthesis. Research has identified the transcription factors Sp1, Rb, TEF4, Ets-1 and E2F as enhancing the expression of the cytidylyltransferase and Net as a factor that represses cytidylyltransferase expression. Key transcription factors involved in cholesterol or fatty acid metabolism (SREBPs, LXRs, PPARs) do not have a major role in transcriptional regulation of the cytidylyltransferase. Rather than being linked to cholesterol or energy metabolism, regulation of the cytidylyltransferase is linked to the cell cycle, cell growth and differentiation. Transcriptional regulation of phospholipid biosynthesis is more elegantly understood in yeast and involves responses to inositol, choline and zinc in the culture medium.

Phosphatidylcholine Biosynthesis during Neuronal Differentiation and Its Role in Cell Fate Determination

Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase α (CKα) and choline cytidylyltransferase α (CCTα). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCTα. Later, the transcription of CKα- and CCTα-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CKα or CCTα increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCTα or CKα was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration.

Oral immunization with live Lactococcus lactis expressing rotavirus VP8* subunit induces specific immune response in mice

Rotaviruses are the major cause of worldwide infectious diarrhea in children and vaccination is considered to be the most effective way to control these infections. The development of a mucosal live vaccine using the food-grade lactic acid bacteria Lactococcus lactis as antigen vehicle is an attractive and safe vaccination strategy against rotavirus. In this study, the construction of recombinant L. lactis strains able to produce the rotavirus spike-protein subunit VP8 in cytoplasmic, secreted and cell wall-anchored forms is reported. Evaluation of the immune response generated after immunization was conducted in a mouse model. The present study shows that animals inoculated orally with the L. lactis strain producing the cytoplasmic form of VP8 (LL1) developed significant levels of intestinal IgA antibodies while animals receiving L. lactis producing the cell wall-anchored VP8 form (LL3) exhibited anti-VP8 antibodies at both intestinal and systemic levels. Furthermore, it was observed that intestinal antibodies of the LL1-treated group and serum antibodies of the LL3-treated group were able to block rotavirus infection by 50% and 100%, respectively. These encouraging results represent a step towards the development of a new and safe mucosal vaccine against rotavirus.

Signalling pathways controlling fatty acid desaturation.

Microorganisms, plants and animals regulate the synthesis of unsaturated fatty acids (UFAs) during changing environmental conditions as well as in response to nutrients. Unsaturation of fatty acid chains has important structural roles in cell membranes: a proper ratio of saturated to UFAs contributes to membrane fluidity. Alterations in this ratio have been implicated in various disease states including cardiovascular diseases, immune disorders, cancer and obesity. They are also the major components of triglycerides and intermediates in the synthesis of biologically active molecules such as eicosanoids, which mediates fever, inflammation and neurotransmission. UFAs homeostasis in many organisms is achieved by feedback regulation of fatty acid desaturases gene transcription. Here, we review recently discovered components and mechanisms of the regulatory machinery governing the transcription of fatty acid desaturases in bacteria, yeast and animals.

Role of Phosphatidylcholine during Neuronal differentiation

Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate.

Rab27a controls the recruitment of phosphatidylinositol 4-kinase type 2α from endosomes to the plasma membrane, which promotes high levels of PI(4)P, fuels PI(4,5)P2 production, and favors the recruitment of Pr55Gag and HIV-1 assembly.

Sp-1 binds promoter elements that are regulated by retinoblastoma and regulate CTP:Phosphocholine cytidylyltransferase-α transcription

The retinoblastoma (Rb) protein is implicated in transcriptional regulation of at least five cellular genes. Co-transfection of Rb and truncated promoter constructs has defined a discrete element (retinoblastoma control element (RCE)) within the promoters of each of these genes as being necessary for Rb-mediated transcriptional control. In the present report we demonstrate that two RCEs identified within the CTP:phosphocholine cytidylyltransferase-α (CTα) proximal promoter are essential to promote transcription. Mutations that abolished each RCE markedly decreased CTα transcription. Co-transfection of Rb and truncated promoter constructs demonstrated that Rb regulates CTα expression by different mechanisms depending on the phase of the cell cycle. The regulation of CTα expression by Rb required both the Sp1 and the RCEs. Maximal expression occurred when both Rb and Sp1 were overexpressed. Moreover, RCEs were required for Rb association with the DNA. This regulatory mechanism alters CTα activity and thereafter changes PC availability and cell physiology. This is the first report demonstrating not only that surrounding Sp1 binding sites alter regulation mediated by Rb, but also that the expression of a gene involved in PC biosynthesis shares a common regulatory pathway with genes responsible for cell growth and differentiation.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.

Induction of Hepatic Multidrug Resistance-Associated Protein 3 by Ethynylestradiol Is Independent of Cholestasis and Mediated by Estrogen Receptor

Multidrug resistance–associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1–10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17β-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.

Role of Histone Deacetylase in the Expression of CTP:Phosphocholine Cytidylyltransferase α

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of CTP:phosphocholine cytidylyltransferase α (CTα) are not fully understood. In this study, we investigated whether or not histone deacetylation is involved in repression of CTα expression in quiescent C3H10T1/2 mouse embryo fibroblasts. We have examined the contributions of the Sp1 and E2F binding sites in the repression of CTα gene expression. Immunoprecipitation experiments showed that histone deacetylase 1 (HDAC1) and HDAC activity are associated with Sp1 in serum-starved cells or during serum stimulation. However, HDAC1 association with E2F was only detected in serum-starved cells. By chromatin immunoprecipitation assays, we detected both direct and indirect association of HDAC1 with the CTα promoter. Treatment with the HDAC inhibitor trichostatin A induced CTα expression. Our data suggest that HDAC1 plays a critical role in CTα repression and that Sp1 and E2F may serve as key targets for HDAC1-mediated CTα repression in fibroblasts.