RESUMO
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.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Quimiocina CCL5/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Regiões Promotoras Genéticas/genética , Linhagem Celular , Quimiocina CCL5/genética , Simulação por Computador , Dimerização , Genes Reporter/genética , Humanos , Monócitos/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Quimiocina CCL5/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Fator 3 Ativador da Transcrição , Sequência de Bases , Células Cultivadas , DNA/genética , DNA/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Genes Reporter/genética , Humanos , Monócitos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Deleção de Sequência/genética , Especificidade por Substrato , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The chemokine RANTES is an important mediator of inflammatory processes. In this report, we describe the DNA sequence and transcription factor requirements for interleukin-1beta (IL-1beta) induction of the RANTES promoter in the human astrocytoma line CH235. RANTES promoter sequences between -278 and +55 are sufficient for IL-1beta-inducibility. In vitro DNA binding assays demonstrate constitutive binding of Sp1, HMG, Ets domain, and bZIP family members to their cognate sites in the RANTES promoter, whereas NF-kappaB and IRF-1 bind in an IL-1beta-inducible manner. IL-1beta-inducibility of the RANTES promoter requires both constitutive and inducible transcription factors. The formation of a higher order nucleoprotein complex, or 'enhanceosome', may be critical for IL-1beta induction of the RANTES promoter.