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

Abstract

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

Nitro-fatty Acid Reaction with Glutathione and Cysteine

“…The apparent second-order rate constants determined herein show that reactions occur at biologically-relevant rates ( Table 2) and similar to a previous report (44). While some data were obtained from reports where concentrations of reactants may have differed from herein, rate constant comparisons still allow for nitroalkene reactivity to be examined in the context of other biological oxidants and electrophiles.…”

“…Nitroalkylation of proteins promotes membrane association and influences the function of proteins having Cys and His residues critical for structure or catalysis (35). The nitroalkylation of protein thiols is GSH-reversible, yielding a GS-nitrofatty acid exchange reaction product detectable in healthy human blood (35) and cell culture systems (44). The extracellular transport of GS-nitroalkene conjugation products can be mediated by the multi-drug resistance protein-1, a reaction that would be expected to mitigate cell signaling actions of nitroalkenes (44).…”

“…Biological and chemical nitration of unsaturated fatty acids yields an array of regioisomers (28,43,44). There was no significant difference in apparent second-order rate constants for the reaction of GSH with mixed regioisomers of OA-NO 2 and the specific regioisomer C 9 -OA-NO 2 (Table 1), while the rate constant for LNO 2 reaction with GSH was 1.9 times faster than the mixed regioisomers of OA-NO 2 and C 9 -OA-NO 2 .…”

“…GSH conjugates with xenobiotics and cellular electrophiles (35,38,39) and reacts with nitrated fatty acids to form, GS-OA-NO 2 and GS-LNO 2 derivatives detectible at 3.3 and 1.3 nM, respectively. Additionally, GS-LNO 2 was detected in cultured MCF7/WT cells, with the in vivo levels of adducts dependent on expression levels of MRP1, an efflux transporter of GSH conjugates (44). Collision-induced dissociation (CID) of nitroalkylated GSH and structural analysis by MS/MS/MS affirm that nitroalkenes are bonded to nucleophilic Cys residues (35).…”

Modulation of Nitro-fatty Acid Signaling

“…15, 49, 52, 69). The formation of nitroalkene-glutathione adducts not only reverses electrophilic modifications but also promotes cellular export via the multidrug resistance protein 1 (MRP-1) and excretion in urine (10,70).…”