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
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
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DOI：
10.1074/jbc.m110.130377
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发表时间：
2010-10-22
影响因子：
4.8
通讯作者：
Sandell, Linda J.
中科院分区：
文献类型：
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作者：
Zhang, Zhiqi;Bryan, Jennifer L.;Sandell, Linda J.
大量趋化因子在人软骨细胞中响应于IL-1 β而高度上调（Sandell，L. J.，Xing，X.，弗朗茨角，澳-地Davies，S.，昌湖，澳-地W.，和Patra，D.（2008）Osteoarthr.卡蒂尔16，1560-1571）。为了研究转录调节的机制，将所选趋化因子基因启动子的缺失构建体，人CCL 3（MIP-1 α）和CCL 4（MIP-1 β），转染到有或没有IL-1 β的人软骨细胞中。结果显示IL-1 β-应答元件位于CCL 3启动子的bp-300和-140之间以及CCL 4启动子的bp-222和-100之间。由于这两个元件都含有CCAAT/增强子结合蛋白β（C/EBP β）基序，因此检查了C/EBP β的功能。IL-1 β刺激了C/EBP β的表达，并且通过EMSA和ChIP分析证实了C/EBP β与C/EBP β基序的直接结合。通过与C/EBP β表达载体共转染，-300 bp CCL 3启动子和-222 bp CCL 4启动子被强烈上调。C/EBP β基序的突变和siRNA对C/EBP β表达的降低降低了上调。此外，另一种细胞因子相关转录因子NF-κ B也被证明参与趋化因子响应IL-1 β的上调，并确定了结合位点。通过C/EBP β和NF-κ B的抑制以及通过在存在或不存在IL-1 β的情况下用C/EBP β和NF-κ B表达载体转染来证实C/EBP β和NF-κ B B的调节。综上所述，我们的研究结果表明，C/EBP β和NF-κ B B都参与了IL-1 β反应性上调人类软骨细胞中的趋化因子基因。时程实验表明，C/EBP β逐渐和稳定地诱导趋化因子上调，而NF-κ B活性在趋化因子上调的早期阶段最高。
A large set of chemokines is highly up-regulated in human chondrocytes in response to IL-1 beta (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-1 alpha) and CCL4 (MIP-1 beta), were transfected into human chondrocytes with or without IL-1 beta. The results show that an IL-1 beta-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 beta (C/EBP beta) motifs, the function of C/EBP beta was examined. IL-1 beta stimulated the expression of C/EBP beta, and the direct binding of C/EBP beta to the C/EBP beta 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/EBP beta expression vector. Mutation of the C/EBP beta motif and reduction of C/EBP beta expression by siRNA decreased the up-regulation. Additionally, another cytokine-related transcription factor, NF-kappa B, was also shown to be involved in the up-regulation of chemokines in response to IL-1 beta, and the binding site was identified. The regulation of C/EBP beta and NF-kappa B was confirmed by the inhibition by C/EBP beta and NF-kappa B and by transfection with C/EBP beta and NF-kappa B expression vectors in the presence or absence of IL-1 beta. Taken together, our results suggest that C/EBP beta and NF-kappa B are both involved in the IL-1 beta-responsive up-regulation of chemokine genes in human chondrocytes. Time course experiments indicated that C/EBP beta gradually and steadily induces chemokine up-regulation, whereas NF-kappa B activity was highest at the early stage of chemokine up-regulation.