TERC promotes cellular inflammatory response independent of telomerase

Abstract

TERC is an RNA component of telomerase. However, TERC is also ubiquitously expressed in most human terminally differentiated cells, which don't have telomerase activity. The function of TERC in these cells is largely unknown. Here, we report that TERC enhances the expression and secretion of inflammatory cytokines by stimulating NK-κB pathway in a telomerase-independent manner. The ectopic expression of TERC in telomerase-negative cells alters the expression of 431 genes with high enrichment of those involved in cellular immunity. We perform genome-wide screening using a previously identified 'binding motif' of TERC and identify 14 genes that are transcriptionally regulated by TERC. Among them, four genes (LIN37, TPRG1L, TYROBP and USP16) are demonstrated to stimulate the activation of NK-κB pathway. Mechanistically, TERC associates with the promoter of these genes through forming RNA-DNA triplexes, thereby enhancing their transcription. In vivo, expression levels of TERC and TERC target genes (TYROBP, TPRG1L and USP16) are upregulated in patients with inflammation-related diseases such as type II diabetes and multiple sclerosis. Collectively, these results reveal an unknown function of TERC on stimulating inflammatory response and highlight a new mechanism by which TERC modulates gene transcription. TERC may be a new target for the development of anti-inflammation therapeutics.

Figure 1.

TERC promotes inflammatory response. (A) Enriched biological processes after TERC overexpression. Gene ontology was analyzed for upregulated genes in TERC-U2OS compared to pBabe-U2OS. GO terms in blue boxes were immune related biological processes. (B) Enriched signaling pathways after TERC overexpression. KEGG pathway enrichment was analyzed with upregulated genes in TERC-U2OS compared to pBabe-U2OS. Different categories of pathways were boxed and labeled. (C) The mRNA levels of cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated with TNF-α for 1 h, and cells were collected for qPCR. (D) The secreted cytokines after TERC overexpression in U2OS cells. The stable cell lines pBabe-U2OS and TERC-U2OS were stimulated by TNF-α for 6 h, and culture medium was collected for ELISA. (E) The mRNA levels of cytokines after TERC knockdown in B2–17 cells. The cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 1 h of transfection. Cells were then collected for qPCR. (F) The secreted cytokines in B2–17 cells after TERC or TERT knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with LPS during the last 6 h of transfection. Culture medium was then collected for ELISA. (G) The knockdown efficiency of TERT. The B2–17 cells were transfected with TERT siRNAs for 72 h, and cells were collected for qPCR. (H) The secreted cytokines in BJ after TERC knockdown. Cells were transfected with indicated siRNAs for 72 h and treated with TNF-α during the last 6 h of transfection. Culture medium was collected for ELISA. All values are means ± SEM of more than three independent experiments (*P< 0.05, **P< 0.01,***P< 0.001).

Figure 2.

TERC activates the NF-κB pathway. (A) Inhibition of NF-κB or STAT3 activation by inhibitors in TERC-U2OS. TERC-U2OS cells were treated with inhibitors of AKT, IκB and STAT3 (Perifosine, Bay11–7082 and Stattic, respectively) for 6h, and cells were collected for Western blotting of indicated proteins. (B-D) The TERC promotion of cytokine secretion was counteracted by inhibitors of p65 cofactors IκB and STAT3. Stable cell lines pBabe-U2OS and TERC-U2OS were treated with TNF-α plus indicated inhibitor for 6 h, and culture medium was collected for IL-6, IL-8 and CSF2 detection by ELISA. (E) Total and phosphorylated p65 were upregulated by TERC. PBabe-U2OS and TERC-U2OS cells were treated with TNF-α for indicated times. Total and phosphorylated levels of p65 were determined by Western blotting. (F) Quantification of (E). Protein levels in (E) were quantified and normalized by actin. (G) Inhibited nuclear translocation of p65 in TERC depleted cells. B2–17 cells were transfected with TERC siRNA or NC for 72 h and then treated with TNF-α (10 ng/ml) for 20 min. Immunofluorescence (IF) using p65 antibody was performed to determine the amount of p65 in nucleus and cytoplasm. (H) Quantification of (G). Nuclear p65 (Fluorescence intensity) was quantified as a percentage of overall p65 in cell. ∼100 cells were counted for each experiment. (I, J) NF-κB luciferase reporter assay for analysis of activation of NF-κB in TERC overexpressed (I) and depleted cells (J). HEK293T cells were transfected with TERC/pBabe (I) or siTERC/NC (J) together with NF-κB-luc and pRL-TK. Luciferase activity was determined 48 h after transfection. All values are means ± SEM of more than three independent experiments (*P < 0.05, **P< 0.01, ***P< 0.001).

Figure 3.

TERC targets gene's promoters using its binding motif. (A) Schematic diagram of TERC targeting to gene's promoters that contain the sequence of TERC binding motif. (B). Distribution of TERC targeted fragments on genome. The fragment was classified according to its distance to the nearest TSS. (C) The mRNA levels of 30 genes with TERC binding motif in their promoters in TERC-U2OS vs pBabe-U2OS cells. (D–F). Determination of mRNA levels of top 6 genes in (C) that have the highest fold change (up- and downregulation) in response to TERC expression. Gene expression was tested in indicated cells with or without TERC knockdown. Each panel represents different cells as indicated. All values are means ± SEM of more than three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 4.

RNA–DNA triplex formation between TERC and gene's promoters. (A) TERC binds to the promoter of indicated gene in vivo. TERC ChIRP was performed using TERC probe. LacZ probe was used as control. Promoters of the indicated genes were detected by PCR. (B) TERC binds to the promoters of indicated genes in vitro. EMSA was performed to detect the triplex formation between synthesized gene promoters containing TERC binding motif and TERC. RNA with random sequence was used as control. Products were analyzed on 2% agarose gels. (C) Triplex formation in a dose dependent manner. Two picomoles of indicated gene promoters were incubated with increasing amounts of TERC. Products were analyzed on 2% agarose gels. (D) Expression levels of indicated genes after RNH1 knockdown. U2OS cells were transfected with siRNH1 for 72 h, and corresponding mRNAs were detected by qPCR. (E) Melting temperature decreased after triplex formation at neutral pH. Melting temperatures of gene's promoters were detected in the presence or absence of TERC or TERC1–49. All values are means ± SEM of more than three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 5.

TERC activates NF-κB pathways through targeting immune-related genes. (A) The knockdown efficiency of indicated genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h, and corresponding mRNAs were detected by qPCR. (B) P65 and STAT3 were downregulated by knockdown of TERC targeted genes. The TERC-U2OS cells were transfected with indicated siRNAs for 72 h. Total and phosphorylated p65 and STAT3 expressions were determined by western blotting. (C) The TERC promotion of IL-6 secretion was counteracted by knockdown of TERC targeted genes. PBabe-U2OS and TERC-U2OS cells were transfected with indicated siRNAs for 72 h and treated with TNF-α during the last 6 h of transfection. Culture medium was collected for IL-6 detection by ELISA. All values are means ± SEM of more than three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 6.

The expression levels of TERC and TERC targeted genes in inflammation related diseases. (A) TERC was upregulated in CD14⁺ cells of diabetic patients. Data were downloaded from the GEO database (GSE54350 & GSE65561), and TERC expression levels were analyzed (n ≥ 10, *P< 0.05, **P< 0.01). (B) TERC was upregulated in CD14⁺ cells of multiple sclerosis patients. Data were downloaded from the GEO database (GSE66988), and TERC expression levels were analyzed (n ≥ 10, *P< 0.05, **P< 0.01). (C) Expression levels of TERC targeted genes in CD14⁺ cells of diabetic patients. Data analysis is the same as (A). (D) Expression levels of TERC targeted genes in CD14⁺ cells of multiple sclerosis patients. Data analysis is the same as (B). (E) Expression levels of cytokines in CD14⁺ cells of diabetic patients. Data analysis is the same as (A), except that ‘n’ of IL6 is 6 because IL6 is missing in GSE65561. (F) Expression levels of cytokines in CD14⁺ cells of multiple sclerosis patients. Data analysis is the same as (B).