Abstract
A large set of chemokines is highly up-regulated in human chondrocytes in response to IL-1β (Sandell, L. J., Xing, X., Franz, C., Davies, S., Chang, L. W., and Patra, D. (2008) Osteoarthr. Cartil. 16, 1560-1571). To investigate the mechanism of transcriptional regulation, deletion constructs of selected chemokine gene promoters, the human CCL3 (MIP-1α) and CCL4 (MIP-1β), were transfected into human chondrocytes with or without IL-1β. The results show that an IL-1β-responsive element is located between bp -300 and -140 of the CCL3 promoter and between bp -222 and -100 of the CCL4 promoter. Because both of these elements contain CCAAT/enhancer-binding protein β (C/EBPβ) motifs, the function of C/EBPβ was examined. IL-1β stimulated the expression of C/EBPβ, and the direct binding of C/EBPβ to the C/EBPβ motif was confirmed by EMSA and ChIP analyses. The -300 bp CCL3 promoter and -222 bp CCL4 promoter were strongly up-regulated by co-transfection with the C/EBPβ expression vector. Mutation of the C/EBPβ motif and reduction of C/EBPβ expression by siRNA decreased the up-regulation. Additionally, another cytokine-related transcription factor, NF-κB, was also shown to be involved in the up-regulation of chemokines in response to IL-1β, and the binding site was identified. The regulation of C/EBPβ and NF-κB was confirmed by the inhibition by C/EBPβ and NF-κB and by transfection with C/EBPβ and NF-κB expression vectors in the presence or absence of IL-1β. Taken together, our results suggest that C/EBPβ and NF-κB are both involved in the IL-1β-responsive up-regulation of chemokine genes in human chondrocytes. Time course experiments indicated that C/EBPβ gradually and steadily induces chemokine up-regulation, whereas NF-κB activity was highest at the early stage of chemokine up-regulation.
Highlights
Mokines are known to be important in inflammatory diseases, including rheumatoid arthritis, osteoarthritis (OA),2 inflammatory bowel disease, multiple sclerosis and transplant rejection [1, 2]
IL-1 Stimulates the Expression of Chemokine Genes CCL3 and CCL4 in Normal Human Articular Chondrocytes and the Chondrocyte-derived Cell Line, T/C-28—We have reported that a large set of chemokines that are up-regulated by the IL-1 in adult normal cartilage and from patients with OA [6]
We demonstrate a critical role for C/EBP in the increased expression of chemokine genes in response to IL-1 in human chondrocytes
Summary
Materials—The materials used in this work were purchased as follows. Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 medium were from Mediatech Inc. (Herndon, VA). RNA Isolation and Real Time Quantitative PCR—Total RNA was isolated from T/C-28 cells and human primary articular chondrocytes with an RNeasy minikit with DNase I treatment, following the protocol recommended by the manufacturer (Qiagen). A 10-l aliquot of the sonicated chromatin sample (“input”) was removed for PCR analysis, and the remainder was used for IPs. To each IP was added either 5 g of C/EBP antibody or normal rabbit IgG, and reactions were incubated overnight with constant mixing at 4 °C. The secondary antibodies, Alexa fluor 488 dye-labeled goat antimouse IgG diluted to 1:250 and Alexa fluor 594 dye-labeled goat anti-rabbit IgG diluted to 1:400 in 2% normal goat serum in PBS, were added to the cells for 1 h at room temperature.
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