Genomic organization, promoter activity, and expression of the human choline transporter-like protein 1.

Choline transporter-like (CTL) proteins of the CTL1 family are novel transmembrane proteins implicated in choline transport for phospholipid synthesis. In this study, we characterized the 5'-flanking region of the human (h)CTL1 gene and examined some of the possible mechanisms of its regulation, including promoter activity, splicing, and expression. The transcription start site of the hCTL1 gene was mapped by 5'-rapid amplification of cDNA ends (RACE), and the presence of two splice variants, hCTL1a and hCTL1b, was investigated using isoform-specific PCR and 3'-RACE. The hCTL1 promoter region of approximately 900 bp was isolated from MCF-7 human breast cancer cells. The promoter was TATA-less and driven by a long stretch of GC-rich sequence in accordance with widespread expression of hCTL1 at both mRNA and protein levels. Deletion analyses demonstrated that a very strong promoter is contained within 500 bp of the transcription start site, and more upstream regions did not increase its activity. The core promoter that conferred the minimal transcription is within the -188/+27-bp region, and its activity varied in human breast cancer and mouse skeletal muscle cells. Multiple motifs within the promoter regulatory region bound nuclear factors from both cultured cells and normal human skeletal muscle. The motifs within the three regions [S1 (-92/-61 bp), S2 (-174/-145 bp), and S3 (-289/-260 bp)] contained overlapping binding sites for hematopoietic transcription factors and ubiquitous transcription factors, in line with the expected gene function. Genomic analyses demonstrated a high conservation of hCTL1 and mouse CTL1 proximal promoters. Accordingly, mRNA profiles demonstrated that human splice variants were expressed ubiquitously, as demonstrated for the mouse transcripts; however, they differed from the profiles of rat CTL1 transcripts, which were more restricted to neurons and intestinal tissues. The shorter hCTL1b variant contained the cytosolic COOH-terminal motif L651KKR654 for endoplasmic reticulum retrieval/retention. This retention signal was conserved in hCTL1b and rat and mouse CTL1b and is typical for transmembrane proteins of type 1 topology.

Characterization of transcription factors and cis-acting elements that regulate human CTP: phosphoethanolamine cytidylyltransferase (Pcyt2).

CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) promoter was isolated from human breast cancer MCF-7 cells and its activity delineated by luciferase reporter assays and gel-shift analysis. The Pcyt2 promoter is driven by a functional CAAT box (-90/-73) and by negative (-385/-255) and positive regulatory elements (-255/-153) in the upstream regions.

Choline transport for phospholipid synthesis.

Choline is an essential nutrient for all cells because it plays a role in the synthesis of the membrane phospholipid components of the cell membranes, as a methyl-group donor in methionine metabolism as well as in the synthesis of the neurotransmitter acetylcholine. Choline deficiency affects the expression of genes involved in cell proliferation, differentiation, and apoptosis, and it has been associated with liver dysfunction and cancer. Abnormal choline transport and metabolism have been implicated in a number of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Therefore, the study of choline transport and the characteristics of choline transporters are of central importance to understanding the mechanisms that underlie membrane integrity and cell signaling in such disorders. Kinetic studies with radiolabeled choline and inhibitors distinguish three systems for choline transport: (i) low-affinity facilitated diffusion, (ii) high-affinity, Na+-dependent transport, and (iii) intermediate-affinity, Na+-independent transport. It is only recently, however, that the proteins having transport characteristics of at least one of these systems have been identified. They include (i) polyspecific organic cation transporters (OCTs) with low affinity for choline, (ii) high-affinity choline transporters (CHT1s), and (iii) intermediate-affinity choline transporter-like (CTL1) proteins. CHT1 and CTL1 but not OCT transporters are selectively inhibited with hemicholinium-3 and essentially display characteristics of specialized transporters for targeted choline metabolism. CHT1 is abundant in neurons and almost exclusively supplies choline for acetyl-choline synthesis. The focus here is more on newly-discovered CTL1 choline transporters. They are expressed in different organisms and cell types, apparently not for the biosynthesis of acetylcholine but for the production of the most abundant metabolite of choline, the membrane lipid phosphatidylcholine.

Identification and expression of a mouse muscle-specific CTL1 gene.

In this study, a mouse gene and cDNA encoding for a novel skeletal muscle-specific choline transporter-like protein 1 (mCTL1) were identified and mCTL1 mRNA and protein expression characterized. The mCTL1 cDNA is 2888-bp long; consisting of a 653-amino-acid open-reading frame, 8-11 putative transmembrane domains, three N-glycosylation sites and seven protein kinase C phosphorylation sites. The mCTL1 gene is localized to chromosome 4B2, at 182 kb in length, and encoded by 17 exons. Although the mCTL1 mRNA was expressed in several mouse tissues such as muscle, brain, heart and testis, the protein analyses of multiple tissues and membrane vesicles reveal that mCTL1 is exclusively expressed in skeletal muscle. Expression of His-tagged mCTL1 in Cos-7 cells produces an increase in saturable choline uptake that is sensitive to a Na(+)-ion gradient, ethanolamine and the Ca(2+)-channel blocker verapamil, and insensitive to low concentrations of hemicholinium-3.

Increased microRNA-34c abundance in Alzheimer's disease circulating blood plasma.

Circulating microRNAs, present either in the cellular component, peripheral blood mononuclear cells (PBMC), or in cell-free plasma, have emerged as biomarkers for age-dependent systemic, disease-associated changes in many organs. Previously, we have shown that microRNA (miR)-34a is increased in circulating PBMC of Alzheimer's disease (AD) patients. In the present study, we show that this microRNA's sister, miR-34c, exhibits even greater increase in both cellular and plasma components of AD circulating blood samples, compared to normal age-matched controls. Statistical analysis shows the accuracy of levels of miR-34c assayed by receiver operating characteristic (ROC) analysis: the area under the curve is 0.99 (p < 0.0001) and the 95% confidence level extends from 0.97 to 1. Pearson correlation between miR-34c levels and mild and moderate AD, as defined by the mini-mental state examination (MMSE), shows an r-value of -0.7, suggesting a relatively strong inverse relationship between the two parameters. These data show that plasma levels of microRNA 34c are much more prominent in AD than those of its sister, miR-34a, or than its own level in PBMC. Transfection studies show that miR-34c, as does its sister miR-34a, represses the expression of several selected genes involved in cell survival and oxidative defense pathways, such as Bcl2, SIRT1, and others, in cultured cells. Taken together, our results indicate that increased levels of miR-34c in both PBMC and plasma may reflect changes in circulating blood samples in AD patients, compared to age-matched normal controls.

Docosahexaenoic acid increases cellular adiponectin mRNA and secreted adiponectin protein, as well as PPARγ mRNA, in 3T3-L1 adipocytes.

Adiponectin, a protein secreted from adipose tissue, has been shown to have anti-diabetic and anti-inflammatory effects, but its regulation is not completely understood. Long-chain n-3 fatty acids eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) may be involved in adiponectin regulation as they are potential ligands for peroxisome proliferator-activated receptor-γ (PPARγ), a key transcription factor for the adiponectin gene. To examine this, 3T3-L1 adipocytes were incubated with 125 µmol·L-1 EPA, DHA, palmitic, or oleic acids complexed to albumin, or with albumin alone (control) for 24 h. Adipocytes were also incubated for 24 h with EPA and DHA plus bisphenol-A-diglycidyl ether (BADGE), a PPARγ antagonist. Both EPA and DHA increased (p < 0.05) secreted adiponectin concentration compared with the control (44% and 102%, respectively), but did not affect cellular adiponectin protein content. Incubation with BADGE and DHA inhibited increases in secreted adiponectin protein, suggesting that DHA may act through a PPARγ-dependent mechanism. However, BADGE had no effect on EPA-induced increases in secreted adiponectin protein. Only DHA enhanced (p < 0.05) PPARγ and adiponectin mRNA expression compared wtih the control. Our results demonstrate that DHA increases cellular adiponectin mRNA and secreted adiponectin protein in 3T3-L1 adipocytes, possibly by a mechanism involving PPARγ. Moreover, DHA increased adiponectin concentration to a greater extent (40% more, p < 0.05) compared with EPA, emphasizing the need to consider the independent actions of EPA and DHA in adipocytes.

Generation of epidermal growth factor-expressing Lactococcus lactis and its enhancement on intestinal development and growth of early-weaned mice.

BACKGROUND: Epidermal growth factor (EGF) plays an important role in intestinal proliferation and differentiation. Previous studies by others have shown that administration of EGF into the ileum lumen enhances intestinal development. OBJECTIVE: The objective was to examine the feasibility of expressing and delivering EGF via Lactococcus lactis to early-weaned mice to enhance intestinal development at this critical transition stage. DESIGN: EGF-expressing L. lactis (EGF-LL) was generated with a recombinant approach. Early-weaned mice were orally gavaged with the recombinant bacteria. Body weight, mean villous height, and crypt depth in the intestine were measured to examine the influence of EGF-LL on the intestinal development of early-weaned mice in vivo. RESULTS: Populations of EGF-LL were shown to survive throughout the intestinal tract, and the recombinant EGF protein was also detected in intestinal contents. Weight gain was significantly greater in mice that received EGF-LL than in control mice fed phosphate-buffered saline or L. lactis transformed with the empty vector backbone but was comparable with that of the positive control mice that received recombinant human EGF. EGF-LL increased mean villous height and crypt depth in the intestine. Immunohistochemistry also confirmed that enterocyte proliferation was enhanced in mice that received EGF-LL, as evidenced by the greater number of cells stained with proliferative cell nuclear antigen in the intestine. CONCLUSIONS: This study showed that EGF-LL had beneficial effects on the intestinal growth of newly weaned mice. The combination of growth factor delivery and a probiotic approach may offer possibilities for formulating dietary supplements for children during their weaning transition stage.

Guar gum consumption increases hepatic nuclear SREBP2 and LDL receptor expression in pigs fed an atherogenic diet.

To gain insight into the regulation of hepatic sterol-responsive genes that are thought to mediate the hypocholesterolemic effects of guar gum (GG) consumption, the mRNA and protein expression of sterol regulatory element binding protein 2 (SREBP2), LDL receptor (LDLr), and scavenger receptor class B, type 1 (SR-B1) were examined in pigs consuming an atherogenic control diet or the control diet supplemented with 10% GG. Compared with the control group, GG consumption reduced (P < 0.05) plasma total cholesterol and LDL cholesterol concentrations by 27 and 37%, respectively. Furthermore, hepatic free cholesterol concentration was lower (P < 0.05) in the GG-fed pigs in comparison with the control group. GG consumption increased hepatic LDLr mRNA (1.5-fold of the control, P = 0.09) and protein (2-fold of the control, P < 0.05) expression in comparison with the control group. However, GG consumption reduced hepatic SR-B1 mRNA to 36% of the control (P < 0.05) expression but did not affect (P = 0.19) SR-B1 protein abundance in comparison with the control group. Although SREBP2 mRNA expression was similar (P = 0.89) in the 2 groups, GG consumption increased (P < 0.05) the expression of the cytoplasmic precursor (3-fold of the control) and nuclear active forms (1.5-fold of the control) of SREBP2. We conclude that the hypocholesterolemic effects of GG consumption are related to a reduction in hepatic free cholesterol concentration and associated increases in nuclear active SREBP2 expression and hepatic LDLr abundance.

Impaired trafficking of choline transporter-like protein-1 at plasma membrane and inhibition of choline transport in THP-1 monocyte-derived macrophages.

The present study investigates choline transport processes and regulation of choline transporter-like protein-1 (CTL1) in human THP-1 monocytic cells and phorbol myristate 13-acetate (PMA)-differentiated macrophages. Choline uptake is saturable and therefore protein-mediated in both cell types, but its transport characteristics change soon after treatments with PMA. The maximal rate of choline uptake intrinsic to monocytic cells is greatly diminished in differentiated macrophages as demonstrated by alterations in V(max) values from 1,973 +/- 118 to 380 +/- 18 nmol x mg(-1) x min(-1), when the binding affinity did not change significantly (K(m) values 56 +/- 8 and 53 +/- 6 microM, respectively). Treatments with hemicholinim-3 effectively inhibit most of the choline uptake, establishing that a choline-specific transport protein rather than a general transporter is responsible for the observed kinetic parameters. mRNA screening for the expression of various transporters reveals that CTL1 is the most plausible candidate that possesses the described kinetic and inhibitory properties. Fluorescence-activated cell sorting analyses at various times after PMA treatments further demonstrate that the disappearance of CTL1 protein from the cell surface follows the same trend as the reduction in choline uptake. Importantly, the loss of functional CTL1 from the cell surface occurs without significant changes in total CTL1 protein or its mRNA level indicating that an impaired CTL1 trafficking is the key contributing factor to the reduced choline uptake, subsequent to the PMA-induced THP-1 differentiation to macrophages.

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis).

In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles--plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine.

14-3-3 binds to and mediates phosphorylation of microtubule-associated tau protein by Ser9-phosphorylated glycogen synthase kinase 3beta in the brain.

In mammalian brain, tau, glycogen synthase kinase 3beta (GSK3beta), and 14-3-3, a phosphoserine-binding protein, are parts of a multiprotein tau phosphorylation complex. Within the complex, 14-3-3 simultaneously binds to tau and GSK3beta (Agarwal-Mawal, A., Qureshi, H. Y., Cafferty, P. W., Yuan, Z., Han, D., Lin, R., and Paudel, H. K. (2003) J. Biol. Chem. 278, 12722-12728). The molecular mechanism by which 14-3-3 connects GSK3beta to tau within the complex is not clear. In this study, we find that GSK3beta within the tau phosphorylation complex is phosphorylated on Ser(9). From extracts of rat brain and rat primary cultured neurons, Ser(9)-phosphorylated GSK3beta precipitates with glutathione-agarose beads coated with glutathione S-transferase-14-3-3. Similarly, from rat brain extract, Ser(9)-phosphorylated GSK3beta co-immunoprecipitates with tau. In vitro, 14-3-3 binds to GSK3beta only when the kinase is phosphorylated on Ser(9). In transfected HEK-293 cells, 14-3-3 binds to Ser(9)-phosphorylated GSK3beta and does not bind to GSK3beta (S9A). Tau, on the other hand, binds to both GSK3beta (WT) and GSK3beta (S9A). Moreover, 14-3-3 enhances the binding of tau with Ser(9)-phosphorylated GSK3beta by approximately 3-fold but not with GSK3beta (S9A). Similarly, 14-3-3 stimulates phosphorylation of tau by Ser(9)-phosphorylated GSK3beta but not by GSK3beta (S9A). In transfected HEK-293 cells, Ser(9) phosphorylation suppresses GSK3beta-catalyzed tau phosphorylation in the absence of 14-3-3. In the presence of 14-3-3, however, Ser(9)-phosphorylated GSK3beta remains active and phosphorylates tau. Our data indicate that within the tau phosphorylation complex, 14-3-3 connects Ser(9)-phosphorylated GSK3beta to tau and Ser(9)-phosphorylated GSK3beta phosphorylates tau.

14-3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubule-associated tau phosphorylation complex.

In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3beta and tau are parts of an approximately 400-500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 zeta co-elutes with the tau phosphorylation complex components tau and GSK3 beta. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3 zeta, GSK3 beta, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3 beta, 14-3-3 zeta co-precipitates with GSK3 beta, indicating that GSK3 beta binds to 14-3-3 zeta. From HEK-293 cells transfected with tau, GSK3 beta, and 14-3-3 zeta in different combinations, tau co-immunoprecipitates with GSK3 beta only in the presence of 14-3-3 zeta. In vitro, approximately 10-fold more tau binds to GSK3 beta in the presence of than in the absence of 14-3-3 zeta. In transfected HEK-293 cells, 14-3-3 zeta stimulates GSK3 beta-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 zeta dimer simultaneously binds and bridges tau and GSK3 beta and stimulates GSK3 beta-catalyzed tau phosphorylation.