Characterization of Synergistic Induction of CX3CL1/Fractalkine by TNF-α and IFN-γ in Vascular Endothelial Cells: An Essential Role for TNF-α in Post-Transcriptional Regulation of CX3CL1

Abstract

CX3CL1/Fractalkine, a chemokine specific to monocytes and NK cells, is induced synergistically by TNF-α and IFN-γ in vascular endothelial cells. However, the mechanism for this synergism remains unclear. This study explored the hypothesis that the CX3CL1 expression is regulated at a posttranscriptional level, which may responsible for the synergism between TNF-α and IFN-γ. Brief exposure of HUVECs to TNF-α led to a robust increase in IFN-γ–induced CX3CL1 production. We found that TNF-α stabilized CX3CL1 mRNA in HUVECs stimulated with IFN-γ. Cloning of 3′untranslated region (UTR) of CX3CL1 mRNA revealed the presence of a single copy of nonametric AU-rich element in its 3′UTR, and a luciferase reporter assay showed that a single AU-rich element is a crucial cis-element in the posttranscriptional regulation of CX3CL1. TNF-α treatment resulted in the phosphorylation of p38 MAPK and its downstream target, MAPK-activated protein kinase-2, but IFN-γ did not affect the levels of MAPK and MAPK-activated protein kinase-2 phosphorylation induced by TNF-α. Treatment of the cells with an inhibitor of p38 MAPK accelerated the decay of CX3CL1 mRNA induced by TNF-α or the combination of TNF-α and IFN-γ. Immunoprecipitation assay revealed that mRNA stabilizer HuR directly binds to 3′UTR of CX3CL1 mRNA. CX3CL1 expression is under control of posttranscriptional regulation, which is involved in the synergistic induction of CX3CL1 in response to the combined stimulation with TNF-α and IFN-γ.

Arterial and Venous Endothelia Display Differential Functional Fractalkine (CX 3 CL1) Expression by Angiotensin-II

“…28,30,31,45 Using a pharmacological approach, we have consistently demonstrated that Ang-II-induced CX 3 CL1 arterial expression and mononuclear cell adhesiveness are regulated through ERK1/2 and p38MAPK activation and that both pathways are involved in endothelial CX 3 CL1 upregulation. 33,34 Both ROS and MAPKs can regulate the transcription of many genes through their action on downstream targets, such as NF-κB 31,32 Indeed, disruption of NFκB transactivation effectively blocked Ang-II-induced responses in our study, as previously observed for other CX 3 CL1-inducing cytokines. 34,43 Taken together all these findings and although Ang-II-induced signal transduction mechanisms converge with many of TNFα, 45 it is likely that Ang-II-induced fractalkine synthesis is secondary to TNFα formation because increased CX 3 CL1 mRNA expression was detected after 4 hours of Ang-II stimulation but not at 1 hour.…”

“…31,32 In addition, some of these vascular signaling pathways have been implicated in CX 3 CL1 expression. 33,34 Thus, we examined the potential involvement of these signaling pathways in Ang-II-induced responses. Preincubation of arterial endothelial cells with an ERK1/2 or a p38MAPK inhibitor but not with a JNK inhibitor undermined the CX 3 CL1 expression induced by Ang-II stimulation by 82% and 93%, respectively ( Figure 5A).…”

“…19,34,35 Such a scenario can be envisaged in both acute and chronic inflammatory processes such as atherosclerosis, where the concomitant presence of both cytokines with Ang-II is very likely to occur. As illustrated in Figure 6A, compared with the signal detected in unstimulated cells, an increase in the mean fluorescence intensity was observed when HUAEC were stimulated independently with Ang-II, TNFα, or IFNγ for 24 hours ( Figure 6A).…”

Effect of the Plant Derivative Compound A on the Production of Corticosteroid-Resistant Chemokines in Airway Smooth Muscle Cells

“…Our silencing experiments confirmed the key role of IRF-1 in ASM cells in driving the expression of GC-resistant chemokines via transcriptional (CCL5) and posttranscriptional mechanisms (CX3CL1). The post-transcriptional role of IRF-1 is interesting because a previous report performed in vascular endothelial cells showed the importance of posttranscriptional pathways in the synergistic induction of CX3CL1 by the same cytokine combination (TNF-a/IFN-g), although the implication of IRF-1 was not investigated (44). More importantly, we made the unique finding that IRF-1 was not involved in the regulation of CXCL10 expression induced by TNF-a/IFN-g ( Figure 5).…”

Double-Stranded RNA Induces Biphasic STAT1 Phosphorylation by both Type I Interferon (IFN)-Dependent and Type I IFN-Independent Pathways

“…Immunofluorescence staining was performed as reported previously (32). Briefly, A549 or U5A cells were grown on glass coverslips and then fixed with 4% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin for 1 h. The cells were then incubated for 1 h with mouse monoclonal anti-IFN-␤ (R&D systems Minneapolis, MN) (1:100) or rabbit polyclonal anti-STAT1 antibody (Santa Cruz Biotechnology) (1:200), respectively.…”