ABSTRACT
The chemokine RANTES/CCL5 is a proinflammatory agent produced by a variety of tissues in response to specific stimuli. In human monocytes, RANTES/CCL5 transcription is up-regulated rapidly and transiently in response to LPS. We describe here two regions that help control LPS-driven transcription from the human RANTES/CCL5 promoter in monocytic cells. These sites were analyzed by using DNase I footprinting, transient transfection assays, site-directed mutagenesis, and EMSA. RANTES site E (R(E), -125/-99) constitutively binds C/EBP proteins in monocytic Mono Mac 6 cells. Mutation of region R(E) led to a significant (40%-50%) reduction in LPS-induced promoter reporter activity. Region R(AB) is composed of tandem kB-like elements R(A) and R(B) (-73/-34). These sites working in concert act as an LPS-responsive promoter module. R(A) constitutively binds Sp1, and Rel p50/p65 following LPS stimulation. Either factor can mediate transcriptional effects at R(A). Induced Rel p50/p50 binding to site R(B) is required for LPS regulation of RANTES/CCL5 transcription. A series of computer models based on the RANTES/CCL5 promoter were generated to represent the organization of these functional elements. The models could identify LPS-regulated promoters in human, other vertebrate, and viral sequences in various databases.
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , Chemokine CCL5/metabolism , Lipopolysaccharides/pharmacology , Monocytes/metabolism , Promoter Regions, Genetic/genetics , Cell Line , Chemokine CCL5/genetics , Computer Simulation , Dimerization , Genes, Reporter/genetics , Humans , Monocytes/drug effects , NF-kappa B/genetics , NF-kappa B/metabolism , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , TransfectionABSTRACT
The chemokine RANTES is produced by a variety of tissues, including cells of the monocyte/macrophage lineage. RANTES expression is rapidly and transiently up-regulated in primary monocytes and the monocytic cell line Mono Mac 6 in response to stimulation by the bacterial product lipopolysaccharide (LPS). Transient transfection of Mono Mac 6 cells with RANTES reporter-promoter deletion constructs, in conjunction with DNase I footprinting and heterologous reporter gene assays, allowed identification of an LPS-responsive region within the RANTES promoter. Electrophoretic mobility shift assays (EMSA), methylation interference and EMSA supershift experiments were used to characterize sequences and transcription factors responsible for this LPS inducibility. The region, termed RANTES site G [R(G)], contains consensus sites for Ets and CRE/AP-1-like elements. Site-directed mutagenesis of the Ets site resulted in a loss of only 15 % of promoter activity, while mutation of the CRE/AP-1 site led to a loss of 40 % of LPS-induced promoter activity. The Ets site constitutively binds the Ets family member PU.1. LPS stimulation leads to an induction of ATF-3 and JunD factor binding to the CRE/AP-1 site. Thus, LPS induction of RANTES transcription is mediated, in part, through the activation and selective binding of ATF and Jun nuclear factors to the R(G) promoter module.