Abstract

Macrophages stimulated by lipopolysaccharide (LPS) from gram negative bacteria undergo activation of a group of immediate early genes including Rantes. The mouse Rantes gene promoter region contains an LPS responsive element (LRE). We detected 3 specific bands, termed B1, B2, and B3, formed by the interaction of the LRE and proteins found in LPS-stimulated RAW 264.7 cells. An additional band, B4, was determined to be an AP-1 binding protein. The B1 band appears within 1 hour of LPS stimulation. The observed binding pattern could be changed by a specific heparin column fraction of nuclear extracts from LPS-stimulated cells. We have determined that a Ser/Thr-specific protein kinase is activated by LPS stimulation, and this protein kinase enhances B1 band formation. The B1 band can be converted to band B2/B3 by adding specific heparin column fraction. Purified Ser/Thr-specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by Ser/Thr protein kinase and one or more Ser/Thr specific phosphatases. At least four proteins are involved in the regulation of the LRE-dependent Rantes expression: two binding factors that bind directly to the target sequences, and two factors that control their binding. The future purification and characterization of these binding proteins will reveal in detail the mechanism of Rantes gene activation after LPS stimulation.

• Keywords: mouse Rantes gene promoter; LPS responsive element; protein kinase and phosphatase