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.

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Type I interferons (IFNs) are a family of cytokines that exhibit various biological activities. Besides their roles in immune response, IFNs have been known to modulate cell proliferation and to induce apoptosis. Thus, IFNs are used as an antitumor agent against certain types of cancer, but it is unclear why many other cancers are not influenced by IFNs. Here, we found that IFN-alpha2b, a subfamily of IFN-alpha, enhanced proliferation of HeLa cells, a cell line derived from human cervical cancer. IFN-alpha2b was rather inhibitory on the growth of other types of cervical cancer cells including those positive for HPV. Among the proliferation- and the apoptosis-related genes, p21(cip1/waf1) (p21) was upregulated by IFN-alpha2b, whereas p53, p27 or BCL-2 associated X protein (BAX) was not affected. IFN-alpha2b did not alter promoter activities of p21 but did prolong the decay of p21 mRNA. In contrast, the level of p21 protein was lowered by IFN-alpha2b, and half-life analysis of p21 protein revealed that IFN-alpha2b enhances p21 protein instability in HeLa cells. Pretreatment of the cells with MG132, a proteasome inhibitor, abolished the IFN-alpha2b-mediated p21 degradation, suggesting that IFN-alpha2b accelerated the ubiquitin-proteasome dependent degradation of p21. Consistent with these results, IFN-alpha2b increased S-phase cell cycle distribution in HeLa cells. In addition, IFN-alpha2b liberated the cells from G(1)-phase arrest by 5-fluorouracil (5-FU) and from G(2)-phase arrest by paclitaxel. These results provide a novel role of Type I IFNs in cell cycle regulation and may define an importance of individualized IFN-based therapy against specific types of cancer.