Multidrug Resistance Protein 4 (MRP4/ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation

Sabrina Copsel,Corina Garcia,Federico Diez,Monica Vermeulem,Alberto Baldi,Liliana G Bianciotti,Frans G.M Russel,Carina Shayo,Carlos Davio

doi:10.1074/jbc.m110.166868

Abstract

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Highlights

The first second messenger discovered, cAMP, plays a critical role in the cellular response to numerous extracellular stimuli
The aim of the present study was to characterize cAMP extrusion, that mediated by MRP4, in three different acute myeloid leukemia (AML) cell lines to assess the role of this transporter in cell proliferation and differentiation
Because intracellular cAMP levels are critical for leukemic cell differentiation, its regulation by MRP4 is to our knowledge the first direct evidence that these proteins may represent new potential targets for leukemia differentiation therapy

Summary

Introduction

The first second messenger discovered, cAMP, plays a critical role in the cellular response to numerous extracellular stimuli. Despite a large increase in cAMP levels mediated by H2 receptor activation, U937 cells trigger PDE4 activation and G-protein-coupled receptor kinase 2 (GRK2)-mediated receptor desensitization. These mechanisms regulate the duration and intensity of cAMP signaling, thereby partially conditioning cell differentiation failure (8 –10). The aim of the present study was to characterize cAMP extrusion, that mediated by MRP4, in three different AML cell lines to assess the role of this transporter in cell proliferation and differentiation. Because intracellular cAMP levels are critical for leukemic cell differentiation, its regulation by MRP4 is to our knowledge the first direct evidence that these proteins may represent new potential targets for leukemia differentiation therapy

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Conclusion