Trail (TNF-related apoptosis-inducing ligand) induces an inflammatory response in human adipocytes

Abstract

High serum concentrations of TNF-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor protein family, are found in patients with increased BMI and serum lipid levels. In a model of murine obesity, both the expression of TRAIL and its receptor (TRAIL-R) is elevated in adipose tissue. Accordingly, TRAIL has been proposed as an important mediator of adipose tissue inflammation and obesity-associated diseases. The aim of this study was to investigate if TRAIL regulates inflammatory processes at the level of the adipocyte. Using human Simpson-Golabi-Behmel syndrome (SGBS) cells as a model system, we found that TRAIL induces an inflammatory response in both preadipocytes and adipocytes. It stimulates the expression of interleukin 6 (IL-6), interleukin 8 (IL-8) as well as the chemokines monocyte chemoattractant protein-1 (MCP-1) and chemokine C-C motif ligand 20 (CCL-20) in a time- and dose-dependent manner. By using small molecule inhibitors, we found that both the NFκB and the ERK1/2 pathway are crucial for mediating the effect of TRAIL. Taken together, we identified a novel pro-inflammatory function of TRAIL in human adipocytes. Our findings suggest that targeting the TRAIL/TRAIL-R system might be a useful strategy to tackle obesity-associated adipose tissue inflammation.

Figure 1

TRAIL induces a pro-inflammatory expression profile in human preadipocytes and adipocytes. SGBS preadipocytes and adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (+)(30 ng/ml) or vehicle (−) as indicated. After 12 hours, RNA was harvested and subjected to mRNA array analysis (GeneChip Human Gene 1.0 ST Array; Affymetrix). Heatmaps display the TRAIL-regulated inflammatory genes in preadipocytes (A) and adipocytes (B). An evidence-based STRING 10 analysis was performed to visualize the network of TRAIL-regulated genes in preadipocytes and in adipocytes (C).

Figure 2

TRAIL regulates the production of cytokines and chemokines in a time- and dose-dependent manner. (A–D) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle for 6, 12 and 24 hours. The mRNA expression of IL-6 (A), IL-8 (B), MCP-1 (C) and CCL-20 (D) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. Two-way ANOVA and Sidak’s multiple comparison were used to test for statistical significance. (E–H) SGBS adipocytes on day 14 of adipogenic differentiation were treated with increasing doses of TRAIL or vehicle for 6 hours. The mRNA expression of IL-6 (E), IL-8 (F), MCP-1 (G) and CCL-20 (H) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 3 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. (I–L) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle. After 24 hours, media supernatants were collected and IL-6 (I), IL-8 (J), MCP-1 (K) and CCL-20 (L) concentrations were determined by ELISA. Means and SEM of 3–7 independent experiments are shown. Unpaired Student’s t-tests were used to test for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

TRAIL induces the expression of cytokines and chemokines in human primary adipocytes. Human primary adipose stromal cells were isolated from white adipose tissue (n = 7) and adipogenesis was induced ex vivo. On day 14 of adipogenesis, the cells were treated with TRAIL (30 ng/ml) or vehicle for 6 hours and the gene expression of IL-6 (A), IL-8 (B), MCP-1 (C) and CCL-20 (D) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Values are means and SEM of 7 independent experiments. Unpaired Student’s t-test was used to test for statistical significance. *p < 0.05, **p < 0.01.

Figure 4

TRAIL triggers caspase activation. (A) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). Cleavage of caspase-8 and caspase-3 was analyzed by Western blot. α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. (B–E) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the pan-caspase inhibitor zVAD.fmk (20 μM). After 6 hours, IL-6 (B), IL-8 (C), MCP-1 (D) and CCL-20 (E) expression was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 3 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

TRAIL induces the phosphorylation of IκBα. (A) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). Cells stimulated with macrophage-conditioned medium (MaCM) were used as a positive control. The phosphorylation of IκBα was analyzed by Western blot. α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. (B) SGBS adipocytes on day 14 of adipogenic differentiation were treated for 2 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and nuclear extracts were prepared. DNA binding activity of NFκB was analyzed by electrophoretic mobility shift assay (EMSA). One representative experiment out of three performed experiments is presented. (C) SGBS adipocytes on day 7 of adipogenic differentiation were transfected with NFκB Firefly luciferase reporter vector and Renilla luciferase control reporter vector. On day 9, cells were treated for 24 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and luciferase activity was determined. Values are means and SEM of 3 different experiments. Unpaired Student´s t-test was used to test for statistical significance. (D-H) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the IKK inhibitor SC-514 (100 μM). After 6 hours, the phosphorylation of IκBα was analyzed by Western blot (D). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, the expression of IL-6 (E), IL-8 (F), MCP-1 (G) and CCL-20 (H) was assessed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

TRAIL induces the phosphorylation of ERK1/2. (A) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). The phosphorylation of ERK1/2, JNK and AKT was determined by Western blot. (B–F) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the MEK1/2 inhibitor PD-0325901 (100 nM). After 6 hours, the phosphorylation of ERK1/2 and IκBα was analyzed by Western blot (B). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, IL-6 (C), IL-8 (D), MCP-1 (E) and CCL-20 (F) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.