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Interleukin (IL)-18, formerly known as interferon (IFN)-γ-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-γ, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-γ. Mutational analysis revealed that a proximal γ-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-γ-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-γ-stimulated DLD-1 colon carcinoma cells.
Highlights
• TAM polarization induces CP RNA.
• CP RNA expression is regulated by HIF-2 and STAT1.
• CP RNA is transferred from TAMs to HT1080 cells.
• CP RNA is translated by HT1080 cells and protects from ferroptosis.
• Co-cultured HT1080 cells decrease iron and lipid peroxidation.
Abstract
Solid tumors are characterized by hypoxic areas, which are prone for macrophage infiltration. Once infiltrated, macrophages polarize to tumor associated macrophages (TAM) to support tumor progression. Therefore, the crosstalk between TAMs and tumor cells is of current interest for the development of novel therapeutic strategies. These may comprise induction of an iron- and lipid peroxidation-dependent form of cell death, known as ferroptosis. To study the macrophage - tumor cell crosstalk we polarized primary human macrophages towards a TAM-like phenotype, co-cultured them with HT1080 fibrosarcoma cells, and analyzed the tumor cell response to ferroptosis induction. In TAMs the expression of ceruloplasmin mRNA increased, which was driven by hypoxia inducible factor 2 and signal transducer and activator of transcription 1. Subsequently, ceruloplasmin mRNA was transferred from TAMs to HT1080 cells via extracellular vesicles. In tumor cells, mRNA was translated into protein to protect HT1080 cells from RSL3-induced ferroptosis. Mechanistically this was based on reduced iron abundance and lipid peroxidation. Interestingly, in naïve macrophages also hypoxia induced ceruloplasmin under hypoxia and a co-culture of HT1080 cells with hypoxic macrophages recapitulated the protective effect observed in TAM co-cultures. In conclusion, TAMs provoke tumor cells to release iron and thereby protect them from lipid peroxidation/ferroptosis.