title: Functional analysis of the fractalkine gene promoter in human aortic smooth muscle cells exposed to proinflammatory conditions.
abstract: Fractalkine (Fk) and its receptor CX3C receptor 1 contribute effectively to the atherosclerotic process, mediating the recruitment of leukocytes and promoting the interactions between monocytes/macrophages and smooth muscle cells (SMCs). As Fk expression is significantly increased in SMCs during atherogenesis, we aimed to uncover the detailed molecular mechanisms of transcriptional regulation of the Fk gene. For this purpose, we cloned and characterized the human Fk promoter, and studied the specific roles of different transcription factors in its regulation in human aortic SMCs activated by interferon-γ. In silico analysis of the Fk promoter indicated the presence of binding sites for various inflammatory modulators, such as nuclear factor-κB (NF-κB), signal transducer and activator of transcription (STAT)1/STAT3, and activator protein-1. Using a luciferase reporter plasmid, we identified a 2046-bp region spanning the transcriptional start point of the Fk gene, which has strong constitutive promoter activity in SMCs. The effects of interferon-γ on both Fk reporter activity and endogenous transcription were abolished by silencing NF-κB, STAT1, and STAT3. Transient overexpression of p65/NF-κB and STAT1/STAT3 increased Fk promoter activity, whereas c-Jun/activator protein-1 overexpression had no effect. The results obtained with chromatin immunoprecipitation assays revealed the existence of physical interactions of p65 and STAT1/STAT3 with the predicted elements of the Fk promoter. Moreover, Fk-promoted monocyte chemotaxis was dependent on the janus kinase-STAT pathway. Investigation of the detailed molecular mechanisms by cloning and characterizing potential transcriptional response elements has identified the Fk regulatory mechanism in activated human SMCs.
authors.affiliation: Institute of Cellular Biology and Pathology 'Nicolae Simionescu' of the Romanian Academy, Bucharest, Romania.
authors.forename: Ana-Maria
authors.initials: AM
authors.lastname: Gan
authors.forename: Elena
authors.initials: E
authors.lastname: Butoi
authors.forename: Adrian
authors.initials: A
authors.lastname: Manea
authors.forename: Monica Madalina
authors.initials: MM
authors.lastname: Pirvulescu
authors.forename: Daniela
authors.initials: D
authors.lastname: Stan
authors.forename: Viorel
authors.initials: V
authors.lastname: Simion
authors.forename: Manuela
authors.initials: M
authors.lastname: Calin
authors.forename: Maya
authors.initials: M
authors.lastname: Simionescu
authors.forename: Ileana
authors.initials: I
authors.lastname: Manduteanu
mesh_terms: D001011:Aorta / Q000378:metabolism; D001483:Base Sequence; D054428:Chemokine CX3CL1 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D007249:Inflammation / Q000503:physiopathology; D007371:Interferon-gamma / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D050794:STAT1 Transcription Factor / Q000502:physiology; D050796:STAT3 Transcription Factor / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000502:physiology
chemical_list: D054428:Chemokine CX3CL1; D016328:NF-kappa B; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; D018808:Transcription Factor AP-1; D007371:Interferon-gamma
keywords: fractalkine promoter; interferon-γ; nuclear factor-κB (NF-κB); signal transducer and activator of transcription (STAT)1/STAT3; smooth muscle cells