The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs

Abstract

Prostaglandins are involved in a wide variety of physiological and pathophysiological processes, but the mechanism of prostaglandin release from cells is not completely understood. Although poorly membrane permeable, prostaglandins are believed to exit cells by passive diffusion. We have investigated the interaction between prostaglandins and members of the ATP-binding cassette (ABC) transporter ABCC [multidrug resistance protein (MRP)] family of membrane export pumps. In inside-out membrane vesicles derived from insect cells or HEK293 cells, MRP4 catalyzed the time- and ATP-dependent uptake of prostaglandin E1 (PGE1) and PGE2. In contrast, MRP1, MRP2, MRP3, and MRP5 did not transport PGE1 or PGE2. The MRP4-mediated transport of PGE1 and PGE2 displayed saturation kinetics, with Km values of 2.1 and 3.4 microM, respectively. Further studies showed that PGF1alpha, PGF2alpha, PGA1, and thromboxane B2 were high-affinity inhibitors (and therefore presumably substrates) of MRP4. Furthermore, several nonsteroidal antiinflammatory drugs were potent inhibitors of MRP4 at concentrations that did not inhibit MRP1. In cells expressing the prostaglandin transporter PGT, the steady-state accumulation of PGE1 and PGE2 was reduced proportional to MRP4 expression. Inhibition of MRP4 by an MRP4-specific RNA interference construct or by indomethacin reversed this accumulation deficit. Together, these data suggest that MRP4 can release prostaglandins from cells, and that, in addition to inhibiting prostaglandin synthesis, some nonsteroidal antiinflammatory drugs might also act by inhibiting this release.

Fig. 1.

Transport of PGE₁ and PGE₂ into membrane vesicles from Sf9 or HEK293 cells. Membrane vesicles (20 μg) were incubated with 41 nM [³H]PGE₁ (A and B) or 6.6 nM [³H]PGE₂ (C and D) for 10 min in the presence or absence of 4 mM ATP. The vesicles were derived from Sf9 cells infected with a WT or MRP1, 2, 3, or 4 cDNA-containing baculovirus (A and C), or MRP4- or MRP5-overexpressing and parental HEK293 cells (B and D). Values are the average ± SE of two independent determinations done in triplicate.

Fig. 2.

Time and concentration dependence of PGE₁ and PGE₂ transport by MRP4. Membrane vesicles (100 μg of protein) from WT (circles) or MRP4-containing (diamonds) Sf9 cells were incubated at 37°C with 41 nM [³H]PGE₁ (A) or 8.7 nM [³H]PGE₂ (C) in the presence (filled symbols) or absence (open symbols) of ATP, and aliquots (20 μg of protein) were taken at the times indicated. To analyze the concentration dependence of uptake, MRP4 vesicles were incubated at 37°C for 3 min with 41 nM [³H]PGE₁ (B) or 13 nM [³H]PGE₂ (D) with unlabeled substrate added to the final concentration. The samples were incubated in the presence or absence of 4 mM ATP, and only the ATP-dependent uptake is plotted. Values are the average ± SE of representative experiments done in triplicate. (Insets) Lineweaver–Burk transformation.

Fig. 3.

Leakage of E₂17βG and PGE₁ from inside-out membrane vesicles. Membrane vesicles derived from HEK293/4.63 cells were incubated with 1 μM E₂17βG (A) or 51 nM PGE₁ (B) at 37°C (as in Fig. 2, except that the MgCl₂ concentration was reduced to 5 mM), and aliquots corresponding to 15 μgof protein were filtered at the indicated times (filled symbols). To determine the permeability of the vesicles, a combination of 5 mM orthovanadate and 5 mM EDTA was added to stop ATP-dependent uptake (open symbols), either at the beginning of the reaction or after 5 or 10 min (asterisks), and the reaction was followed until 20 min. Values are the average ± SE of two experiments done in triplicate.

Fig. 4.

Effect of MRP4 expression on PGE₁ and PGE₂ accumulation in HEK293 cells expressing PGT. HEK293 parental cells and those stably overexpressing MRP4 (4.3, medium overexpression; 4.63, high overexpression) were transfected with a PGT construct. On day 4 after transfection, cells were incubated with [³H]PGE₁ (A) or [³H]PGE₂ (B), and intracellular prostaglandin was determined at the indicated times (a representative of three independent experiments is shown). The effect of 25 μM indomethacin or 50 μM bromosulfophthalein on PGE₂ accumulation (C) was determined after 10 min and is expressed as the percentage of PGE₂ accumulated in the absence of these compounds (values represent the mean ± SE of three independent experiments).

Fig. 5.

Effect of RNAi on MRP4 expression and PGE₂ accumulation. (A) The effect of RNAi expression on the level of MRP4. (Upper) MRP4 expression in HEK293 (30-min exposure) and HEK293/4.63 (5-min exposure) cells 3 days after transfection with 7.5 μg of control pS vector (C) or RNAi construct pS42. (Lower) HEK293/4.63 cells were transduced with pRS or RNAi construct pRS42 and, after selection with puromycin, vector (C) and RNAi clones (53, 59, 66, and 71) were analyzed for MRP4 expression. (B) Cells were cotransfected with PGT and pS42, and 4 days after transfection the accumulation of PGE₂ was determined after 10 min and is expressed as a percentage of PGE₂ accumulated in the vector control. (C) Accumulation of PGE₂ in HEK293/4.63 cells and clones stably transfected with pRS or pRS42 was determined (a representative experiment is shown).