Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA.
J Bone Miner Res. 2010 Apr;25(4):819-29. doi: 10.1359/jbmr.091019.
Murine MC3T3-E1 and MC-4 cells were stably transfected with -371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at -57/-52 bp, (2) the activating protein-1 (AP-1)-binding site at -69/-63 bp, (3) the nuclear factor of activated T-cells (NFAT)-binding site at -77/-73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)-stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.
将鼠 COX-2（环氧化酶-2）启动子的-371/+70bp 与荧光素酶报告基因（Pluc371）融合，稳定转染鼠 MC3T3-E1 和 MC-4 细胞，或用 Pluc371 携带定点突变。突变位于（1）-57/-52bp 的 cAMP 反应元件（CRE），（2）-69/-63bp 的激活蛋白-1（AP-1）结合位点，（3）-77/-73bp 的核因子活化 T 细胞（NFAT）结合位点，以及（4）AP-1 和 NFAT 位点，这些位点构成了 NFAT/AP-1 的复合共识序列。单独突变 CRE、AP-1 或 NFAT 位点可使甲状旁腺激素（PTH）刺激的 COX-2 启动子活性降低 40%至 60%，而 NFAT 和 AP-1 的联合突变则可阻断诱导。电泳迁移率变动分析显示，PTH 刺激磷酸化 CREB 与跨越 CRE 的寡核苷酸结合，以及 NFATc1、c-Fos 和 c-Jun 与跨越 NFAT/AP-1 复合位点的寡核苷酸结合。NFAT 位点的突变在与复合元件的结合竞争中不如 AP-1 位点的突变有效，这表明 NFATc1 和 AP-1 的协同相互作用更依赖于 NFAT 而不是 AP-1。PTH 和 forskolin（一种激活腺苷酸环化酶的物质）均可刺激 NFATc1 核转位。钙螯合或钙调神经磷酸酶抑制剂可抑制 PTH 和 forskolin 刺激的 COX-2 启动子活性 56%至 80%，PKA 抑制剂可抑制 60%至 98%。这些结果表明，钙-钙调神经磷酸酶-NFAT 信号通路在 PTH 诱导 COX-2 中起重要作用，并表明 cAMP/PKA 通路与钙-钙调神经磷酸酶-NFAT 通路之间的交叉对话可能在 PTH 在成骨细胞中的其他功能中发挥作用。
