Regulation of Lipopolysaccharide-Induced Translation of Tumor Necrosis Factor-Alpha by the Toll-Like Receptor 4 Adaptor Protein TRAM

Abstract

Lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-alpha requires the recruitment of two pairs of adaptors to the Toll-like receptor 4 cytoplasmic domain. The contribution of one pair - Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) and TRIF-related adaptor molecule (TRAM) - to TNF-alpha expression is not well understood. To clarify this issue, we studied TRAM knockout bone marrow-derived macrophages (BMDM). LPS-stimulated TRAM-deficient BMDM had decreased TNF-alpha protein expression even at times when TNF-alpha mRNA levels were normal, suggesting impaired translation. Consistent with this idea, knockdown of TRAM in RAW264.7 macrophages decreased translation of a reporter controlled by the TNF-alpha 3' untranslated region, while transfection of TRAM in HEK293T cells increased translation of this reporter. Also consistent with a role for TRAM in TNF-alpha translation, LPS-induced activation of MK2, a kinase involved in this process, was impaired in TRAM-deficient BMDM. TRIF did not increase translation of the TNF-alpha 3' untranslated region reporter when expressed in HEK293T cells. However, BMDM that lacked functional TRIF produced reduced levels of TNF-alpha protein in response to LPS despite normal amounts of the mRNA. Unlike BMDM, LPS-stimulated TRAM-deficient peritoneal macrophages displayed equivalent reductions in TNF-alpha protein and mRNA. Our results indicate that TRAM- and TRIF-dependent signals have a previously unappreciated, cell type-specific role in regulating TNF-alpha translation.