Thioredoxin increases exocytosis by denitrosylating N-ethylmaleimide-sensitive factor

Abstract

Exocytosis involves membrane fusion between granules and the plasma membrane. Nitric oxide (NO) inhibits exocytosis by chemically modifying N-ethylmaleimide-sensitive factor (NSF), a key component of the exocytic machinery. However, cells recover the ability to release messenger molecules within hours of exposure to NO through unknown mechanisms. We now identify thioredoxin (TRX1) as a denitrosylase that reverses NO inhibition of exocytosis. Endogenously synthesized NO increases S-nitrosylated NSF levels, but S-nitrosylated NSF levels decrease within 3 h after exposure to NO. We found that NO increases the interaction between TRX1 and NSF, and endogenous TRX1 removes NO from S-nitrosylated NSF. Knockdown of TRX1 increases the level of S-nitrosylated NSF, prolongs the inhibition of exocytosis, and suppresses leukocyte adhesion. Taken together, these data show that TRX1 promotes exocytosis by denitrosylating NSF. Our findings suggest that TRX1 might regulate exocytosis in a variety of physiological settings, such as vascular inflammation, thrombosis, and insulin release.

FIGURE 1.

Nitric oxide inhibition of exocytosis is reversible. A, NO inhibition of VWF release. HUVEC were pretreated with the NO donor SNAP or its control AP for 6 h, stimulated with histamine, and the amount of VWF released into the media over 45 min was measured by an ELISA (n = 3, mean ± S.D.). The NO donor SNAP decreases endothelial exocytosis. B, endogenous NO inhibits VWF release. HUVEC were pretreated with the NOS inhibitor l-NAME for 4 h, stimulated with histamine, and the VWF released into the media was measured as above (n = 3, mean ± S.D.). The NOS inhibitor l-NAME increases endothelial exocytosis. C, NO inhibition of leukocyte adhesion. HUVEC were pretreated with SNAP for 4 h, stimulated with histamine for 20 min, then co-cultured with calcein-labeled HL-60 cells for 15 min. The upper panels show bright field images of HUVEC and HL-60, and the lower panels show calcein-labeled HL-60 co-cultured with HUVEC. Scale bars = 100 μm. D, quantitation of NO inhibition of leukocyte adhesion in C (n = 3, mean ± S.D.). The NO donor SNAP decreases leukocyte adhesion to endothelial cells. E, NO inhibition of exocytosis is reversible. HUVEC were pretreated with SNAP (20 μm) for 6 h, and then after recovery for 0, 0.5, 1, or 3 h the cells were stimulated with histamine (20 μm). Exocytosis was measured as above (n = 4, mean ± S.D.). The effect of NO upon exocytosis diminishes over time. *, p < 0.05; **, p < 0.01; NS, not significant.

FIGURE 2.

S-nitrosylation of NSF is reversible. A, S-nitrosylation of NSF in HeLa cells. HeLa cells were treated with the NO donor SNAP for 6 h. Then, cell lysates were harvested and levels of NSF-SNO were measured by the biotin switch assay. The NO donor SNAP increases NSF-SNO in a dose-dependent manner. B, S-nitrosylation of NSF decreases over time in HeLa cells. HeLa cells were treated with SNAP for 6 h and then washed. After 0–3 h of recovery, cell lysates were harvested, and levels of NSF-SNO were measured by the biotin switch assay. After exposure to an NO donor, the amount of S-nitrosylated NSF decreases over time. C, endogenous S-nitrosylation of NSF decreases over time in endothelial cells. HUVEC were treated with the NOS inhibitor l-NAME for 0–3 h, and then levels of NSF-SNO were measured as above. After NO synthase is inhibited, the amount of endogenous S-nitrosylated NSF decreases over time. D, quantitation of C (mean ± S.D). **, p < 0.01.

FIGURE 3.

Thioredoxin interacts with NSF in the presence of NO. A, interaction between endogenous NSF and endogenous TRX1. HUVEC were treated with SNAP (500 μm) or l-NAME (500 μm) for 4 h. Cell lysates were immunoprecipitated with antibody to NSF and immunoblotted for TRX1. NSF interacts with TRX1 in an NO-dependent manner. B, quantitation of A (mean ± S.D). **, p < 0.01. C, stable interaction between NSF and TRTX1 depends upon TRX1 residue Cys-35. HeLa cells were transfected with a vector expressing wild-type Myc-TRX1(WT) or mutant Myc-TRX1(C35S) and treated with SNAP for 4 h. Cell lysates were immunoprecipitated with antibody to Myc and immunoblotted for NSF. TRX1 interacts with NSF, through the CXXC disulfide reductase domain of TRX1. D, HeLa cells were transfected with a vector expressing mutant Myc-TRX1(C35S) and treated with increasing amounts of SNAP (5–500 μm) for 4 h. The interaction between NSF and TRX1 was analyzed as above. SNAP increases the interaction in a dose-dependent manner.

FIGURE 4.

Thioredoxin denitrosylates NSF. A, TRX1 denitrosylates NSF in HeLa cells. HeLa cells were transfected with siRNA control or siRNA to TRX1. Cells were treated with the NO donor SNAP (20 μm) for 6 h, washed, and harvested during 0–3 h recovery. Levels of NSF-SNO and GAPDH-SNO were measured as above. Knockdown of TRX1 increases levels of NSF-SNO and GAPDH-SNO. B, TRX1 denitrosylates NSF in endothelial cells. HUVEC were transfected with siRNA control or siRNA to TRX1. Cells were treated with the NOS inhibitor l-NAME for 0–3 h. Levels of NSF-SNO were measured as above. Knockdown of TRX1 increases levels of NSF-SNO. C, quantitation of B (mean ± S.D.). **, p < 0.01.

FIGURE 5.

Thioredoxin restores exocytosis after NO inhibition. A, thioredoxin restores VWF release. HUVEC were transfected with siRNA control or siRNA to TRX1 (insert shows immunoblot of TRX1), pretreated with the NO donor SNAP (20 μm) for 6 h, and washed. After 0–3 h of recovery, HUVEC were stimulated with histamine (20 μm) for 45 min, and exocytosis was measured as above (n = 4, mean ± S.D.). Knockdown of TRX1 delays the recovery of exocytosis from NO inhibition. B, thioredoxin restores leukocyte adhesion after NO inhibition. HUVEC were transfected with siRNA control or siRNA to TRX1, pretreated with SNAP (20 μm) for 4 h, and washed. After 0–1 h of recovery, HUVEC were stimulated with histamine (20 μm) for 20 min, and leukocyte adhesion was evaluated as above. The upper panels show bright field images of HUVEC and HL-60, and the lower panels show fluorescent images of calcein-labeled HL-60. Scale bars = 100 μm. C, quantification of B. Knockdown of TRX1 delays the recovery of leukocyte adhesion from NO inhibition. (n = 3, mean ± S.D.). *, p < 0.05; **, p < 0.01.