Transcriptional roles of CCAAT/enhancer binding protein-β, nuclear factor-κB, and C-promoter binding factor 1 in interleukin (IL)-1β-induced IL-6 synthesis by human rheumatoid fibroblast-like synoviocytes

The involvement of interleukin (IL)-6 in the pathogenesis of rheumatoid arthritis (RA) has been recently demonstrated. IL-1 beta stimulated rheumatoid fibroblast-like synoviocytes (FLSs) to produce IL-6 in a concentration-and time-dependent manner. In the present study we investigated how the IL-6 promoter is transcriptionally regulated in rheumatoid FLSs in response to a physiologically relevant mediator of inflammation, IL-1 beta. Deletion analysis showed that the IL-6 promoter is regulated by two positive elements (located at -159 to -142 base pairs (bp) and -77 to -59 bp). Electrophoretic mobility shift assays revealed that CCAAT/enhancer binding protein-beta (C/EBP beta) binding to nucleotides -159 to -142 bp was constitutively present. The probe corresponding to nucleotides -77 to -59 bp gave three positive bands. The two slower migrating bands were induced by IL-1 beta and comprised an nuclear factor (NF)-kappa B p50/p65 heterodimer and a p65/p65 homodimer. The faster migrating band was constitutively expressed and identified as Epstein-Barr virus C-promoter binding factor 1, CBF1. Site-specific mutagenesis analysis demonstrated that the NF-kappa B and CBF1 binding elements regulated inducible activity of the IL-6 promoter in response to IL-1 beta stimulation, whereas the C/EBP beta binding element mainly regulated basal activity. We also provide the first evidence that CBF1 functions as a positive regulator of human IL-6 gene transcription.