Molecular andin silicocharacterization of a promoter module and C/EBP element that mediate LPS‐induced RANTES/CCL5 expression in monocytic cells

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.

Activation of Monocytic Cells Through Fcγ Receptors Induces the Expression of Macrophage-Inflammatory Protein (MIP)-1α, MIP-1β, and RANTES

“…Many different monocytic cell lines, including THP-1 cells, show constitutive nuclear B activity (31), and unlike MIP-1␣, RANTES has two B sites near the TATA signal, thus explaining possible interactions with components of the general transcription machinery that could account for the expression of RANTES detected in resting cells. In contrast, the delayed pattern of RANTES mRNA induction by IC agrees with the characterization of RANTES as an unusual gene induced late after T lymphocyte activation, the expression of which has been related to a novel transcription factor termed RANTES factor of late activated T lymphocytes, which belongs to the Kruppel-like family of transcription factors (32), although other factors have been associated with the cell-specific pattern of transcriptional regulation of RANTES, among them NF-B (33), C/EBP␤ (34), and IFN regulatory factor (35).…”

Functional Cooperation of Simian Virus 40 Promoter Factor 1 and CCAAT/Enhancer-Binding Protein β and δ in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages

“…Because mutations at the C/EBP binding site exhibited a more significant inhibition of LPS response than did mutations at Sp1 binding site, the C/EBP binding site seems to play a more important role in the LPS response. According to related literature, it is known that C/EBP is important for some gene expressions, e.g., endotoxin-induced cyclooxygenase-2 gene (24), IL-1␤-induced IL-6 gene (25), and LPS-induced IL-1␤ gene (26) and RANTES/chemokine (C-C motif) ligand 5 gene (27). Recently it was reported that C/EBP␣ and ␤ are critical in both basal and cAMP/stress-dependent induction of human IL-10 expression during monocytic differentiation (28).…”

TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-κB pathway by TRAF-binding TNFRs

“…Nuclear extracts were prepared 26 h later as described in ref. 22. Samples of the nuclear extract or the cytoplasmic fraction were run on SDS͞PAGE under reducing conditions and electrophoretically transferred onto poly(vinylidene difluoride) membranes.…”

“…The cells were harvested 26 h after transfection, and nuclear extracts were prepared as described in ref. 22. Five micrograms of protein of each extract was analyzed by Western blot.…”