TauT Activity Research Articles

Impact of the histone deacetylase inhibitor trichostatin A on active uptake, volume-sensitive release of taurine, and cell fate in human ovarian cancer cells.

The histone deacetylase inhibitor trichostatin A (TSA) reduces cell viability in cisplatin-sensitive (A2780WT) and cisplatin-resistant (A2780RES) human ovarian cancer cells due to progression of apoptosis (increased caspase-9 activity), autophagy (increased LC3-II expression), and cell cycle arrest (increased p21 expression). The TSA-mediated effect on p21 and caspase-9 is mainly p53 independent. Cisplatin increases DNA-damage (histone H2AX phosphorylation) in A2780WT cells, whereas cisplatin, due to reduced uptake [inductively coupled-plasma-mass spectrometry (Pt) analysis], has no DNA-damaging effect in A2780RES cells. TSA has no effect on cisplatin accumulation or cisplatin-induced DNA-damage in A2780WT/A2780RES cells. Tracer technique indicates that TSA inhibits the volume-sensitive organic anion channel (VSOAC) in A2780WT/A2780RES cells and that the activity is restored by exogenous H2O2. As TSA reduces NOX4 mRNA accumulation and concomitantly increases catalase mRNA/protein accumulation, we suggest that TSA increases the antioxidative defense in A2780 cells. Inhibition of the kinase mTOR (rapamycin, palomid, siRNA), which is normally associated with cell growth, reduces VSOAC activity synergistically to TSA. However, as TSA increases mTOR activity (phosphorylation of 4EBP1, S6 kinase, S6, ULK1, SGK1), the effect of TSA on VSOAC activity does not reflect the shift in mTOR signaling. Upregulation of the protein expression and activity of the taurine transporter (TauT) is a phenotypic characteristic of A2780RES cells. However, TSA reduces TauT protein expression in A2780RES cells and activity to values seen in A2780WT cells. It is suggested that therapeutic benefits of TSA in A2780 do not imply facilitation of cisplatin uptake but more likely a synergistic activation of apoptosis/autophagy and reduced TauT activity.

Effects of taurine depletion on human placental syncytiotrophoblast renewal and susceptibility to oxidative stress.

Taurine is conditionally essential in human pregnancy as fetal tissues do not express the biosynthetic enzyme. Taurine demand by both fetus and placenta must be met by sufficient uptake from maternal blood into syncytiotrophoblast (STB), the transport epithelium of the placenta, via the taurine transporter (TauT). STB TauT activity is reduced in the pregnancy complication pre-eclampsia (PE). This condition is associated with fetal growth restriction (FGR) and placental pathology comprising abnormal STB renewal, mitochondrial dysfunction, and elevated oxidative stress. In non-placental cells intracellular taurine is cytoprotective and regulates proliferation, differentiation, and apoptosis; key events in STB renewal. Here we test the hypothesis that STB taurine deficiency impairs renewal and increases susceptibility to oxidative stress by compromising mitochondrial function.

Maternal obesity is associated with a reduction in placental taurine transporter activity.

Background/Objectives:Maternal obesity increases the risk of poor pregnancy outcome including stillbirth, pre-eclampsia, fetal growth restriction and fetal overgrowth. These pregnancy complications are associated with dysfunctional syncytiotrophoblast, the transporting epithelium of the human placenta. Taurine, a β-amino acid with antioxidant and cytoprotective properties, has a role in syncytiotrophoblast development and function and is required for fetal growth and organ development. Taurine is conditionally essential in pregnancy and fetal tissues depend on uptake of taurine from maternal blood. We tested the hypothesis that taurine uptake into placental syncytiotrophoblast by the taurine transporter protein (TauT) is lower in obese women (body mass index (BMI)⩾30 kg m−2) than in women of ideal weight (BMI 18.5–24.9 kg m−2) and explored potential regulatory factors.Subjects/Methods:Placentas were collected from term (37–42-week gestation), uncomplicated, singleton pregnancies from women with BMI 19–49 kg m−2. TauT activity was measured as the Na+-dependent uptake of 3H-taurine into placental villous fragments. TauT expression in membrane-enriched placental samples was investigated by western blot. In vitro studies using placental villous explants examined whether leptin or IL-6, adipokines/cytokines that are elevated in maternal obesity, regulates TauT activity.Results:Placental TauT activity was significantly lower in obese women (BMI⩾30) than women of ideal weight (P<0.03) and inversely related to maternal BMI (19–49 kg m−2; P<0.05; n=61). There was no difference in TauT expression between placentas of ideal weight and obese class III (BMI⩾40) subjects. Long-term exposure (48 h) of placental villous explants to leptin or IL-6 did not affect TauT activity.Conclusions:Placental TauT activity at term is negatively related to maternal BMI. We propose that the reduction in placental TauT activity in maternal obesity could lower syncytiotrophoblast taurine concentration, compromise placental development and function, and reduce the driving force for taurine efflux to the fetus, thereby increasing the risk of poor pregnancy outcome.

MTOR ensures increased release and reduced uptake of the organic osmolyte taurine under hypoosmotic conditions in mouse fibroblasts

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that modulates translation in response to growth factors and alterations in nutrient availability following hypoxia and DNA damage. Here we demonstrate that mTOR activity in Ehrlich Lettré ascites (ELA) cells is transiently increased within minutes following osmotic cell swelling and that inhibition of phosphatidylinositol-3-phosphatase (PTEN) counteracts the upstream phosphatidylinositol kinase and potentiates mTOR activity. PTEN inhibition concomitantly potentiates swelling-induced taurine release via the volume-sensitive transporter for organic osmolytes and anion channels (VSOAC) and enhances swelling-induced inhibition of taurine uptake via the taurine-specific transporter (TauT). Chronic osmotic stress, i.e., exposure to hypotonic or hypertonic media for 24 h, reduces and increases mTOR activity in ELA cells, respectively. Using rapamycin, we demonstrate that mTOR inhibition is accompanied by reduction in TauT activity and increase in VSOAC activity in cells expressing high (NIH3T3 fibroblasts) or low (ELA) amounts of mTOR protein. The effect of mTOR inhibition on TauT activity reflects reduced TauT mRNA, TauT protein abundance, and an overall reduction in protein synthesis, whereas the effect on VSOAC is mimicked by catalase inhibition and correlates with reduced catalase mRNA abundance. Hence, mTOR activity favors loss of taurine following hypoosmotic cell swelling, i.e., release via VSOAC and uptake via TauT during acute hypotonic exposure is potentiated and reduced, respectively, by phosphorylation involving mTOR and/or the kinases upstream to mTOR. Decrease in TauT activity during chronic hypotonic exposure, on the other hand, involves reduction in expression/activity of TauT and enzymes in antioxidative defense.

Reduced placental taurine transporter (TauT) activity in pregnancies complicated by pre-eclampsia and maternal obesity.

Taurine is an important nutrient in intrauterine life, being required for fetal organ development and cellular renewal of syncytiotrophoblast (STB), the nutrient transport epithelium of the placenta. As taurine is conditionally essential in human pregnancy, the fetal and placental demand for taurine is met by uptake from maternal blood into STB through the activity of TauT. Pre-eclampsia (PE) and maternal obesity are serious complications of pregnancy, associated with fetal growth restriction (FGR) and abnormal renewal of STB, and maternal obesity is a major risk factor for PE. Here we test the hypothesis that STB TauT activity is reduced in maternal obesity and PE compared to normal pregnancy.STB TauT activity, measured in fragments of placental tissue, was negatively related to maternal BMI over the range 18-46 kg/m(2) in both the first trimester (7-12 weeks gestation) and at term (p < 0.01; linear regression). Neither TauT activity nor expression in the first trimester differed to normal pregnancy at term. STB TauT activity was significantly lower in PE than normal pregnancy (p < 0.01). Neuropeptide Y (NPY), a protein kinase C (PKC) activator which is elevated in PE and obesity, reduced STB TauT activity by 20% (50 pM-50 nM: 2 h) (p < 0.03). Activation of PKC by phorbol 12-myristate-13-acetate (1 μM) reduced TauT activity by 18% (p < 0.05). As TauT activity is inhibited by phosphorylation, we propose that NPY activates PKC in the STB which phosphorylates TauT in PE and maternal obesity.Reduced TauT activity could contribute to dysregulated renewal of STB and FGR that are common to PE and maternal obesity.

Downregulation of the Taurine Transporter TauT During Hypo-Osmotic Stress in NIH3T3 Mouse Fibroblasts

The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na+ concentration constant. TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions. In contrast, TonEBP activity and TauT transcription are significantly increased following hyperosmotic exposure. Swelling-induced ROS production in NIH3T3 fibroblasts is generated by NOX4 and by increasing total ROS, by either exogenous application of H2O2 or overexpressing NOX4, we demonstrate that TonEBP activity and taurine influx are regulated negatively by ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low-sodium/hypo-osmotic conditions by direct modulation of TauT.Electronic supplementary materialThe online version of this article (doi:10.1007/s00232-012-9416-8) contains supplementary material, which is available to authorized users.

Pinpointing Differences in Cisplatin-induced Apoptosis in Adherent and Non-adherent Cancer Cells

Platinum compounds are used in the treatment of cancer. We demonstrate that cisplatin-induced (10 µM) apoptosis (caspase-3 activity) is pronounced within 18 hours in non-adherent Ehrlich ascites tumour cells (EATC), whereas there is no increase in caspase-3 activity in the adherent Ehrlich LettrÉ ascites tumour cells (ELA). Loss of KCl and cell shrinkage are hallmarks in apoptosis and has been shown in EATC. However, we find no reduction in cell volume and only a minor loss of K⁺ which is accompanied by net uptake of Na⁺ following 18 hours cisplatin exposure in ELA. Glutathione and taurine have previously been demonstrated to protect cells from apoptosis. We find, however, that increase or decrease in the cellular content of glutathione and taurine has no effect on cisplatin-induced cell death in EATC and ELA. Nevertheless, knock-down of the taurine transporter TauT leads to a significant increase in apoptosis in ELA following cisplatin exposure. We find that cytosolic accumulation of cisplatin is similar in EATC and ELA. However, the nuclear accumulation and DNA-binding of cisplatin is significant lower in ELA compared to EATC. We suggest three putative reasons for the observed cisplatin insensitivity in the adherent tumor cells (ELA) compared to the non-adherent tumor cells (EATC): less nuclear cisplatin accumulation, increased TauT activity, and decreased anion and water loss.

Regulation of amino acid transporters by glucose and growth factors in cultured primary human trophoblast cells is mediated by mTOR signaling

Inhibition of mammalian target of rapamycin (mTOR) signaling in cultured human primary trophoblast cells reduces the activity of key placental amino acid transporters. However, the upstream regulators of placental mTOR are unknown. We hypothesized that glucose, insulin, and IGF-I regulate placental amino acid transporters by inducing changes in mTOR signaling. Primary human trophoblast cells were cultured for 24 h with media containing various glucose concentrations, insulin, or IGF-I, with or without the mTOR inhibitor rapamycin, and, subsequently, the activity of system A, system L, and taurine (TAUT) transporters was measured. Glucose deprivation (0.5 mM glucose) did not significantly affect Thr172-AMP-activated protein kinase phosphorylation or REDD1 expression but decreased S6 kinase 1 phosphorylation at Thr389. The activity of system L decreased in a dose-dependent manner in response to decreasing glucose concentrations. This effect was abolished in the presence of rapamycin. Glucose deprivation had two opposing effects on system A activity: 1) an "adaptive" upregulation mediated by an mTOR-independent mechanism and 2) downregulation by an mTOR-dependent mechanism. TAUT activity was increased after incubating cells with glucose-deprived media, and this effect was largely independent of mTOR signaling. Insulin and IGF-I increased system A activity and insulin stimulated system L activity, effects that were abolished by rapamycin. We conclude that the mTOR pathway represents an important intracellular regulatory link between nutrient and growth factor concentrations and amino acid transport in the human placenta.

Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts

The cellular level of the organic osmolyte taurine is a balance between active uptake and passive leak via a volume sensitive pathway. Here, we demonstrate that NIH3T3 mouse fibroblasts express a saturable, high affinity taurine transporter (TauT, Km = 18 microm), and that taurine uptake via TauT is a Na+- and Cl(-)-dependent process with an apparent 2.5 : 1 : 1 Na+/Cl-/taurine stoichiometry. Transport activity is reduced following acute administration of H2O2 or activators of protein kinases A or C. TauT transport activity, expression and nuclear localization are significantly increased upon serum starvation (24 h), exposure to tumour necrosis factor alpha (TNFalpha; 16 h), or hyperosmotic medium (24 h); conditions that are also associated with increased localization of TauT to the cytosolic network of microtubules. Conversely, transport activity, expression and nuclear localization of TauT are reduced in a reversible manner following long-term exposure (24 h) to high extracellular taurine concentration. In contrast to active taurine uptake, swelling-induced taurine release is significantly reduced following preincubation with TNFalpha (16 h) but unaffected by high extracellular taurine concentration (24 h). Thus, in NIH3T3 cells, (a) active taurine uptake reflects TauT expression; (b) TauT activity is modulated by multiple stimuli, both acutely, and at the level of TauT expression; (c) the subcellular localization of TauT is regulated; and (d) volume-sensitive taurine release is not mediated by TauT.

Corrigendum

CORRIGENDACorrigendumPublished Online:01 Dec 2004https://doi.org/10.1152/ajpregu.00666.2004Original articleMoreSectionsPDF (34 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations Volume 56, October 2004 Pages R886–R893: Roos S, Powell TL, and Jansson T. “Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation” ( http://ajpregu.physiology.org/cgi/content/full/287/4/R886). Units of measure appeared incorrectly in two sentences. In results, under the subheading TAUT activity, the correct sentence should read “In the third series of experiments, fragments were incubated with PMA (617 ng/ml), CHT (1,920 ng/ml), leptin (500 ng/ml), GH (500 ng/ml), and IGF-I (250 ng/ml).” In the legend to Fig. 4C, the correct sentence should read “Final concentrations were as follows: 617 ng/ml PMA, 1,920 ng/ml CHT, 250 ng/ml IGF-I, 500 ng/ml leptin, and 500 ng/ml growth hormone (GH).”This article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Related ArticlesHuman placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation 01 Oct 2004American Journal of Physiology-Regulatory, Integrative and Comparative Physiology More from this issue > Volume 287Issue 6December 2004Pages R1517-R1517 Copyright & PermissionsCopyright © 2004 the American Physiological Societyhttps://doi.org/10.1152/ajpregu.00666.2004History Published online 1 December 2004 Published in print 1 December 2004 Metrics

Osmoregulation of taurine transporter function and expression in retinal pigment epithelial, ganglion, and müller cells.

To determine whether taurine transporter (TauT) activity and expression are regulated by hyperosmolarity in RPE, ganglion, and Müller cells. Uptake of taurine was measured in ARPE-19 cells cultured in DMEM-F12 medium without or with the addition of 50 mM NaCl or 100 mM mannitol. The kinetics of the transport were analyzed. RT-PCR and Northern and Western blot analyses were used to assess TauT mRNA and protein levels. The influence of hyperosmolarity on the uptake of taurine, myo-inositol, and gamma-aminobutyric acid GABA was studied in RPE, RGC-5, and rMC1 cells. TauT activity was abundant in RPE and was stimulated (3.5-fold) when the cells were exposed to hyperosmolar conditions (DMEM-F12 culture medium plus 50 mM NaCl or 100 mM mannitol). Peak stimulation of taurine uptake occurred after 17 hours of exposure to hyperosmolar medium. Kinetic analysis revealed that the hyperosmolarity-induced stimulation was associated with an increase in V(max) of TauT with no change in K(m). TauT mRNA and protein levels increased in RPE cells exposed to hyperosmolar conditions. Hyperosmolarity also stimulated the uptake of myo-inositol ( approximately 15-fold); GABA uptake was influenced less markedly. Immunofluorescence and functional studies showed that TauT is present in cultured RGC-5 and rMC1 cells. TauT activity was robust in these cells in normal osmolar conditions and increased by approximately twofold in hyperosmolar conditions. These studies provide the first evidence that hyperosmolarity regulates TauT activity and expression in RPE and that TauT is present in ganglion and Müller cells and is regulated by hypertonicity. The data are relevant to diseases such as diabetes, macular degeneration, and neurodegeneration, in which retinal cell volumes may fluctuate dramatically.

Taurine biosynthetic enzymes and taurine transporter: molecular identification and regulations.

Many biological effects of taurine rely upon its cellular concentration, which is primarily controlled by taurine biosynthetic enzymes cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD) and taurine transporter (TauT). The cloning of CDO, CSD and TauT in various species provided first-hand information on these proteins, as well as molecular tools to investigate their regulations. CDO upregulation in hepatocytes in response to high sulfur amino acids appears clearly as the most spectacular among the regulations of the biosynthetic enzymes. Downregulation of TauT activity by activation of PKC appears particularly well documented. A unique serine residue could be identified as a phosphorylation site that leads to an inactive form of TauT. The previously revealed downregulation of TauT expression by taurine and hypertonicity-induced upregulation of TauT expression were shown to result from a modified transcription rate of TauT gene, but the precise molecular mechanisms are not yet formally established. Other regulations of taurine transporter expression were more recently reported, which involve glucose, tumor suppressor protein p53, tumor necrosis factor-alpha, and nitric oxide. This review reports the experimental models and data that support these various regulations but also points out the aspects that remain poorly understood or unknown concerning their molecular basis and physiological significance.

Regulation of the cellular content of the organic osmolyte taurine in mammalian cells.

Change in the intracellular concentration of osmolytes or the extracellular tonicity results in a rapid transmembrane water flow in mammalian cells until intracellular and extracellular tonicities are equilibrated. Most cells respond to the osmotic cell swelling by activation of volume-sensitive flux pathways for ions and organic osmolytes to restore their original cell volume. Taurine is an important organic osmolyte in mammalian cells, and taurine release via a volume-sensitive taurine efflux pathway is increased and the active taurine uptake via the taurine specific taurine transporter TauT decreased following osmotic cell swelling. The cellular signaling cascades, the second messengers profile, the activation of specific transporters, and the subsequent time course for the readjustment of the cellular content of osmolytes and volume vary from cell type to cell type. Using Ehrlich ascites tumor cells, NIH3T3 mouse fibroblasts and HeLa cells as biological systems, it is revealed that phospholipase A2-mediated mobilization of arachidonic acid from phospholipids and subsequent oxidation of the fatty acid via lipoxygenase systems to potent eicosanoids are essential elements in the signaling cascade that is activated by cell swelling and leads to release of osmolytes. The cellular signaling cascade and the activity of the volume-sensitive taurine efflux pathway are modulated by elements of the cytoskeleton, protein tyrosine kinases/phosphatases, GTP-binding proteins, Ca2+/calmodulin, and reactive oxygen species and nucleotides. Serine/threonine phosphorylation of the active taurine uptake system TauT or a putative regulator, as well as change in the membrane potential, are important elements in the regulation of TauT activity. A model describing the cellular sequence, which is activated by cell swelling and leads to activation of the volume-sensitive efflux pathway, is presented at the end of the review.