Modulation of nitrated lipid signaling by multidrug resistance protein 1 (MRP1):: Glutathione conjugation and MRP1-mediated efflux inhibit nitrolinoleic acid-induced, PPARγ-dependent transcription activation

Recent data has shown that nitrolinoleic acid (LNO2), an electrophilic derivative of linoleic acid, has several important bioactivities including antiinflammatory, antiplatelet, vasorelaxation, and as a novel potent ligand of PPAR gamma-transcription regulating activities. Moreover, LNO2 is formed in abundance in vivo at levels sufficient to mediate these bioactivities. In order to investigate the role of glutathione conjugation and MRP1-mediated efflux in the regulation of PPAR gamma-dependent LNO2 signaling, regioisomers of LNO2 were synthesized and characterized. Analysis by 1D and 2D H-1 and C-13 NMR revealed that the LNO2 preparation consisted of four, rather than two, nitrated regioisomers in approximately equal abundance. At physiologic pH and intracellular glutathione levels, LNO2 was rapidly and quantitatively converted to glutathione conjugates (LNO2-SG) via Michael addition. MRP1 mediated efficient ATP-dependent transport of LNO2-SG. Using a PPRE-containing reporter gene transiently transfected into MRP-poor MCF7/WT cells, we verified that the LNO2 mixture was a potent activator of PPAR gamma-dependent transcription. However, expression of MRP1 in the stably transduced MCF7 derivative, MCF7/MRP1-10, resulted in strong inhibition of LNO2-induced transcription activation. Taken together, these results suggest that glutathione conjugation and MRP1-mediated conjugate transport can attenuate LNO2 bioactivity and thereby play important roles in the regulation of cellular signaling by LNO2.