Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway
Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway
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DOI：
10.1359/jbmr.2002.17.2.266
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发表时间：
2002-02-01
影响因子：
6.2
通讯作者：
Pilbeam, CC
中科院分区：
文献类型：
--
作者：
Wadhwa, S;Godwin, SL;Pilbeam, CC
骨的机械负荷可通过间质液流动在细胞表面产生的剪切应力传递给骨细胞和成骨细胞。机械负荷对骨的某些作用是通过骨细胞中的诱导型环氧合酶2（考克斯-2）介导的。我们在用考克斯-2 5 '-侧翼DNA的-371/+70碱基对（bp）稳定转染的永生化MC 3 T3-E1成骨细胞（PIuc 371）和来自Pluc 371转基因小鼠的颅骨的原代成骨细胞（POB）中检测了流体剪切应力（FSS）对考克斯-2基因表达的诱导。将细胞接种在胶原包被的载玻片上，并在平行板流动室中进行稳定的层流FSS。FSS在0.14 ~ 10 dynes/cm（2）范围内可诱导考克斯-2 mRNA和蛋白表达。FSS（10达因/cm 2）在30分钟内诱导考克斯-2 mRNA，MC 3 T3-E1细胞中的峰值效应在4小时，POB中的峰值效应在大于或等于8小时。新蛋白合成抑制剂嘌呤霉素可阻断FSS对考克斯-2 mRNA的诱导峰。考克斯-2启动子活性，以荧光素酶活性测量，与考克斯-2在MC 3 T3-E1和POB细胞中的mRNA表达相关。FSS诱导MC 3 T3-E1细胞中细胞外信号调节激酶（ERK）磷酸化，在5分钟时达到峰值。用特异性抑制剂PD 98059抑制ERK磷酸化，在MC 3 T3-E1和POB细胞中，抑制了55-70%的考克斯-2 mRNA的FSS诱导和大于或等于80%的荧光素酶活性的FSS刺激。我们的结论是，FSS转录诱导成骨细胞中的考克斯-2基因表达，最大的诱导需要新的蛋白质合成，诱导主要通过ERK信号通路发生。
Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo-oxygenase 2 (COX-2) in bone cells. We examined the fluid shear stress (FSS) induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells stably transfected with -371/+70 base pairs (bp) of the COX-2 5'-flanking DNA (PIuc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen-coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber. FSS, from 0.14 to10 dynes/cm(2), induced COX-2 messenger RNA (mRNA) and protein. FSS (10 dynes/cm2) induced COX-2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3-E1 cells and at greater than or equal to8 h in POBs. An inhibitor of new protein synthesis puromycin blocked the peak induction of COX-2 mRNA by FSS. COX-2 promoter activity, measured as luciferase activity, correlated with COX-2 mRNA expression in both MC3T3-E1 and POB cells. FSS induced phosphorylation of extracellular signal-regulated kinase (ERK) in MC3T3-E1 cells, with peak effects at 5 minutes. Inhibiting ERK phosphorylation with the specific inhibitor PD98059 inhibited FSS induction of COX-2 mRNA by 55-70% and FSS stimulation of luciferase activity by greater than or equal to80% in both MC3T3-E1 and POB cells. We conclude that FSS transcriptionally induces COX-2 gene expression in osteoblasts, that the maximum induction requires new protein synthesis, and that induction occurs largely via an ERK signaling pathway.