LPS induction of Egr-1 expression is mediated by the Ras-Raf-MEK-ERK pathway.

(A) THP-1 cells were preincubated with PD98059 (25 μM) for 30 minutes prior to stimulation with LPS for 2 hours. Nuclear extracts were analyzed by Western blotting using an anti–Egr-1 antibody. (B) Total RNA was extracted from THP-1 cells that were unstimulated or stimulated with LPS (10 μg/mL) for 1 hour at 37°C with or without PD98059 (25 μM). Egr-1 mRNA levels were determined by Northern blotting using a radiolabeled human Egr-1 cDNA probe. The membrane was reprobed with a G3PDH probe as a measure of loading. (C) THP-1 cells were transiently transfected with pEgr-1–LUC (3 μg). After transfection, cells were preincubated in the presence or absence of PD98059 (25 μM) for 30 minutes before incubation with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and results were expressed as fold induction (n = 3). (D) THP-1 cells were cotransfected with pEgr-1–LUC (1.5 μg) and either pcDNA3 (5.5 μg) or plasmids expressing a dominant-negative version of Ras, Raf-1, ERK1, ERK2, or MEKK-1. Transfected cells were treated with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and results were expressed as percentage of control induction (n = 3).