Molecular and in silico characterization of a promoter module and C/EBP element that mediate LPS-induced RANTES/CCL5 expression in monocytic cells.
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.
Fessele
S
S
Medizinische Poliklinik, Klinikum Innenstadt, Ludwig-Maximilians-University of Munich, Germany.
Boehlk
S
S
Mojaat
A
A
Miyamoto
N G
NG
Werner
T
T
Nelson
E L
EL
Schlondorff
D
D
Nelson
P J
PJ
D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D019281:Dimerization; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D018946:Chemokine CCL5; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016329:Sp1 Transcription Factor