T. Merrill Ayres

Bacterial lipopolysaccharide (LPS) induces activation of the transcription factor nuclear factor kappaB (NF-kappaB) in host cells upon infection. LPS binds to the glycosylphosphatidylinositol (GPI)- anchored membrane protein CD14, which lacks an intracellular signaling domain. Here we investigated the role of mammalian Toll-like receptors (TLRs) as signal transducers for LPS. Overexpression of TLR2, but not TLR1, TLR4, or CD14 conferred LPS inducibility of NF-kappaB activation in mammalian 293 cells. Mutational analysis demonstrated that this LPS response requires the intracellular domain of TLR2. LPS signaling through TLR2 was dependent on serum which contains soluble CD14 (sCD14). Coexpression of CD14 synergistically enhanced LPS signal transmission through TLR2. In addition, purified recombinant sCD14 could substitute for serum to support LPS-induced TLR2 activation. LPS stimulation of TLR2 initiated an interleukin 1 receptor-like NF-kappaB signaling cascade. These findings suggest that TLR2 may be a signaling component of a cellular receptor for LPS.

The mammalian tumor necrosis factor receptor (TNFR) family consists of 10 cell-surface proteins that regulate development and homeostasis of the immune system. Based on an expressed sequence tag, we have cloned a cDNA encoding a novel member of the human TNFR family. A closely related protein, designated HVEM (for herpesvirus entry mediator), was identified independently by another group as a mediator of herpesvirus entry into mammalian cells (Montgomery, R., Warner, M., Lum, B., and Spear, P. (1996) Cell 87, 427-436). HVEM differed from our clone by two amino acid residues, suggesting that the two proteins represent polymorphism of a single HVEM gene. We detected HVEM mRNA expression in several human fetal and adult tissues, although the predominant sites of expression were lymphocyte-rich tissues such as adult spleen and peripheral blood leukocytes. The cytoplasmic region of HVEM bound to several members of the TNFR-associated factor (TRAF) family, namely TRAF1, TRAF2, TRAF3, and TRAF5, but not to TRAF6. Transient transfection of HVEM into human 293 cells caused marked activation of nuclear factor-kappaB (NF-kappaB), a transcriptional regulator of multiple immunomodulatory and inflammatory genes. HVEM transfection induced also marked activation of Jun N-terminal kinase, and of the Jun-containing transcription factor AP-1, a regulator of cellular stress-response genes. These results suggest that HVEM is linked via TRAFs to signal transduction pathways that activate the immune response.

Plasmid clones containing a human Alu family repeat can be transcribed efficiently by RNA polymerase III in HeLa cell extract. This generated three RNA species, all of which initiated from the first base (+1) of the repeat. By studying the transcriptional properties of deletion clones, subclones, and topologically different DNA templates, we demonstrated that: supercoiled DNA templates are transcribed 3- to 5-fold more efficiently than are linear or nicked circular DNA molecules; a contiguous DNA helix in the transcription complexes that extends into the 5' flanking region of positions -30 to -85 is absolutely required for initiation to occur (this interaction does not involve recognition of specific DNA sequences); and similar to the adenovirus VAI RNA and tRNA genes, the Alu repeat 3' to the alpha 1-globin gene (designated 3'-alpha 1 Alu) contains a split intragenic promoter: an anterior element (positions +4 to +37) and a posterior element (positions +70 to +82). However, the promoter of the Alu repeat functions in distinctive ways in comparison to those of other RNA polymerase III-dependent genes. The posterior promoter element alone is sufficient and necessary for an accurate initiation to occur. The presence of the anterior promoter element, which by itself does not initiate transcription, enhances the transcriptional efficiency by a factor of 10- to 20-fold. Furthermore, the distance between the initiation sites and the posterior promoter element, but not the anterior promoter element, remains constant. These results suggest that the promoter of this Alu family repeat consists of at least two functionally different domains: a "directing element" (the posterior promoter element) that determines the accuracy of initiation and an "enhancing element" (the anterior promoter element) that is mainly responsible for the transcriptional efficiency.