VSports最新版本 - Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2005 Oct 15;391(Pt 2):399-408.
doi: 10.1042/BJ20050795.

Tumour necrosis factor alpha induces co-ordinated activation of rat GSH synthetic enzymes via nuclear factor kappaB and activator protein-1

Heping Yang  1 , Nathaniel Magilnick, Xiaopeng Ou, Shelly C Lu
Affiliations

Tumour necrosis factor alpha induces co-ordinated activation of rat GSH synthetic enzymes via nuclear factor kappaB and activator protein-1

Heping Yang et al. Biochem J. .

Abstract

GSH synthesis occurs via two enzymatic steps catalysed by GCL [glutamate-cysteine ligase, made up of GCLC (GCL catalytic subunit), and GCLM (GCL modifier subunit)] and GSS (GSH synthetase). Co-ordinated up-regulation of GCL and GSS further enhances GSH synthetic capacity. The present study examined whether TNFalpha (tumour necrosis factor alpha) influences the expression of rat GSH synthetic enzymes. To facilitate transcriptional studies of the rat GCLM, we cloned its 1 VSports手机版. 8 kb 5'-flanking region. TNFalpha induces the expression and recombinant promoter activities of GCLC, GCLM and GSS in H4IIE cells. TNFalpha induces NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein 1) nuclear-binding activities. Blocking AP-1 with dominant negative c-Jun or NF-kappaB with IkappaBSR (IkappaB super-repressor, where IkappaB stands for inhibitory kappaB) lowered basal expression and inhibited the TNFalpha-mediated increase in mRNA levels of all three genes. While all three genes have multiple AP-1-binding sites, only GCLC has a NF-kappaB-binding site. Overexpression with p50 or p65 increased c-Jun mRNA levels, c-Jun-dependent promoter activity and the promoter activity of GCLM and GSS. Blocking NF-kappaB also lowered basal c-Jun expression and blunted the TNFalpha-mediated increase in c-Jun mRNA levels. TNFalpha treatment resulted in increased c-Jun and Nrf2 (nuclear factor erythroid 2-related factor 2) nuclear binding to the antioxidant response element of the rat GCLM and if this was prevented, TNFalpha no longer induced the GCLM promoter activity. In conclusion, both c-Jun and NF-kappaB are required for basal and TNFalpha-mediated induction of GSH synthetic enzymes in H4IIE cells. While NF-kappaB may exert a direct effect on the GCLC promoter, it induces the GCLM and GSS promoters indirectly via c-Jun. .

PubMed Disclaimer

Figures (VSports手机版)

Figure 1
Figure 1. Nucleotide sequence of the 5′-flanking region of the rat GCLM gene
Sequence is numbered relative to the translational start site. The putative regulatory elements are indicated in bold letters above the underlined sequences. The arrows denote transcriptional start sites. HSF, heat-shock transcription factor; C/EBP, CCAAT/enhancer-binding protein; STATx, signal transducer and activator of transcription x; MZF1, myeloid zinc finger 1; Sp1, stimulating protein 1; AML-1a, acute myeloid leukaemia-1a.
Figure 2
Figure 2. Alignment of the rat and mouse GCLM 5′-flanking regions [15]
The numbers above the rat sequence denote position relative to the translational start site (ATG). Note the similarity in the 5′-flanking sequence and transcriptional start sites (shown as forward arrows, top for rat and bottom for mouse).
Figure 3
Figure 3. Transient transfection analysis of the rat GCLM promoter–luciferase constructs in H4IIE cells
Progressive 5′-deletions of the GCLM promoter extending from −1803 to +3 bp were generated and fused to the promoterless luciferase pGL-3 basic vector as described in the Materials and methods section. Numbering is defined relative to the translational start site. Results represent means±S.E.M. from four independent experiments performed in triplicates. TNFα treatment (15 ng/ml) was only during the last 4 h of the transfection. Data are expressed as relative luciferase activity compared with that of pGL-3 basic vector control, which is assigned a value of 1.0. *P<0.05 versus respective control GCLM (CON-GCLM) constructs (ANOVA followed by Fisher's test).
Figure 4
Figure 4. Effect of TNFα on expression of GCLC, GCLM and GSS in H4IIE cells
(A) H4IIE cells were treated with varying concentrations (0–15 ng/ml) of TNFα for 8 h. Total RNA (20 μg/lane) was subjected to Northern-blot analysis using cDNA probe for GCLC as described in the Materials and methods section. The same membrane was sequentially rehybridized with cDNA probes for GCLM, GSS and β-actin to ensure equal loading. (B) H4IIE cells were treated with TNFα (7.5 ng/ml) for varying duration (0–12 h) and processed for Northern-blot analysis as described above.
Figure 5
Figure 5. Effect of TNFα on luciferase activity driven by rat GCLC promoter constructs (A) or GSS promoter constructs (B)
H4IIE cells were transiently transfected with rat GCLC or GSS promoter constructs and treated with TNFα (15 ng/ml during the last 4 h) or vehicle control as described in the Materials and methods section. Positions of the consensus AP-1 and NF-κB sites present in the rat GCLC and GSS 5′-flanking regions are shown. Results represent means±S.E.M. from four independent experiments performed in triplicates. Data are expressed as relative luciferase activity compared with that of pGL-3 enhancer vector control, which is assigned a value of 1.0. *P<0.05 versus respective control GCLC (CON-GCLC) or GSS (CON-GSS) constructs (ANOVA followed by Fisher's test).
Figure 6
Figure 6. TNFα induces apoptosis in H4IIE cells
H4IIE cells were treated with TNFα (0–60 ng/ml)±IκBSR±Z-VAD-FMK (0–30 μM) for 8 h, and apoptosis was determined by DNA fragmentation as described in the Materials and methods section. Note that there is no apoptosis at 15 ng/ml dose (first lane from the right). IκBSR greatly potentiated apoptosis (see DNA fragmentation at the 3.75 ng/ml dose, second lane from the left). This can be blocked in a dose-dependent manner by Z-VAD-FMK so that no fragmentation occurs at 15 ng/ml TNFα + IκBSR in the presence of 30 μM Z-VAD-FMK.
Figure 7
Figure 7. Role of NF-κB in TNFα-mediated up-regulation of GSH synthetic enzymes
H4IIE cells were infected with IκBSR or empty vector and treated with TNFα (15 ng/ml, 8 h) with or without Z-VAD-FMK. Northern-blot analysis was performed for GCLC, GCLM (A) and GSS (B) as described in the Materials and methods section. Z-VAD-FMK treatment alone had no influence on the mRNA levels of GCLC, GCLM and GSS. IκBSR treatment alone resulted in a significant lowering of baseline mRNA levels of all three genes by approx. 40–45%. It also blunted but did not eliminate the TNFα-mediated increase in mRNA levels of all three genes. Representative Northern blots from three independent experiments are shown.
Figure 8
Figure 8. Role of c-Jun in TNFα-mediated up-regulation of GSH synthetic enzymes
H4IIE cells were infected with dominant negative c-Jun (TAM67) or empty vector and treated with TNFα (15 ng/ml, 8 h) or vehicle control. Northern-blot analysis was performed for GCLC (A), GCLM and GSS (B) as described in the Materials and methods section. TAM67 treatment alone resulted in a significant lowering of baseline mRNA levels of all three genes by approx. 30–75%. It also blocked significantly the TNFα-mediated increase in mRNA levels of all three genes. Representative Northern blots from three independent experiments are shown.
Figure 9
Figure 9. NF-kB positively regulates rat GCLM and GSS promoter activities, c-Jun expression and c-Jun-dependent promoter activity, and mediates c-Jun induction by TNFa
Effect of p50 and p65 overexpression on GCLM and GSS promoter activity (A), c-Jun mRNA levels and c-Jun-dependent promoter activity (B). (C) The effect of blocking NF-κB with or without TNFα treatment on c-Jun mRNA level is shown. H4IIE cells were co-transfected with GCLM promoter construct −902/+3-LUC, GSS promoter construct −1164/+2-LUC or Jun2-Luc construct and p50 or p65 expression vector or empty vector control as described in the Materials and methods section. Overexpression of either p50 or p65 more than doubled the GCLM, GSS and c-Jun-dependent promoter activity (A, B), as well as increasing the steady state c-Jun mRNA levels (B). Baseline c-Jun level was 36% lower when NF-κB was blocked by IκBSR (C). TNFα treatment also increased the steady-state c-Jun mRNA level by 108%, which was significantly blocked by IκBSR (C). *P<0.05 versus respective controls (GCLM, GSS or Jun2-Luc). Con, control.
Figure 10
Figure 10. Effect of TNFα on c-Jun and Nrf2 binding to the GCLM ARE (A, B) and importance of the ARE in TNFα-mediated induction of GCLM promoter activity (C)
H4IIE cells were treated with TNFα (15 ng/ml, 4 h) or vehicle control and subjected to EMSA with supershift (A) or ChIP analysis (B) as described in the Materials and methods section. (A) EMSA with supershift results: note that TNFα treatment increased the nuclear binding activity of both Nrf2 and c-Jun to the ARE of the rat GCLM. Arrows in (A) point to supershifted bands. 100X Comp, 100-fold unlabelled probe. (B) H4IIE cells were treated with TNFα or vehicle control, then processed for ChIP assay as described in the Materials and methods section. PCR products from amplification of the ARE site after immunoprecipitation with antisera against Nrf2 or c-Jun demonstrate that TNFα treatment led to increased Nrf2 and c-Jun binding to the ARE site. Input genomic DNA (gDNA input) was used as a positive control and a no antibody immunoprecipitation (no Ab) was used as a negative control. Representative results from two experiments are shown. (C) The functional significance of the ARE element. To confirm functionality of the ARE, EMSA was first performed to document the effect of site-directed mutagenesis on nuclear-binding activity (C, left panel, the number of mutated bases is shown on top; note that nuclear binding was completely abolished only when three bases were mutated). The right panel shows the effect of preventing ARE binding on baseline and TNFα-mediated increase in GCLM promoter activity. H4IIE cells were transfected with wild-type (WT) or mutated (MUT, mutated in three bases) GCLM −329/+3-LUC constructs and treated with TNFα (15 ng/ml, 4 h). Results represent means±S.E.M. from five to nine experiments (C, right panel). Data are expressed as relative luciferase activity compared with that of pGL-3 basic vector control, which is assigned a value of 1.0. Note that the mutant construct had lower baseline promoter activity and TNFα no longer was able to induce its reporter activity. *P<0.001 versus WT −329/+3-LUC (ANOVA followed by Fisher's test).
Figure 11
Figure 11. Proposed model for the interactions between NF-κB, c-Jun, AP-1, ARE and their roles in TNFα-mediated induction of rat GCLC, GCLM and GSS
⊕ indicates activation or induction (NF-κB induces the expression of c-Jun, whereas all other effects are transactivation of the respective binding sites).
See this image and copyright information in PMC

References

    1. Lu S. C. Regulation of hepatic glutathione synthesis: current concept and controversies. FASEB J. 1999;13:1169–1183. - PubMed (VSports最新版本)
    1. Suthanthiran M., Anderson M. E., Sharma V. K., Meister A. Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens. Proc. Natl. Acad. Sci. U.S.A. 1990;87:3343–3347. - PMC - PubMed
    1. Poot M., Teubert H., Rabinovitch P. S., Kavanagh T. J. De novo synthesis of glutathione is required for both entry into and progression through the cell cycle. J. Cell. Physiol. 1995;163:555–560. - PubMed
    1. Richman P. G., Meister A. Regulation of γ-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J. Biol. Chem. 1975;250:1422–1426. - PubMed
    1. Yan N., Meister A. Amino acid sequence of rat kidney γ-glutamylcysteine synthetase. J. Biol. Chem. 1990;265:1588–1593. - V体育ios版 - PubMed

V体育平台登录 - Publication types

MeSH terms