Abstract
Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-kappaB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-kappaB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.
Highlights
Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis
Pharmacological inhibition or antisense ablation of aldose reductase prevented protein kinase C, NF-B, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN
In the culture media, we have observed a time-dependent increase (30, 60, and 120 min, and 24 h) in the GS-HNE and GS-DHN levels and a time-dependent decrease in GS-HNE- and GS-DHN-esters (Table-1). These results suggest that GS-DHN does not dissociate, whereas some dissociation of GS-HNE could occur in vascular smooth muscle cell (VSMC)
Summary
Materials—Dulbecco’s modified Eagle’s medium (DMEM), phosphatebuffered saline, penicillin/streptomycin solution, trypsin, and fetal bovine serum (FBS) were purchased from Invitrogen. The GS-HNE-ester conjugate was purified by reverse phase HPLC as described below. At the end of the incubation, the GS-DHN-ester conjugate was separated from GS-HNE-ester by reverse phase HPLC as described below. To examine the role of RLIP76 (76-kDa Ral-binding, Rho/RacGAP, and Ral effector protein) and MRP-2 (multidrug resistance-associated protein-2) in mediating cell growth, growth-arrested VSMC were treated with peptide-specific antibodies raised against RLIP76 or MRP-2 for 1 h followed by incubation with GS-DHN-ester (0 –5 M) and the rates of cell proliferation or apoptosis were determined as described above. The cells were continuously cultured in the 0.1% FBS media without or with BSO in the absence or presence of HNE (1 M), GS-HNE-ester (0.75 M), or GS-DHN-ester (0.75 M) for another 24 h. The growth-arrested VSMC (2 ϫ 106/well in six-well plates) were incubated with 1 M radiolabeled GS-HNE-ester or GS-DHN-ester containing ϳ51,000 cpm in serum-free medium. Peak 1 eluted at fractions 22–25 corresponded to GS-HNE/GS-DHN, peak 2 eluted at fractions 28 –31 corresponded to GS-HNE-ester/GS-DHN-ester, and peak 3 eluted at fractions 35–38 corresponded to DHN/HNA elution times
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