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流体剪切应力诱导的环氧合酶-2表达是由成骨细胞MC3T3-E1细胞中的C/EBPβ、cAMP反应元件结合蛋白和AP-1介导的。

Fluid shear stress-induced cyclooxygenase-2 expression is mediated by C/EBP beta, cAMP-response element-binding protein, and AP-1 in osteoblastic MC3T3-E1 cells.

作者信息

Ogasawara A, Arakawa T, Kaneda T, Takuma T, Sato T, Kaneko H, Kumegawa M, Hakeda Y

机构信息

Department of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama, Japan.

出版信息

J Biol Chem. 2001 Mar 9;276(10):7048-54. doi: 10.1074/jbc.M008070200. Epub 2000 Nov 22.

Abstract

Mechanical loading is crucial for maintenance of bone integrity and architecture, and prostaglandins are an important mediator of mechanosensing. Cyclooxygenase-2 (COX-2), an inducible isoform of prostaglandin G/H synthase, is induced by mechanical loading-derived fluid shear stress in bone-forming cells such as osteoblasts and osteocytes. In this study, we investigated transcription factor and transcriptional regulatory elements responsible for the shear stress-induced COX-2 expression in osteoblastic MC3T3-E1 cells. When the cells were transfected with luciferase-reporter plasmids including the 5'-flanking region of the murine cox-2 gene, the fluid shear stress increased the luciferase activities, consistent with the induction of COX-2 mRNA and protein expression. Deletion analysis of the promoter region revealed that the shear stress-induced luciferase responses were regulated by two regions, -172 to -100 base pair (bp) and -79 to -46 bp, of the cox-2 promoter, in which putative cis-elements of C/EBP beta, AP-1, cAMP-response element-binding protein (CREB), and E box are included. Mutation of sites of C/EBP beta, AP-1, and/or cAMP-response element decreased the shear stress-induced luciferase activities, whereas mutation of the E box did not affect the responses. In an electrophoretic mobility shift assay, shear stress enhanced nuclear extract binding to double-stranded oligonucleotide probes containing C/EBP beta and AP-1-binding motifs, and the bands of the complexes were supershifted by the addition of antibody specific for each regulator. Although the binding activity of CREB toward its probe was unaffected by shear stress, the phosphorylation of CREB was enhanced by the stress. These data suggest that C/EBP beta, AP-1, and CREB play crucial roles in the shear stress-induced cox-2 expression in osteoblasts.

摘要

机械负荷对于维持骨的完整性和结构至关重要,而前列腺素是机械传感的重要介质。环氧化酶-2(COX-2)是前列腺素G/H合酶的一种诱导型同工酶,在成骨细胞和骨细胞等骨形成细胞中,由机械负荷衍生的流体剪切应力诱导产生。在本研究中,我们调查了负责剪切应力诱导成骨细胞MC3T3-E1细胞中COX-2表达的转录因子和转录调控元件。当用包含小鼠cox-2基因5'-侧翼区的荧光素酶报告质粒转染细胞时,流体剪切应力增加了荧光素酶活性,这与COX-2 mRNA和蛋白表达的诱导一致。启动子区域的缺失分析表明,剪切应力诱导的荧光素酶反应受cox-2启动子的两个区域调控,即-172至-100碱基对(bp)和-79至-46 bp,其中包含C/EBPβ、AP-1、cAMP反应元件结合蛋白(CREB)和E盒的假定顺式元件。C/EBPβ、AP-1和/或cAMP反应元件位点的突变降低了剪切应力诱导的荧光素酶活性,而E盒的突变不影响反应。在电泳迁移率变动分析中,剪切应力增强了核提取物与包含C/EBPβ和AP-1结合基序的双链寡核苷酸探针的结合,并且通过添加针对每个调节因子的特异性抗体使复合物条带发生超迁移。尽管CREB对其探针的结合活性不受剪切应力影响,但应力增强了CREB的磷酸化。这些数据表明,C/EBPβ、AP-1和CREB在剪切应力诱导成骨细胞中cox-2表达中起关键作用。

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