Interferon-inducible IFI16, a negative regulator of cell growth, down-regulates expression of human telomerase reverse transcriptase (hTERT) gene

Abstract

Background: Increased levels of interferon (IFN)-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22) in human normal prostate epithelial cells and diploid fibroblasts (HDFs) are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT) gene.

Methodology/principal findings: We optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP) assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT.

Conclusions/significance: These data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases.

Figure 1. Reduced expression levels of IFI16 protein in aphidicolin-treated human normal diploid fibroblasts are associated with increased expression levels of the hTERT and telomerase activity.

(A and B) Total RNA isolated from untreated (control) or aphidicolin (5 µg/ml for 24 h) treated young WI-38 fibroblasts was subjected cDNA synthesis followed by quantitative real-time PCR using the TaqMan assay for the IFI16 gene (A) or hTERT gene (B). Results are mean values of triplicate experiments and the error bars represent standard deviation (^* p<0.05). (C) Total protein extracts prepared from untreated (lane 1) or aphidicolin (5 µg/ml for 24 h) treated (lane 2) young WI-38 fibroblasts were subjected immunoblotting using antibodies specific to the indicated proteins. (D) Extracts containing the indicated amounts (µg) of proteins from control (lanes 2–4) or aphidicolin-treated (lanes 5–7) young WI-38 cells were subjected to TRAPeze assays without any treatment (lanes 2, 3, 5, and 6) or after heat treatment (lanes 4 and 7) to detect the telomerase activity. As controls, extracts from HeLa cells (lane 8) or buffer alone (lane 9) were also included in the assay. The reaction products were subjected to native polyacrylamide gel electrophoresis along with DNA fragments of increasing length as size markers (DNA-ladder).

Figure 2. Reduced expression levels of IFI16 protein in human normal diploid fibroblasts after treatment with histone deacetylase inhibitor are associated with increased expression of hTERT and increased telomerase activity.

(A) Total RNA isolated from untreated (control, lane 1) or CGK1026 (10 µM for 24 h, lane 2) treated young WI-38 fibroblasts was subjected cDNA synthesis followed by semi-quantitative PCR using a pair of primer specific to the IFI16, hTERT, or actin. As a positive control, we used RNA from human HT1080, a human fibrosarcoma cell line. (B) Total RNA isolated from untreated (control) or CGK1026 (10 µM for 24 h; treated) treated young WI-38 fibroblasts was subjected cDNA synthesis, followed by quantitative real-time PCR using the TaqMan assay for the hTERT gene. Results are mean values of triplicate experiments and error bars represent standard deviation (^** p<0.005). (C and D) Total protein extracts prepared from untreated (lane 1) or CGK1026 (10 µM for 24 h; treated) treated young WI-38 fibroblasts were subjected to immunoblotting using antibodies specific to the indicated proteins.

Figure 3. IFI16 inhibits c-Myc-stimulated transcription and hTERT expression in HeLa cells.

(A) Total protein extracts prepared from HeLa cells infected with control retrovirus (lane 1) or a virus encoding IFI16 protein (lane 2) were subjected to immunoblotting using antibodies specific to the indicated proteins. (B) Sub-confluent cultures of HeLa cells were transfected with pMyc-TA-luc reporter plasmid (1.0 µg) along with a second pRL-TK reporter plasmid (0.2 µg) and an empty plasmid (pCMV; column 1), a plasmid encoding c-Myc (column 2 and 4), a plasmid encoding IFI16 (column 3), or both plasmids encoding c-Myc and increasing amounts of the plasmid encoding IFI16 protein (column 5 and 6). After 44–48 h of transfections, cells were lysed and the lysates were analyzed for dual luciferase activity. Normalized relative luciferase activity in control cells is indicated as 1.0. (C) Total protein extracts prepared from HeLa cells infected with control retrovirus (lane 1) or a virus encoding antisense to IFI16 mRNA (lanes 2) were subjected to immunoblotting using antibodies specific to the indicated proteins.

Figure 4. Increased levels of IFI16 protein in WI-38 cells reduce hTERT levels and inhibit telomerase activity.

(A) Total protein extracts prepared from WI-38 cells transfected with control siRNA (lane 1) or IFI16 siRNA RNA (lane 2) were subjected to immunoblotting using antibodies specific to the indicated proteins. (B) Total protein extracts prepared from WI-38 cells, either transfected with control (pCMV) vector (lane 1) or pCMV-IFI16 plasmid (lane 2), were subjected to immunoblotting using antibodies specific to the indicated proteins. (C) Total protein extracts prepared from WI-38 cells treated with lipofectamine (mock, lane 1), transfected with control (pCMV) vector (lane 2) or pCMV-IFI16 plasmid (lane 3) were subjected to immunoblotting using antibodies specific to the indicated proteins. (D) Extracts containing the indicated amounts (µg) of proteins from mock transfected (lanes 2 and 3)CMV transfected (lanes 4 and 5), or pCMV-IFI16 transfected (lanes 6 and 7) young WI-38 cells were subjected to TRAPeze assays to detect the telomerase activity. As controls, extracts from HeLa cells (lane 8) or buffer alone (lane 9) were also included in the assay. The reaction products were subjected to native polyacrylamide gel electrophoresis along with DNA fragments of increasing lengths as size markers (DNA-ladder, lanes 1 and 10). (E) Quantitation (using the Bio-Rad imager) of the intensities of the DNA fragments on the gel (in lanes 4, 5, 6, and 7) that were generated during the TRAPeze assays, which is shown in the panel (D).

Figure 5. The IFI16 protein inhibits c-Myc-induced transcription of the hTERT gene.

Sub-confluent cultures of young WI-38 cells were transfected with hTERT-luc reporter plasmid (1.8 µg) along with a second reporter pRL-TK (0.2 µg) plasmid and an empty vector (pCMV), c-Myc encoding plasmid, or IFI16 encoding plasmid as described in methods. 40–44 h after transfections, firefly luciferase and Renilla luciferase activities were assayed using dual-luciferase reporter assay kit. Relative luciferase activity was expressed as the ratio of the firefly luciferase and Renilla luciferase activity. The numbers indicate fold change in the activity of the firefly luciferase.

Figure 6. Increased levels of IFI16 protein in human cells inhibit c-Myc-induced expression of the hTERT gene.