Activation of the JAK/STAT3 and PI3K/AKT pathways are crucial for IL-6 trans-signaling-mediated pro-inflammatory response in human vascular endothelial cells

Abstract

Background: IL-6 classic signaling is linked to anti-inflammatory functions while the trans-signaling is associated with pro-inflammatory responses. Classic signaling is induced via membrane-bound IL-6 receptor (IL-6R) whereas trans-signaling requires prior binding of IL-6 to the soluble IL-6R. In both cases, association with the signal transducing gp130 receptor is compulsory. However, differences in the downstream signaling mechanisms of IL-6 classic- versus trans-signaling remains largely elusive.

Methods: In this study, we used flow cytometry, quantitative PCR, ELISA and immuno-blotting techniques to investigate IL-6 classic and trans-signaling mechanisms in Human Umbilical Vein Endothelial Cells (HUVECs).

Results: We show that both IL-6R and gp130 are expressed on the surface of human vascular endothelial cells, and that the expression is affected by pro-inflammatory stimuli. In contrast to IL-6 classic signaling, IL-6 trans-signaling induces the release of the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1) from human vascular endothelial cells. In addition, we reveal that the classic signaling induces activation of the JAK/STAT3 pathway while trans-signaling also activates the PI3K/AKT and the MEK/ERK pathways. Furthermore, we demonstrate that MCP-1 induction by IL-6 trans-signaling requires simultaneous activation of the JAK/STAT3 and PI3K/AKT pathways.

Conclusions: Collectively, our study reports molecular differences in IL-6 classic- and trans-signaling in human vascular endothelial cells; and elucidates the pathways which mediate MCP-1 induction by IL-6 trans-signaling.

Keywords: Endothelium; HUVECs; Interleukin-6 signaling; Monocyte chemoattractant Protein-1; Pro-inflammatory cytokines.

Fig. 1

Expression and regulation of IL-6R and gp130 in human vascular endothelial cells. Flow cytometry analyses of EDTA detached endothelial cells stained for a IL-6R and b gp130. The light grey histograms show respective FMO (fluorescence minus one) controls and the dark grey histograms show IL-6R or gp130 stained cells. ELISA analyses of the levels of c IL-6R and d gp130 in cell lysates and medium after 48 h of IL-6 knockdown. ELISA analyses of the levels of e IL-6R and f gp130 in cell lysates and medium after 48 h of treatment with TNF-α (50 ng/ml) and LPS (100 ng/ml). Data is presented as mean ± SEM of at least 3–4 experiments each run-in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control

Fig. 2

MCP-1 mRNA expression and release from human vascular endothelial cells. a qPCR analyses depicting the relative expression of MCP-1 mRNA after exposure to IL-6 (100 ng/ml) alone or in combination with sIL-6R (200 ng/ml). b ELISA data showing the MCP-1 release into medium (48 h) induced by increasing concentrations of IL-6 alone or in combination with sIL-6R. Data is presented as mean ± SEM of 3 experiments each run-in duplicate. *p < 0.05, **p < 0.01 compared to its respective control

Fig. 3

Western blot analyses showing phosphorylation of a STAT3^Tyr705, b AKT^Ser473 and c ERK1/2^{Thr202/Tyr204} in human vascular endothelial cells treated with IL-6 alone (50 ng/ml) or in combination with sIL-6R (100 ng/ml). One representative blot containing the phosphorylated protein, total protein and β-tubulin (loading control) is shown for each pathway (left column). The signals from the phosphorylated proteins and total proteins are first normalized to β-tubulin, and the ratio of the phosphorylated proteins and the total proteins are calculated. The graphs show arbitrary units (a.u., control is set to 1) compiled from 3 independent experiments presented as mean ± SEM for each pathway (right column). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control

Fig. 4

The effect of different inhibitions on IL-6 trans-signaling induced MCP-1 mRNA expression. The effect of a CP960550 (JAK inhibitor, 10 μM), b STAT3 knockdown, c LY294002 (PI3K inhibitor, 50 μM) and d PD98059 (MEK inhibitor, 10 μM) on trans-signaling (IL-6 = 100 ng/ml, sIL-6R = 200 ng/ml) induced MCP-1 expression is presented as mean ± SEM (n = 3 for each). Western blots show effect of CP960550, STAT3 knockdown, LY294002 and PD98059 on IL-6 trans-signaling (IL-6 = 50 ng/ml, sIL-6R = 100 ng/ml) induced phosphorylation of downstream pathway proteins. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control