Interferon regulatory factor 7 mediates obesity-associated MCP-1 transcription

Abstract

Hypertrophy, associated with adipocyte dysfunction, causes increased pro-inflammatory adipokine, and abnormal glucose and lipid metabolism, leading to insulin resistance and obesity-related-health problems. By combining DNA microarray and genomic data analyses to predict DNA binding motifs, we identified the transcription factor Interferon Regulatory Factor 7 (IRF7) as a possible regulator of genes related to adipocyte hypertrophy. To investigate the role of IRF7 in adipocytes, we examined gene expression patterns in 3T3-L1 cells infected with a retrovirus carrying the IRF7 gene and found that enforced IRF7 expression induced the expression of monocyte chemoattractant protein-1 (MCP-1), a key initial adipokine in the chronic inflammation of obesity. CRISPR/Cas9 mediated-suppression of IRF7 significantly reduced MCP-1 mRNA. Luciferase assays, chromatin immunoprecipitation PCR analysis and gel shift assay showed that IRF7 transactivates the MCP-1 gene by binding to its proximal Interferon Stimulation Response Element (ISRE), a putative IRF7 binding motif. IRF7 knockout mice exhibited lower expression of MCP-1 in epidydimal white adipose tissue under high-fat feeding conditions, suggesting the transcription factor is physiologically important for inducing MCP-1. Taken together, our results suggest that IRF7 transactivates MCP-1 mRNA in adipocytes, and it may be involved in the adipose tissue inflammation associated with obesity.

Fig 1. Identification of interferon regulatory factor 7.

A and B: DNA microarray analysis was conducted in 3T3-L1 cells at 2, 8, and 20 days after inducing adipogenic differentiation. Hierarchical clustering analyses identified 503 genes that were upregulated in hypertrophied adipocytes. C: The identified gene set was submitted to TransFind, a software tool to predict the affinity of the transcription factor to promoter regions (−800 to +300 bp from transcription start site). A transcription matrix was defined as significant when the corresponding p-value was < 0.01. The output of the TransFind analysis was listed according to the p-value order. IRF: interferon regulatory factor, ICSBP: interferon consensus sequence-binding protein, AP-1: activator protein 1, PAX-5: paired box protein-5 and NRF-1: nuclear respiratory factor 1. FDR: false discovery rate.

Fig 2. Expression pattern of IRFs in adipocytes during adipogenic differentiation and hypertrophy.

A–I: The IRF1−9 mRNAs during adipogenesis and lipid accumulation in 3T3-L1 adipocytes (n = 4) were quantified by real-time qPCR. J and K: The mRNA levels of IRF7 and IRF9 in eWAT were determined by real-time qPCR. White adipose tissue was excised from C57BL/6J mice fed with normal chow (NC) or a high-fat diet (HFD) for 8 weeks starting at 4 weeks of age. *p < 0.05 and **p < 0.01. All values are means ± SEM.

Fig 3. Identification of IRF7 target gene in adipocytes.

A: The set of upregulated genes by IRF7 in 3T3-L1 adipocytes was obtained by DNA microarray analysis; 97 genes were overlapped with hypertrophy-induced genes. B: MCP-1 mRNA level changes during adipogenesis and lipid accumulation in 3T3-L1 adipocytes (n = 4). C–G: Empty- or IRF7-retrovirus infected 3T3-L1 pre-adipocytes were differentiated into adipocytes. 7 days after the induction of the differentiation, cells were harvested and analyzed by real-time qPCR (n = 4). H and I: Empty- or IRF7-retrovirus infected 3T3-L1 pre-adipocytes were differentiated into adipocytes. At day 6, cells were serum-starved for 5 hours and thereafter incubated in DMEM containing LPS at 10 ng/ml for 24 hours. Cells were harvested and analyzed by real-time qPCR (n = 4). J and K: IRF7 in 3T3-L1 cells were silenced by transfecting IRF7-PX459 into them and selected with puromycin. The resulting 3T3-L1 pre-adipocytes were differentiated into adipocytes. After 20 days of the induction into adipocytes, total RNA was isolated, and IRF7 and MCP-1 mRNA levels were examined (n = 5–6). *p < 0.05 and **p < 0.01. All values are means ± SEM.

Fig 4. Effects of IRF7 expression on promoter activity of murine MCP-1.

A: The promoter −2933nt to +71nt of the mouse MCP-1, upstream of the transcription start site, was inserted into the pGL4.19 reporter vector. Putative binding motifs are shown. B: The activity of MCP-1 promoter was measured in HEK293 cells with various lengths of 5’-fragments. Results are shown as fold inductions relative to the values in the cells transfected with Empty-pcDNA vector (n = 4). C: Luciferase assay performed in HEK 293 cells with MCP-1 promoter- pGL4.19 vector carrying one or two proximal ISRE mutations. Results are shown as fold inductions relative to the values in the cells transfected with Empty-pcDNA vector (n = 4). *p < 0.05 and **p < 0.01. All values are means ± SEM.

Fig 5. ChIP-PCR analysis of IRF7-myc-C binding to the mouse MCP-1 promoter.

A: Quantitative, real-time PCR was performed with the indicated primer pair using an anti-myc antibody immunoprecipitated DNA fragment as template. Normal mouse IgG was used as negative control (n = 4). B: PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining. C: DNA and nuclear protein interaction was examined by EMSA. DNA probes for ISRE (−228 to −204 nt) (WT) and its mutant (Mut) and for ISRE (−178 to −154 nt) and its mutant were used. The DNA probes confirmed to bind to IRF7 in the past [14] was used as positive control (PC). The arrowhead indicates protein-DNA complex. *p < 0.05 and **p < 0.01. All values represent mean ± SEM.

Fig 6. Characteristics of IRF7 knockout mice and effects of short-term high-fat diet (HFD) on IRF7 in adipocytes.

A–J: At the age of 4 weeks, IRF7 KO or WT control mice were started on either a HFD or a NC (n = 6–8). After 3 weeks, subcutaneous white adipose tissue (sWAT), eWAT and liver tissue were excised. Body weights were measured during the HFD feeding (A). Excised tissues were weighed (B) and eWAT was subjected to HE staining (C; wild type mice and D: knockout mice). The effect of short-term HFD on MCP-1 mRNA in eWAT was assessed in WT mice (E). The expressions of several inflammatory adipokines (MCP-1, TNF-α, IL1β) and macrophage marker gene (F4/80) were examined by real-time qPCR in WT and KO mice (F–I). IRF7 mRNA levels in eWAT were also measured in WT mice (J). 3T3-L1 adipocytes (at day 5) were transfected with IRF7-myc expression vector. After 24 hours, the cells were treated with 400 μM palmitic acid (PA), 100 ng/ml LPS, or with a combination of both. Cells were lysed and separated into cytosol and nuclear fractions, and subjected to SDS-PAGE and immune blot analysis. *p < 0.05 and **p < 0.01. All values are means ± SEM.