Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial cells: Role of ubiquitin-proteasome pathway

Abstract

We investigated here the mechanism of cytoprotection of nitric oxide (*NO) in bovine aortic endothelial cells treated with H2O2. NONOates were used as *NO donors that released *NO slowly at a well defined rate in the extracellular and intracellular milieus. H2O2-mediated intracellular dichlorofluorescein fluorescence and apoptosis were enhanced by the transferrin receptor (TfR)-mediated iron uptake. *NO inhibited the TfR-mediated iron uptake, dichlorofluorescein fluorescence, and apoptosis in H2O2-treated cells. *NO increased the proteasomal activity and degradation of nitrated TfR via ubiquitination. Nomega-nitro-L-arginine methyl ester, a nonspecific inhibitor of endogenous *NO biosynthesis, decreased the trypsin-like activity of 26S proteasome. *NO, by activating proteolysis, mitigates TfR-dependent iron uptake, dichlorodihydrofluorescein oxidation, and apoptosis in H2O2-treated bovine aortic endothelial cells. The relevance of biological nitration on redox signaling is discussed.

Fig. 1.

Effect of ^•NO on H₂O₂-induced oxidation of DCFH, cytochrome c release, and caspase-3 activity in BAECs. (A) BAECs treated with glucose (Glu)/GO for 4 h in the presence and absence of ^•NO donor DETA/NO (10–100 μM). Cells were washed with DPBS and incubated with DCFH-DA (10 μM) for 20 min. Cells were washed twice with DPBS and kept in a culture medium. Green fluorescence due to DCF was monitored. In one experiment, cells were treated with AcOM-PYRRO/NO (1 μM), an esterase-specific intracellular NO donor. (B) Average fluorescence intensity (n = 3) of photographs obtained in A. (C) BAECs were treated with glucose/GO for 8 h with and without DETA/NO (100 μM), and the release of cytochrome c from the mitochondria into the cytosol was measured by Western blot analysis. (D) BAECs were treated with glucose/GO (20 milliunits) for 8 h with and without DETA/NO (100 μM), and caspase-3 activity was measured. Data aremean ± SD for three experiments.

Fig. 2.

Effect of ^•NO on H₂O₂-induced changes in GSH, aconitase activity, TfR, and IRP-1 levels in BAECs. (A) BAECs were treated with glucose (Glu)/GO (20 milliunits) for 4 h with and without DETA/NO (50 and 100 μM), and GSH levels were measured. (B and C) Cells were treated with glucose/GO (20 milliunits) for 4 h with and without DETA/NO (100 μM), and aconitase activity was measured in the cytosolic (B) and mitochondrial (C) fractions. (D) BAECs were treated with glucose/GO for 4 h with and without DETA/NO, and TfR protein expression was measured by using 20 μg of protein of the 12,000 × g supernatant. (E) BAECs were treated as described for B, and cytoplasmic extracts were analyzed by gel-shift assay with and without 2-mercaptoethanol (2-ME) to measure IRP/IRE binding. (F) Cells were treated as described for D by using 12 μg of protein of the 12,000 × g supernatant except that the effect of lactacystin (10 μM) on TfR protein expression in glucose/GO-treated cells with and without DETA/NO was determined. *, P < 0.05 vs. control.

Fig. 3.

Effect of proteasomal inhibitors on DCFH oxidation, iron uptake, and apoptosis in BAECs treated with H₂O₂ and ^•NO. (A) BAECs were treated with glucose (Glu)/GO with or without DETA/NO (100 μM), proteasome inhibitors, lactacystin (10 μM), epoxomycin (2 μM) and MG-132 (10 μM), and anti-TfR antibody (IgA class, 42/6) (12 μg/ml). Cells were pretreated with the proteasome inhibitors and DETA/NO for 2 h before treatment with H₂O₂ for 4 h. Cells then were washed in DPBS and incubated with DCFH-DA (10 μM) for 20 min. Green fluorescence due to DCF was monitored with time. (B) ⁵⁵Fe uptake into cells was monitored under the same conditions as described for A and C. Caspase-3 activity was measured under the same conditions described for A except that cells were treated with H₂O₂ for 8 h. 1, control; 2, glucose/GO; 3, DETA/NO alone; 4, glucose/GO + DETA/NO; 5, Lac alone; 6, Lac + glucose/GO; 7, Lac + DETA/NO; 8, Lac + glucose/GO + DETA/NO; 9, glucose/GO + AcOM-PYRRO/NO; 10, Lac + glucose/GO + AcOM-PYRRO/NO. (D) Cells were treated as described for B, and 5 μg of the derivatized total protein was resolved on an SDS/8% PAGE gel and probed with a monoclonal anti-2,4-dinitrophenol antibody to measure protein carbonyl levels by Western blot analysis. (E) BAECs were treated as described for A, and the cell lysate (100 μg of protein) was immunoprecipitated with 6 μg of anti-TfR antibody, resolved on an SDS/8% PAGE, and probed for nitrated TfR with anti-nitrotyrosine antibody by Western blot analysis. *, P < 0.05 vs. control.

Fig. 4.

Effect of ^•NO on proteasomal activity in BAECs: degradation of ubiquitinated proteins. (A and B) BAECs were pretreated with DETA/NO (100 μM) and proteasome inhibitors (10 μM) for 2 h before the addition of glucose (Glu)/GO (20 milliunits) for 6 h. The chymotrypsin-like (A) and trypsin-like (B) activities of 26S proteasome were measured as described in Materials and Methods. (C) Treatment conditions were the same as described for A and B, but the 20S proteasome activity was measured as a function of time from 0 to 6 h by using the fluorogenic peptide, sLLVY-MCA, as the substrate. (D) Total ubiquitination of proteins measured in BAECs treated with glucose/GO for 6 h and resolved on SDS/12% PAGE followed by Western blot analysis with a polyclonal anti-Ub antibody. (E) Densitometric analysis of the data shown in D. (F) BAECs pretreated with DETA/NO and/or proteasome inhibitors for 2 h were treated with glucose/GO for 6 h and immunoprecipitated with anti-TfR antibody, resolved on SDS/8% PAGE, and probed with anti-Ub antibody by Western blot analysis. (G) BAECs were pretreated with l-NAME for 8 h, and the trypsin-like activity of 26S proteasome was measured. *, P < 0.05 vs. control.