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

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 probenecidsensitive, 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.

Blockade of Multidrug Resistance-Associated Proteins Aggravates Acute Pancreatitis and Blunts Atrial Natriuretic Factor’s Beneficial Effect in Rats: Role of MRP4 (ABCC4)

“…MRP4 (ATP-binding cassette sub-family C member 4 [ABCC4]) is a member of the multidrug resistance proteins (MRPs) belonging to the C group of the ATP-binding cassette (ABC) protein superfamily, which functions as an energy-dependent, transmembrane efflux transporter. Compelling evidence supports that cAMP extrusion through MRP4 in several cell types provides a complementary mechanism to the phosphodiesterases (PDEs) as modulators of the intracellular concentration of the second messenger (6)(7)(8)(9)(10). In isolated pancreatic acini, we found that secretin-induced cAMP is extruded by ANF through NPR-C receptors coupled to the PLC/PKC pathway, thus restricting its intracellular accumulation.…”

“…Blood samples were rapidly centrifuged, and plasmas were collected in ethanol and stored at -20°C. Samples were dried and the remaining residue was suspended for cAMP determination, assessed by competition of [ 3 H]cAMP for PKA as previously detailed (9,19). Results were expressed as picomoles per milliliter plasma.…”

The Pharmacological and Physiological Role of Multidrug-Resistant Protein 4

“…MRP4 inhibition induced the differentiation of leukemic stem cells, which are crucial for leukemia initiation, progression, metastasis, and relapse, and one approach to combatting leukemia is to force the cells into differentiation. Moreover, Copsel et al (2011) reported that MRP4 extrusion is directly involved in the regulation of intracellular cAMP in leukemia cell lines. Pharmacological inhibition or small interfering RNA (siRNA) knockdown of MRP4 led to cell cycle arrest and cell differentiation.…”

Semen Promotes the Differentiation of Tolerogenic Dendritic Cells

“…Ethanol was dried and residues were resuspended in 50 mM TrisHCl, pH 7.4, 0.1% BSA, for cAMP determination. cAMP content was determined by competitive radio-binding assay for PKA using [ 3 H]cAMP, as described previously (31). The standard curve was generated using different concentrations of cAMP.…”