Co-localization and interaction of human organic anion transporter 4 with caveolin-1 in primary cultured human placental trophoblasts

Abstract

The human organic anion transporter 4 (hOAT4) has been identified as the fourth isoform of OAT family. hOAT4 contributes to move several negatively charged organic compounds between cells and their extracellular milieu. The functional characteristics and regulatory mechanisms of hOAT4 remain to be elucidated. It is well known that caveolin plays a role in modulating proteins having some biological functions. To address this issue, we investigated the co-localization and interaction between hOAT4 and caveolin-1. hOAT4 and caveolin-1 (mRNA and protein expression) were observed in cultured human placental trophoblasts isolated from placenta. The confocal microscopy of immuno-cytochemistry using primary cultured human trophoblasts showed hOAT4 and caveolin-1 were co-localized at the plasma membrane of the cell. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions and immune-precipitates from the trophoblasts. When synthesized cRNA of hOAT4 along with scrambled- or antisense-oligodeoxynucleotide (ODN) of Xenopus caveolin-1 were co-injected to Xenopus oocytes, the [3H]estrone sulfate uptake was significantly decreased by the co-injection of antisense ODN but not by scrambled ODN. These findings suggest that hOAT4 and caveolin-1 share a cellular expression in the plasma membrane and caveolin-1 up-regulates the organic anionic compound uptake by hOAT4 under the normal physiological condition.

Figure 1

Predicted location of hOAT4 caveolin-binding motifs. The hydropathy plot model of hOAT4 is based upon topological analysis. Transmembrane regions are represented by shaded cylinders and extramembranal loops are black lines. Closed circles designate the approximate location of the hOAT4 caveolin-binding motifs.

Figure 2

Morphology and characterization of primarily cultured placental trophoblasts. (A) Light microscopy of confluently grown placental trophoblasts. (B) Messenger RNA expression of cytokeratin-7, hOAT4 and caveolin-1 in the cultured placental trophoblasts. Total RNA (500 ng) isolated from cultured trophoblasts was reverse transcribed and PCR was performed (35 cycles). PCR products were separated using 1% agarose gel and stained with ethidium bromide. (C) Isolated membrane protein (30 µg/lane) was separated by electrophoresis and presence of each cytokeratin-7, hOAT4 and caveolin-1 was detected with respective antibodies. Data was shown typical result from 3 independent experiments. a; cytokeratin-7, b; hOAT4, c; caveolin-1.

Figure 3

Confocal microscopy of cultured trophoblasts immuno-cytochemically stained with hOAT4 and caveolin-1 antibodies. Cells were double stained with fluorescein isothiocyanate- and Texas Red-conjugated secondary antibodies after an initial treatment with either hOAT4 or caveolin-1 antibodies separately and then examined under a confocal microscope at their respective wavelengths. For each experiment, at least 50 cells were examined and the presented images represent typical staining pattern for the majority of examined cells. A; hOAT4, B; caveolin-1, C; merged images of A and B. D and E, stained only with secondary antibodies without the prior treatment of either hOAT4 or caveolin-1 antibodies to show the images of non-specific bindings.

Figure 4

Western blot analysis using isolated membrane fraction and immunoprecipitates by antibodies in the cultured trophoblasts. (A) The cells were lysed using a teflon/glass homogenizer and a sonicator, and subjected to sucrose gradient centrifugation as described under Materials and Methods. Equal amounts of protein (10 µg) from each of twelve fractions (1-12) were separated by SDS-PAGE 15% acrylamide gel and transferred to membranes, which were blotted using anti-hOAT4 antibody (1:500) and anti-caveolin-1 (1:200). Obtained data from 2 separate experiments were consistent. (B) Cell lysates were immuno-precipitated initially with antibodies of hOAT4 or caveolin-1 and the respective immuno-precipitated (IP) proteins (15 µg) were loaded onto each lane of a 10% SDS-polyacrylamide gel. Cell lysate was immuno-precipitated initially with hOAT4 (left pannel) and was immuno-precipitated initially with caveolin-1 antibodies (right pannel). Obtained data from 2 separate experiments were consistent.

Figure 5

The effects of Xenopus laevis caveolin-1 scrambled or antisense oligodeoxynucleotide on hOAT4-mediated [³H] estrone sulfate uptake in Xenopus laevis oocyte expression system. (A) The expression of caveolin-1 in scrambled or antisense ODN injected oocytes. Membrane protein was extracted from 50 ODN injected oocytes and Western blot analysis was performed. (B) Defolliculated stage VI and V oocyte were injected with water, with hOAT4 cRNA (30 ng/oocyte), with hOAT4+scrambled ODN (3 pmol/oocyte), or with hOAT4 antisense ODN (10 pmol/oocyte). After 3 days, [³H] estrone sulfate (100 nM) uptakes were determined for 1 h. Each bar represents the mean ± standard error for three experiments. Each experiment contains 8-10 oocytes.