CCAAT/enhancer-binding protein β and NF-κB mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to IL-1β.
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.
Zhang
Zhiqi
Z
From the Departments of Orthopaedic Surgery, St. Louis, Missouri 63110; Department of Orthopaedic Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China.
Bryan
Jennifer L
JL
From the Departments of Orthopaedic Surgery, St. Louis, Missouri 63110.
DeLassus
Elizabeth
E
From the Departments of Orthopaedic Surgery, St. Louis, Missouri 63110.
Chang
Li-Wei
LW
Pathology and Immunology, St. Louis, Missouri 63110.
Liao
Weiming
W
Department of Orthopaedic Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China.
Sandell
Linda J
LJ
From the Departments of Orthopaedic Surgery, St. Louis, Missouri 63110; Cell Biology and Physiology, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110. Electronic address: sandelll@wustl.edu.
D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D054405:Chemokine CCL3 / Q000096:biosynthesis* / Q000235:genetics; D054407:Chemokine CCL4 / Q000096:biosynthesis* / Q000235:genetics; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*
D022782:CCAAT-Enhancer-Binding Protein-beta; C515429:CCL3 protein, human; C066507:CCL4 protein, human; C545012:CEBPB protein, human; D054405:Chemokine CCL3; D054407:Chemokine CCL4; C508585:IL1B protein, human; D053583:Interleukin-1beta; D016328:NF-kappa B