Hydrogen sulfide-linked sulfhydration of NF-κB mediates its antiapoptotic actions

Abstract

Nuclear factor κB (NF-κB) is an antiapoptotic transcription factor. We show that the antiapoptotic actions of NF-κB are mediated by hydrogen sulfide (H(2)S) synthesized by cystathionine gamma-lyase (CSE). TNF-α treatment triples H(2)S generation by stimulating binding of SP1 to the CSE promoter. H(2)S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE-deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H(2)S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the coactivator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early after TNF-α treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. In CSE mutant mice, antiapoptotic influences of NF-κB are markedly diminished. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its antiapoptotic transcriptional activity.

Figure 1. CSE^-/- mice are more susceptible to TNF-α induced cell death; TNF-α stimulates hydrogen sulfide production by stimulating CSE transcription via SP1

(A) Treatment withTNF-α elicits more TUNEL positive cells in liver of CSE^-/- than wild type mice. (B) Caspase 3 activation is increased in CSE^-/- mice liver following TNF-α treatment. *p < 0.01, n = 5, one-way ANOVA, mean ± SEM. (C) DNA fragmentation induced by TNF-α in macrophages isolated from CSE^-/- mice is decreased by pretreatment with GYY-4137 in a concentration dependent manner. *p < 0.01, n = 5, one-way ANOVA, mean ± SEM. **p < 0.001, n = 5, one-way ANOVA, mean ± SEM. (D) TNF-α stimulation of hepatic H₂S formation is abolished in CSE^-/- mice. Wild type and CSE^-/- mice were injected with TNF-α and H₂S formation assayed in liver lysates. **p < 0.001, n = 5, one-way ANOVA, mean ± SEM. (E) TNF-α stimulation of H₂S formation in peritoneal macrophages is diminished in CSE^-/- preparations. *p < 0.01, n = 3, oneway ANOVA, mean ± SEM. (F) TNF-α treatment increases hepatic CSE protein level (n=4). (G) TNF-α causes an increase in CSE protein level in peritoneal macrophages in time dependent manner. Densitometric analysis of CSE protein level in TNF-α treated macrophages. *p < 0.01, n = 5, one-way ANOVA, mean ± SEM. (H) TNF-α causes an increase in peritoneal macrophage CSE mRNA levels in time dependent manner. (I) TNF-α-elicited enhancement of SP1 binding to the CSE promoter. (J) SP1 RNAi treatment prevents TNF-α stimulation of CSE mRNA in peritoneal macrophages.(K) Treatment with SP1 RNAi reduces TNF-α-elicited H₂S formation in peritoneal macrophages. **p < 0.001, n = 5, one-way ANOVA, mean ± SEM.

Figure 2. Transcriptional activity of p65 is reduced in CSE^-/- mice

(A) DNA binding of NF-κB is reduced in CSE^-/- mice. Wild type and CSE^-/- mice were injected with TNF-α, and liver lysates subjected to EMSA assay. (B) In peritoneal macrophages TNF-α stimulates DNA binding of NF-κB in a time dependent manner, effects abolished in CSE^-/- mice. (C) Anti-apoptotic gene expression is reduced in CSE^-/- mice. Wild type and CSE^-/- mice were injected with TNF-α, and mRNA levels of anti-apoptotic genes measured by real time RT-PCR. *p < 0.01, n = 5, one-way ANOVA, mean ± SEM. (D) Reduced binding of p65 to promoters of anti-apoptotic genes in CSE^-/- mice treated with TNF-α. (E) Overexpression of wild type CSE in CSE^-/- macrophages can rescue binding of p65 to promoters of anti-apoptotic genes. Overexpressed catalytically dead CSE mutant (CSE CD) in CSE^-/- macrophages fails to rescue binding of p65 to promoters of anti-apoptotic genes. (F) DNA binding of NF-κB is rescued in TNF-α treated CSE^-/- macrophages after treatment with GYY-4137 (200 μM). *p < 0.01, n = 5, one-way ANOVA, mean ± SEM.

Figure 3. Sulfhydration of p65-C38, detected by maleimide assay, enhances its binding to canonical NF-κB DNA sequences

(A) Schematic diagram for detection of p65 sulfhydration using red maleimide. (B) Depletion of red fluorescence intensity in TNF-α treated mice after DTT treatment establishes sulfhydration of p65. Data are presented as mean ± SEM. (C) Time dependent decrease in red fluorescence intensity of p65 protein in TNF-α treated macrophages associated with sulfhydration of p65. Data are presented as mean ± SEM. (D) Concentration dependent decrease in red fluorescence intensity of p65 protein in TNF-α treated macrophages reflecting sulfhydration of p65. Data are presented as mean ± SEM. (E) LC-MS/MS analysis of endogenous full-length p65 protein treated with TNF-α (10 μg/kg) for 4 h reveals sulfhydration of Cys38. (F) Abolition of red fluorescence intensity in C38S mutant of p65 indicates that C38 is the site for sulfhydration. Data are presented as mean ± SEM. (G) Luciferase activity of p65 but not p65-C38S is enhanced by transfection of CSE. *p < 0.01, n = 4, one-way ANOVA, mean ± SEM.

Figure 4. Detection of nitrosylation and sulfhydration of p65 by maleimide assay

(A) Levels of CSE and iNOS in TNF-α treated macrophages upon treatment with TNF-α for 8h. Densitometric analysis of CSE protein level in macrophages after treatment with TNF-α for 8 h. *p < 0.001, n = 3, one-way ANOVA, mean ± SEM. (B) Schematic diagram for detection of sulfhydration and nitrosylation of p65 using red and green maleimide. (C) Time dependent changes in sulfhydration and nitrosylation of p65 in TNF-α treated macrophages. As a positive control, we have measured sulfhydration and nitrosylation of p65 upon treatment with GYY-4137 (800 μM) or GSNO (500 μM) for 0, 2, 5 and 8 h. (D) Assessment of sulfhydration and nitrosylation of p65 at various times in TNF-α treated cells. Data are presented as mean ± SEM. (E) Levels of nitrosylation of p65 in TNF-α treated CSE ^-/- macrophages.

Figure 5. Binding of sulfhydrated p65 to RPS3 is required for transcriptional activity of NF-κB

(A) Treatment with TNF-α enhances binding between p65 and RPS3 in liver of wild type but not CSE^-/- mice. (B) TNF-α elicits binding between RPS3 and wt p65 but not p65-C38S. (C) TNF-α elicits binding between p65 and RPS3 in peritoneal macrophages. (D) H₂S donor GYY-4137 augments p65-RPS3 binding selectively in the presence of. TNF-α but GSNO does not. (E) Formation of p65–RPS3 complex at the Bcl-XL promoter region in RAW264.7 cells. (F) TNF-α elicits a complex of RPS3 at the Bcl-XL promoter region with p65 but not p65-C38S. (G) Depletion of RPS3 abolishes binding of p65 to promoters of the anti-apoptotic genes Bcl-XL, c-IAP2, TRAF and A20. (H) Apoptotic effects of TNF-α in macrophages are prevented by overexpression of wt p65. DNA fragmentation is elevated in RPS3 depleted cells. Overexpression of wt p65 does not reverse cell death in RPS3 depleted cells. *p < 0.01, n = 5, one-way ANOVA, mean ± SEM. **p < 0.001, n = 5, one-way ANOVA, mean ± SEM.