Cellular events mediated by lipopolysaccharide-stimulated toll-like receptor 4 - MD-2 is required for activation of mitogen-activated protein kinasesand Elk-1

Lipopolysaccharide (LPS) stimulates multiple signaling events, including nu clear factor-kappa B (NF-kappa B) activity and the mitogen-activated protei n (MAP) kinases, ERK, JNK, and p38 in LPS-responsive cells, resulting in tr anscriptional activation and cytokine generation. LPS-induced signaling via toll-like receptor 4 (TLR4) results in the activation of the transcription factor NF-kappa B. Since LPS activates other signaling cascades in respons ive cells, the objective of this study was to determine whether such events are mediated by TLR4 in response to LPS, me generated human embryonic kidn ey cells (HEK293) that stably express TLR4 (HEK-TLR4) and examined their re sponsiveness to LPS by measuring NF-kappa B activity and production of inte rleukin-8 (IL-8). A trans-reporting system was used to measure the activity of Elk-1, an ETS-domain transcription factor targeted by MAP kinase pathwa ys. LPS stimulated NF-kappa B reporter activity and IL-8 production but not Elk-1 activity in HEK-TLR4 cells. When MD-2, a protein associated with the extracellular domain of TLR4, was expressed in these cells, there was a ma rked increase in Elk-1 activity as well as ERK, JNK, and p38 MAP kinase pho sphorylation in response to LPS. TLR4-mediated NF-kappa B reporter activity and IL-8 production was enhanced by the expression of MD-2. This study dem onstrates that expression of both TLR4 and MD-2 is required for LPS to acti vate or augment the MAP kinase pathways, Elk-1 stimulation, and IL-8 genera tion.