Figure 1.
Human GLS1 promoter construct and sequence analysis.
(A). Schematic diagram of the human GLS1 promoter-driven luciferase reporter construct. The construct contains the Firefly luciferase coding sequence (black) with regions of the promoter (blank) and the 5' UTR (shaded). The restriction enzymes used for cloning are shown. (B). Sequence analysis of the human GLS1 promoter. The TSS is designated as +1. The 5' UTR is shown in gray. The coding sequence is underlined. Putative transcription factor binding sites along the promoter sequence are highlighted in different colors, with their corresponding transcription factors shown on top of the sequence. Highlighted in red: TATA box, dark green: CAAT box, pink: AP-1, blue: SP-1, bright green: STAT, grey: 5' UTR glutaminase; font color in blue: NF-1.
Figure 2.
IFN-α specifically activates human GLS1 promoter.
HEK 293T cells were co-transfected with the human GLS1 promoter construct and pRL-SV40. 24 hours later, the cells were treated with either various individual cytokines for another 24 hours (A), IFN-α of varying doses for 24 hours (B), or with 100 U/ml IFN-α for varied time lengths (C). In (A), 100 U/ml IFN-α, 100 ng/ml IFN-γ, 10 ng/ml IL-1β, 10 ng/ml IL-6, 25 ng/ml IL-10, 50 ng/ml TRAIL, or 50 ng/ml TNF-α was used. Luciferase activity in the lysates was measured by luminescence detection. Renilla luciferase was used as internal control to normalize transfection efficiency. The data are representative of three independent experiments and are the means of triplicate samples. #, p<0.05, *, p<0.01, **, p<0.001 in comparison to control.
Figure 3.
STAT1 phosphorylation is required for IFN-α to activate the GLS1 promoter and induce glutaminase expression and function.
(A). MDM were treated with different doses of IFN-α for 24 hours, then p-STAT1 (Tyr 701), STAT1, KGA and GAC were detected by Western blot. β-actin was used as a loading control. (B). Intracellular glutamate was detected using the Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data are representative of three independent experiments using three different donors and are the means of triplicate samples. (C-F). Levels of p-STAT1 (C), STAT1 (D), GAC (E) and KGA (F) in Western blot (A) were normalized as a ratio to β-actin and shown as fold change relative to control. Results are shown as the average ± SEM of three independent experiments with three different donors, (G, H). Correlation of p-STAT1 with GAC (G) and p-STAT1 with KGA (H) in representative donor are shown. (I, J). HEK 293T cells were co-transfected with the GLS1 promoter construct and pRL-SV40. 24 hours later, the cells were pretreated with 1 µM fludarabine or 1:10,000 DMSO for 1 hour, then treated with or without 100 U/ml IFN-α for 24 hours. (I). p-STAT1 and STAT1 were detected by Western blot, β-actin was used as a loading control. (J). Luciferase activity in the lysates was measured by luminescence detection. Renilla luciferase was used to normalize transfection efficiency. The data are representative of three independent experiments and are the means of triplicate samples. #, p<0.05. *, p<0.01, **, p<0.001 when compared to control.
Figure 4.
STAT1 binds directly with the GLS1 promoter in IFN-α treated MDM.
(A). The predicted STAT1 binding sites in the human GLS1 promoter, TSS is designated as +1. (B). MDM were treated with 100 U/ml IFN-α for 1 hour, then ChIP assay was performed using digested chromatin, p-STAT1 (Tyr 701) and STAT1 antibodies, or IgG antibody as a negative control. Purified DNA was analyzed by quantitative real-time PCR using specific primers. The amount of immunoprecipitated DNA is represented as signal relative to the total amount of input chromatin. The data are representative of three independent experiments using three different donors. #, p<0.05, *, p<0.01 in comparison with control. (C). MDM were pretreated with 1 µM fludarabine or 1:10,000 DMSO for 1 hour, then treated with or without 100 U/ml IFN-α for another hour. ChIP assay was performed using p-STAT1 antibody as in (B). The data are representative of two independent experiments using two different donors. *, p<0.01 in comparison with control that were treated with DMSO only; #, p<0.05 in comparison to cells treated with IFN-α and DMSO.
Figure 5.
STAT1 binds directly with the GLS1 promoter in HIV-1 infected MDM.
(A-E). MDM were infected with or without HIV-1ADA for 5 days. (A). ChIP was performed using p-STAT1 (Tyr 701) antibody; IgG was used as a negative control. (B). Supernatants were tested for RTase activity. (C). p-STAT1 and STAT1 were detected by Western blot with β-actin used as a loading control. (D). Glutamate was detected in supernatants by HPLC. (E). Intracellular glutamate was detected using the Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data (A-E) are representative of three independent experiments using three different donors. (F) Human MDM were infected with HIV-1 for 5 days, then total RNA was collected. IFN-α and IFN-β mRNA levels were determined by real-time RT-PCR. Results shown are representative of three independent experiments using three different donors and are means of triplicate samples. (G-I) Human MDM were infected with HIV-1 for 5 days in the presence of IgG or IFN-α/IFN-β neutralizing antibodies. Total RNA and cell lysates were collected and GAC mRNA (G) and protein (H and I) levels were determined by real time RT-PCR and Western blot, respectively. For quantification, GAC expression were normalized as a ratio to β-actin and shown as fold change relative to IgG control . Results (F, I) are shown as the average ± SEM of three independent experiments with three different donors, #, p<0.05, *, p<0.01, **, p<0.001, when compared with uninfected MDM or IgG control.
Figure 6.
Significant correlation between STAT1 and GAC mRNA levels in brain tissues of HAD patients.
Total RNA from post-mortem brain tissue collected from HAD patients, HIV serum-positive patients without dementia, and HIV serum negative individuals were extracted and subjected to real time RT-PCR for STAT1, GAC, IFN-α2 and IFN-β. GAPDH expression was used as an internal control. (A) Levels of STAT1 are normalized as a ratio to GAPDH and shown as fold change relative to the average of HIV serum negative controls. Results are shown as the average ± SEM, #, p<0.05. Correlation of GAC with STAT1 (B), IFN-α2 (C) and IFN-β (D) mRNA levels was determined by Spearman correlation.