[Show abstract][Hide abstract] ABSTRACT: Estriol biosynthesis in human placenta requires uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT) 4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium, but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as DHEAS, estrone-3-sulfate, and bromosulfophthalein (BSP), but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [(3)H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells, as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hrs, but was inhibited in the presence of 50 μ M BSP. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in uptake of 16α-OH DHEAS for placental estriol synthesis.
[Show abstract][Hide abstract] ABSTRACT: Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez-/-) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez-/- mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study is to assess the role of the protein kinase A (PKA) in regulating uptake of dehydroepiandrosterone sulfate (DHEAS), an estrogen precursor, by syncytiotrophoblasts. Forskolin, a PKA activator, significantly increased [3H]DHEAS uptake and the mRNA expression levels of organic anion transporter (OAT) 4 and CYP19A1 in choriocarcinoma JEG-3 cells, while other steroid sulfate transporters present in the placenta showed no change in expression level. KT5720, a PKA inhibitor, attenuated these effects of forskolin. Accordingly, the PKA pathway appears to play an important role in estrogen synthesis by cooperatively regulating OAT4 and steroidogenic enzymes in syncytiotrophoblasts.
[Show abstract][Hide abstract] ABSTRACT: Endothelial progenitor cells (EPC) can differentiate into both endothelial cells and contractile smooth muscle cells (SMC). Previously we reported that TR-BME2 cells, a model for EPC, developed contractile SMC-like characteristics in culture medium deprived of endothelial cell growth factors (ECGF). The aim of the present study was to clarify the effect of one of these factors, basic fibroblast growth factor (bFGF) on differentiation of EPC. First it was confirmed that bFGF receptor (FGFR-1) mRNA is expressed in TR-BME2 cultured in both ECGF-rich and ECGF-deprived medium. When TR-BME2 cells were cultured in ECGF-deprived medium, they differentiated into contractile SMC. Expression of an undifferentiated state marker, CD133, and proliferation of TR-BME2 were both reduced by ECGF deprivation, but these changes were diminished in the presence of bFGF. mRNA expression of smooth muscle α-actin (SMA) and smooth muscle protein 22 (SM22), which are contractile SMC markers, was induced by deprivation of ECGF and the induction was suppressed by bFGF. In vascular endothelial cell growth factor (VEGF)-induced tube formation assay, TR-BME2 cells formed tube structures in the presence of bFGF, but not in its absence. Our results indicate that bFGF is essential for the maintenance of EPC phenotype, serving to suppress differentiation to contractile SMC.
[Show abstract][Hide abstract] ABSTRACT: Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [(14)C]betaine uptake by HEK293 cells transiently transfected with human or rat SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3mM and 4.6mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed that the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.
Preview · Article · Jan 2014 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: The purpose of this research was to examine whether the fatigue-relieving efficacy of a revitalizing medicinal product would be increased by counseling (advice to subjects regarding its expected efficacy), using both subjective and objective indicators. Twenty participants took the revitalizing medicinal product (drinkable product: 50 mL, once) on separate occasions, once with and once without counseling, with a 1-week interval. They completed a self-reported questionnaire (covering motivation, feeling of drowsiness, and general fatigue) and multi-dimensional fatigue inventory-20 (MFI-20) as subjective indicators, and blood flow in the inferior frontal cortex was evaluated by means of near-infrared spectroscopy (NIRS) as an objective indicator. The self-reported questionnaire showed that the revitalizing drink with counseling significantly improved general fatigue, as compared to the drink alone. MFI-20 showed no significant difference. Regional blood flow in the inferior frontal cortex was significantly increased by administration of the revitalizing drink with counseling, compared to without counseling (P < 0.05). Overall, the results indicated that counseling intervention increased the effectiveness of the revitalizing medicinal product.