Transcriptional roles of CCAAT/enhancer binding protein-beta, nuclear factor-kappaB, and C-promoter binding factor 1 in interleukin (IL)-1beta-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-1beta 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-1beta. 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/EBPbeta) 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-1beta and comprised an nuclear factor (NF)-kappaB 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-kappaB and CBF1 binding elements regulated inducible activity of the IL-6 promoter in response to IL-1beta stimulation, whereas the C/EBPbeta 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.
Miyazawa
K
K
Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Mori
A
A
Yamamoto
K
K
Okudaira
H
H
D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D007375:Interleukin-1 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D007375:Interleukin-1; D015850:Interleukin-6; D016328:NF-kappa B; D009687:Nuclear Proteins; C497570:RBPJ protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors