Functional differences in steroid sulfate uptake of organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1) in human placenta

Organic anion transporting polypeptide (OATP) 1B3–1B7 (LST-3TM12) is a member of the OATP1B [solute carrier organic anion transporter ( SLCO) 1B] family. This transporter is not only functional but also expressed in the membrane of the smooth endoplasmic reticulum of hepatocytes and enterocytes. OATP1B3–1B7 is a splice variant of SLCO1B3 in which the initial part is encoded by SLCO1B3, whereas the rest of the mRNA originates from the gene locus of SLCO1B7. In this study, we not only showed that SLCO1B3 and the mRNA encoding for OATP1B3–1B7 share the 5′ untranslated region but also that silencing of an initial SLCO1B3 exon lowered the amount of SLCO1B3 and of SLCO1B7 mRNA in Huh-7 cells. To validate the assumption that both transcripts are regulated by the same promoter we tested the influence of the bile acid sensor farnesoid X receptor (FXR) on their transcription. Treatment of Huh-7 and HepaRG cells with activators of this known regulator of OATP1B3 not only increased SLCO1B3 but also OATP1B3–1B7 mRNA transcription. Applying a heterologous expression system, we showed that several bile acids interact with OATP1B3–1B7 and that taurocholic acid and lithocholic acid are OATP1B3–1B7 substrates. As OATP1B3–1B7 is located in the smooth endoplasmic reticulum, it may grant access to metabolizing enzymes. In accordance are our findings showing that the OATP1B3–1B7 inhibitor bromsulphthalein significantly reduced uptake of bile acids into human liver microsomes. Taken together, we report that OATP1B3–1B7 transcription can be modulated with FXR agonists and antagonists and that OATP1B3–1B7 transports bile acids. NEW & NOTEWORTHY Our study on the transcriptional regulation of the novel organic anion transporting polypeptide (OATP) 1B3–1B7 concludes that the promoter of solute carrier organic anion transporter ( SLCO) 1B3 governs SLCO1B3–1B7 transcription. Moreover, the transcription of OATP1B3–1B7 can be modulated by farnesoid X receptor (FXR) agonists and antagonists. FXR is a major regulator in bile acid homeostasis that links OATP1B3–1B7 to this physiological function. Findings in transport studies with OATP1B3–1B7 suggest that this transporter interacts with the herein tested bile acids.

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Estradiol 17 beta-D-glucuronide is a high-affinity substrate for oatp organic anion transporter

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.2.f326 ◽

1996 ◽

Vol 270 (2) ◽

pp. F326-F331 ◽

Cited By ~ 14

Author(s):

N. Kanai ◽

R. Lu ◽

Y. Bao ◽

A. W. Wolkoff ◽

M. Vore ◽

...

Keyword(s):

Signal To Noise Ratio ◽

Organic Anion ◽

Strong Acid ◽

Organic Anion Transporter ◽

Cell Monolayers ◽

Organic Anion Transporting Polypeptide ◽

Anion Transporter ◽

Control Plasmid ◽

Steroid Conjugates

Although substantial evidence indicates that estradiol-17 beta (E2) is conjugated to the glucuronide in the kidney and then excreted by a direct tubular secretory route and that the liver transports E2 glucuronides via carrier-mediated mechanisms, the transporters involved in these processes have not been identified. The so-called "organic anion-transporting polypeptide" (i.e., oatp) has a number of known substrates, including bromosulfophthalein (BSP) and taurocholic acid (TCA) (E. Jacquemin, B. Hagenbuch, B. Stieger, A. W. Wolkoff, and P. J. Meier. Proc. Natl. Acad. Sci. USA 91: 133-137, 1994). In a companion study, we determined that steroid hormones represent a class of hormones that interact strongly with oatp when the latter is transiently expressed in vitro. Here, we studied more extensively steroids and steroid anion conjugates as candidate oatp substrates. In HeLa cell monolayers transfected with a full-length oatp cDNA, [3H]estradiol 17 beta-D-glucuronide ([3H]E2-17G) was transported with a signal-to-noise ratio of 15:1 over that of monolayers transfected with a control plasmid. The affinity of oatp for [3H]E2-17G was significantly higher than that for TCA (K(m) of 3 microM vs. 27 microM, respectively). In contrast to E2-17G, unconjugated estradiol (E2) was not significantly transported by oatp. Several unconjugated steroids and anionic steroid conjugates were tested for their ability to compete with tracer E2-17G for oatp-mediated transport. Conjugation at the 17 or 3 position with the anion of a strong acid (sulfate) resulted in a greater degree of inhibition of tracer E2-17G transport than did conjugation at the 17 or 3 position with an uncharged group (acetate), suggesting that the strength of the negative charge at these positions is an important determinant of the affinity of a given steroid conjugate for oatp. We conclude that the preferred substrates for oatp are steroids with a strong 17- or 3-position anionic group. Since steroid sulfotransferases and glucuronosyltransferases are expressed in the proximal tubule, as is oatp, the transporter may serve as an apical exit pathway for steroids following their conjugation within the tubule cell.

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Role of OAT4 in Uptake of Estriol Precursor 16α-Hydroxydehydroepiandrosterone Sulfate Into Human Placental Syncytiotrophoblasts From Fetus

Endocrinology ◽

10.1210/en.2015-1130 ◽

2015 ◽

Vol 156 (7) ◽

pp. 2704-2712 ◽

Cited By ~ 17

Author(s):

Masatoshi Tomi ◽

Hiromi Eguchi ◽

Mayuko Ozaki ◽

Tomohiro Tawara ◽

Sachika Nishimura ◽

...

Keyword(s):

Fetal Liver ◽

Organic Anion ◽

Dehydroepiandrosterone Sulfate ◽

Cellular Level ◽

Organic Anion Transporter ◽

Organic Anion Transporting Polypeptide ◽

Anion Transporter ◽

Basal Plasma ◽

Good Agreement

Estriol biosynthesis in human placenta requires the 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 [3H]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 [3H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [3H]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 hours but was inhibited in the presence of 50 μM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.

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Characterization and identification of steroid sulfate transporters of human placenta

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00257.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. E390-E398 ◽

Cited By ~ 116

Author(s):

Bernhard Ugele ◽

Marie V. St-Pierre ◽

Monika Pihusch ◽

Andrew Bahn ◽

Peer Hantschmann

Keyword(s):

Organic Anion ◽

Diffusion Constant ◽

Physiological Role ◽

Placental Tissue ◽

Organic Anion Transporter ◽

Uptake Mechanism ◽

Estrone Sulfate ◽

Anion Transporter ◽

S Uptake ◽

Steroid Sulfate

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16α-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-35S uptake by MT revealed a saturable uptake mechanism ( K m = 26 μM, V max = 428 pmol · mg protein−1 · min−1), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 μl · mg protein−1 · min−1). Uptake of [3H]DHEA-S by MT was Na+dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [35S]BSP, [3H]estrone-sulfate, but not 3H-labeled ouabain, estradiol-17β-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na+-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.

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Impact of Organic Anion Transporter 3 (OAT3) on Bendamustine Uptake of Lymphoma Cells.

Blood ◽

10.1182/blood.v110.11.4184.4184 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4184-4184

Author(s):

Yohannes Hagos ◽

Gerald G. Wulf ◽

Vladimir Shnitsar ◽

Philip Hundertmark ◽

Shvangi Gupta ◽

...

Keyword(s):

Cell Lines ◽

Organic Anion ◽

Inhibitory Effect ◽

Lymphoma Cell ◽

Organic Anion Transporter ◽

Hek293 Cells ◽

Sulfate Uptake ◽

Lymphoma Cells ◽

Estrone Sulfate ◽

Anion Transporter

Abstract One main problem of tumor therapy is the resistance of malignant cells to cytostatics due to high expression of efflux transporters. Whereas the role of these efflux transporters for tumor cell resistance is well established, little is known about uptake transporters, which may increase the sensibility of tumor cells for cytostatics. In the present study we addressed the interaction of cytostatics established for the treatment of lymphoma, namely melphalan, chlorambucil or bendamustine with human Organic Anion Transporter (OATs), which belong to the Solute carrier (SLC) gene family. We selected these cytostatics, because they show structural similarity to p-aminohyppurate (PAH), the model substrate of OATs. OATs are mainly expressed in the kidney, where they are responsible for the excretion of endogenous and exogenous organic anions like urate or various drugs e.g. diuretics. Initially, we examined the cis-inhibitory effect of melphalan, chlorambucil and bendamustine on OAT1-mediated [3H]PAH uptake as well as OAT3- and OAT4- mediated [3H]estrone sulfate uptake in HEK293 cells, which were stably transfected with these transporters. Melphalan did not show any significant inhibitory effect on all tested OATs. 100 μM chlorambucil reduced OAT1-, OAT3- and OAT4-mediated uptake of PAH or estrone sulfate down to 14.6 ± 0.17%, 16.3 ± 4.0% and 66.0 ± 1.4%, respectively. 100 μM bendamustine inhibited only OAT3-mediated estrone sulfate uptake up to 91.9 ± 0.5% compared to control cells. OAT1- or OAT4- facilitated transport of PAH and estrone sulfate remained unchanged by bendamustine, suggesting that bendamustine interacts exclusively with OAT3. To determine the affinity of OAT3 for bendamustine and chlorambucil, we performed concentration dependent inhibition of OAT3-mediated estrone sulfate uptake and calculated the Ki values for both cytostatics. Dixon-Plot evaluation confirmed a competitive inhibition of OAT3 by bendamustine as well as chlorambucil. The results demonstrated higher affinity of OAT3 for bendamustine with a Ki value of 2.7 μM than for chlorambucil, showing a Ki value of 38.2 μM. To elucidate the expression of OATs in lymphoma cell lines, we performed RT-PCR experiments. Our data demonstrate high expression of OAT3 in all cell lines compared to lymphocytes isolated from a normal person. No expression of OAT1 and OAT4 was observed any lymphoma cell lines. The expression of OAT3 in B-cell lymphoma cell lines Karpas, Raji, SudHL4 and T-cell lymphoma cell lines L428, Jurkat and Hut78 was quantified by real time PCR. The highest expression of OAT3 was observed in the order Jurkat>Hut78>SudHL4>L428>Raji>Karpas. The expression of OAT3 was confirmed by real time PCR in four patients with chronic lymphocytic leukamia. OAT3- dependent cytostatic effects of bendamustine was examined by [3H] thymidine incorporation. 30 min incubation of OAT3-expressing HEK293 cells with 10, 50 or 100 μM bendamustine significantly reduced the proliferation of transfected versus non-transfected cells. We conclude that the molecular background for the cytostatic efficiency of bendamustine in lymphoma cells is due to 1) the expression of OAT3 in lymphoma cells and 2) a the high affinity of OAT3 for bendamustine.

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