mRNA-binding protein ZFP36 is expressed in atherosclerotic lesions and reduces inflammation in aortic endothelial cells

Abstract

Objective: We studied the expression and function of an mRNA-binding protein, zinc finger protein-36 (ZFP36), in vascular endothelial cells in vivo and in vitro. We tested the hypotheses that ZFP36 regulates inflammation in vascular endothelial cells and that it functions through direct binding to target cytokine mRNAs. We also tested whether ZFP36 inhibits nuclear factor-κB-mediated transcriptional responses in vascular endothelial cells.

Approach and results: ZFP36 was minimally expressed in healthy aorta but was expressed in endothelial cells overlying atherosclerotic lesions in mice and humans. The protein was also expressed in macrophage foam cells of atherosclerosis. ZFP36 was expressed in human aortic endothelial cells in response to bacterial lipopolysaccharide, glucocorticoid, and forskolin, but not oxidized low-density lipoproteins or angiotensin II. Functional studies demonstrated that ZFP36 reduces the expression of inflammatory cytokines in target cells by 2 distinct mechanisms: ZFP36 inhibits nuclear factor-κB transcriptional activation and also binds to cytokine mRNAs, leading to reduced transcript stability.

Conclusions: ZFP36 is expressed in vascular endothelial cells and macrophage foam cells where it inhibits the expression of proinflammatory mRNA transcripts. The anti-inflammatory effects of ZFP36 in endothelial cells occur via both transcriptional and posttranscriptional mechanisms. Our data suggest that enhancing vascular ZFP36 expression might reduce vascular inflammation.

Keywords: AU-rich element; atherosclerosis; cytokines; endothelial cell; inflammation; zinc finger protein-36.

Figure 1

ZFP36 antiserum detects murine and human ZFP36. A) Western blot of murine RAW 264.7 cells with and without LPS stimulation and 293T cells transfected with empty vector or human ZFP36 expression vector. B) Immunoprecipitation (IP) of ZFP36 from ZFP36-expressing 293T cells followed by WB using the same antiserum (with ACTIN loading control from IP-input). Data are representative of experiments performed three times.

Figure 2

ZFP36 Expression in Mouse and Human Arteries. A) Wild-type mouse aorta, regular diet (20x), Zfp36 (red, anti-mouse Zfp36 antiserum) and DAPI-nuclei (blue). B) and C) ApoE null mouse aorta using the same antiserum (B is 10x and C is 20x, colors as above). D) – I) Dual label fluorescent microscopy of human coronary artery. D) H&E stained, E) Pre-immune serum (red), F) DAPI-nuclei (blue), G) ZFP36 (green, anti-human ZFP36 antiserum), H) CD68 (red), and I) ZFP36 and CD68, merged (yellow). Asterisks indicate vessel lumen, arrows indicate strong endothelial and neointimal staining.

Figure 3

A. ZFP36 expression in primary HAEC cells. HAEC cells were stimulated for four hours with LPS, dexamethasone (DEX), and forskolin (all 50 ug protein/lane). Also shown is positive control 293T cells transfected with ZFP36 expressing plasmid (5 ug protein/lane). B. Lentiviral expression of ZFP36 in HAEC cells compared to vector control cells. Data representative of three independent experiments.

Figure 4

A. ZFP36 reduces mRNA expression of inflammatory factors in primary HAEC cells. HAEC cells were transduced with LV control virus (gray) or ZFP36-expressing LV particles (black). Q-RT-PCR was performed and normalized to vector control for each mRNA target. Values represent the means of three independent experiments with SEM, *P < 0.01 relative to vector control. B. ZFP36 inhibits transcriptional activation of nuclear NF-κB. HAECs were transduced with empty vector-containing lentiviral particles (vector) or human ZFP36-expressing lentiviral particles (ZFP36) with and without LPS stimulation for 30 minutes. NF-κB activation assays were performed from nuclear lysates and normalized to total nuclear protein for each sample, shown relative to vector control. Experiments were performed in duplicate, and mean values with SEM are shown for five independent experiments. *P < 0.05.

Figure 5

ZFP36 represses IL-6 and MCP-1 protein expression in basal and LPS-stimulated HAEC cells. Cells were transduced with empty vector-containing lentiviral particles (vector) or human ZFP36-expressing lentiviral particles (ZFP36) with and without LPS stimulation for 24h. Culture medium was collected and IL-6 (A) or MCP-1 (B) proteins were quantitated using ELISA. Data were normalized to total protein in each well. Shown are means with SEM of seven (MCP-1) and eight (IL-6) independent experiments. *P < 0.05.

Figure 6

ZFP36 directly binds to inflammatory mRNA transcripts. RAW cells ± LPS stimulation were subjected to ribonucleoprotein IP (using pre-immune or ZFP36 antiserum) followed by Q-RT-PCR. ZFP36 bound directly to Tnfα, IL-6, and Mcp-1 (but not H2A) transcripts. Means with SEM for three independent experiments normalized to pre-immune control. *P < 0.05.