Endothelial nitric oxide (NO) synthase (eNOS)–derived NO prevents dihydrofolate reductase (DHFR) protein reduction in human umbilical vein endothelial cells (HUVECs). A, eNOS silencing reduced DHFR but not GTP cyclohydrolase I (GTPCH) protein expression. B, eNOS silencing did not alter DHFR mRNA expression. C, NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) treatment reduced DHFR expression in a dose- (0, 75, 150, and 300 μmol/L) and a time- (0, 6, 12, 24, and 48 hours) dependent manner but had no effect on GTPCH expression. D, PTIO (150 μmol/L) had no significant effect on DHFR mRNA expression. E, NO donor S-nitroso-l-glutathione (GSNO; 100 μmol/L) reversed PTIO- (150 μmol/L) induced DHFR reduction. F, GSNO (100 μmol/L) prevents DHFR reduction induced by eNOS silencing (n=3 for each group; *P<0.05 vs scramble [Scr] siRNA in A and F or P<0.05 vs control in E; #P<0.05 vs PTIO in E or P<0.05 vs eNOS siRNA in F).
Nitric oxide (NO) depletion promotes dihydrofolate reductase (DHFR) degradation via ubiquitin–proteasome degradation in human umbilical vein endothelial cells. A, S-Nitroso-l-glutathione (GSNO; 100 μmol/L) supplementation prevented DHFR reduction induced by cycloheximide (CHX; 30 μg/mL) treatment. DHFR degradation induced by endothelial NO synthase (eNOS) silencing (B) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; 150 μmol/L; C) could be inhibited by proteasome inhibitor, MG132 (1 μmol/L). D, PTIO- (150 μmol/L) induced DHFR polyubiquitination, which could be suppressed by GSNO (100 μmol/L; n=3; *P<0.05 vs control in A, C, and D or P<0.05 vs scramble [Scr] siRNA in B; #P<0.05 vs eNOS siRNA in B or P<0.05 vs PTIO in C and D).
Cystein 7 is the site of S-nitrosylation of dihydrofolate reductase (DHFR). A, S-Nitroso-l-glutathione (GSNO; 100 μmol/L) restored DHFR S-nitrosylation in the presence of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; 150 μmol/L) as determined by biotin switch assay. B, Amino acid sequence comparison of DHFR among species. C, C7S mutation blocked GSNO- (100 μmol/L) induced DHFR S-nitrosylation (n=3; *P<0.05 vs control in A or P<0.05 vs wild-type [WT] in C; #P<0.05 vs PTIO in A).
Dihydrofolate reductase (DHFR) S-nitrosylation prevents ubiquitination and degradation. A, C7S mutation in DHFR destabilized the protein compared with the wild-type (WT) DHFR treated with cycloheximide (CHX; 30 μg/mL). S-Nitroso-l-glutathione (GSNO; 100 μmol/L) stabilized DHFR, whereas the C7S mutation abolished the effect. B, GSNO (100 μmol/L) restored DHFR S-nitrosylation, which was suppressed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; 150 μmol/L). Dithiothreitol (DTT; 10 mmol/L) inhibited the effect of GSNO (100 μmol/L). C and D, GSNO (100 μmol/L) prevented DHFR ubiquitination and degradation induced by PTIO (150 μmol/L); this effect could be blocked by DTT (10 mmol/L) supplementation. E, PTIO (150 μmol/L) promoted 26S proteasome activity, which could be reversed by GSNO (100 μmol/L). The addition of DTT (10 mmol/L) did not affect 26S proteasome activity compared with the PTIO+GSNO group (n=3; *P<0.05 vs WT in A or P<0.05 vs control in B–E; #P<0.05 vs C7S in A or P<0.05 vs PTIO in B–E; $P<0.05 vs PTIO+GSNO in B–D).
2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduces aortic endothelial dihydrofolate reductase (DHFR) expression and BH4 content via proteasomal degradation ex vivo. A, Western blot analysis showed reduced DHFR expression in PTIO- (150 μmol/L) treated aortas, which could be blocked by MG132 (1 μmol/L, 6 hours) supplementation. B, Representative immunofluorescence staining of DHFR (red) and endothelium marker CD31 (green) of ex vivo cultured aortas. C, PTIO (150 μmol/L) reduced endothelial DHFR expression, whereas MG132 (1 μmol/L, 6 hours) reversed the effect. D, Representative immunofluorescence staining of GTP cyclohydrolase I (GTPCH; red) and endothelium marker CD31 (green) of ex vivo cultured aortas. E, PTIO (150 μmol/L) had no significant effect on endothelial GTPCH expression. F, PTIO (150 μmol/L) reduced BH4 content, which could be reversed by addition of MG132 (1 μmol/L, 6 hours) in aortas ex vivo (n=4 for each group; *P<0.05 vs control; #P<0.05 vs PTIO).
MG132 prevents aortic endothelial dihydrofolate reductase (DHFR) expression and BH4 content in endothelial nitric oxide synthase (eNOS−/−) mice. A, Representative immunofluorescence staining of DHFR (red) and endothelium marker CD31 (green) of aortas from mice received indicated treatment. B, MG132 (5 mg/kg per day, 3 days) suppressed endothelial DHFR reduction in eNOS−/− mice. C, Representative immunofluorescence staining of GTP cyclohydrolase I (GTPCH; red) and endothelium marker CD31 (green) of aortas from indicated treated mice. D, eNOS deficiency had no significant effect on endothelial GTPCH expression. E, Supplementation of MG132 (5 mg/kg per day, 3 days) suppressed aortic BH4 content reduction in eNOS−/− mice (n=5 for each group; *P<0.05 vs wild-type [WT]; #P<0.05 vs eNOS−/−).
Endothelial nitric oxide (NO) synthase (eNOS)–derived NO prevents dihydrofolate reductase (DHFR) protein reduction in human umbilical vein endothelial cells (HUVECs). A, eNOS silencing reduced DHFR but not GTP cyclohydrolase I (GTPCH) protein expression. B, eNOS silencing did not alter DHFR mRNA expression. C, NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) treatment reduced DHFR expression in a dose- (0, 75, 150, and 300 μmol/L) and a time- (0, 6, 12, 24, and 48 hours) dependent manner but had no effect on GTPCH expression. D, PTIO (150 μmol/L) had no significant effect on DHFR mRNA expression. E, NO donor S-nitroso-l-glutathione (GSNO; 100 μmol/L) reversed PTIO- (150 μmol/L) induced DHFR reduction. F, GSNO (100 μmol/L) prevents DHFR reduction induced by eNOS silencing (n=3 for each group; *P<0.05 vs scramble [Scr] siRNA in A and F or P<0.05 vs control in E; #P<0.05 vs PTIO in E or P<0.05 vs eNOS siRNA in F).
