Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts
Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts
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DOI：
10.1152/ajpcell.2000.278.5.c989
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发表时间：
2000-05-01
影响因子：
5.5
通讯作者：
Duncan, RL
中科院分区：
文献类型：
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作者：
Chen, NX;Ryder, KD;Duncan, RL
成骨细胞受到流体剪切增加早期反应基因c-fos和环氧合酶的诱导型同种型考克斯-2的表达，这两种蛋白质与骨对机械刺激的合成代谢反应有关。这些基因表达的增加依赖于剪切诱导的肌动蛋白应力纤维的形成。在这里，我们证明了MC 3 T3-E1成骨细胞样细胞对剪切力的反应是细胞内Ca 2+浓度（[Ca 2 +]（i））的快速增加，我们假设这对随后的细胞对剪切力的反应是重要的。为了验证这一假设，将MC 3 T3-E1细胞生长在涂有纤连蛋白的载玻片上，并使其经受层流（12达因/厘米（2））。在施加剪切力之前，用两种Ca 2+通道抑制剂或细胞内Ca 2+释放的各种阻断剂处理细胞0.5-1小时。尽管机械敏感性通道阻断剂钆显著降低[Ca 2 +]（i）反应，但钆和L型通道Ca 2+通道阻断剂硝苯地平均不能阻断MC 3 T3-E1细胞中剪切诱导的应力纤维形成以及c-fos和考克斯-2的增加。然而，1，2-双（2-氨基苯氧基）乙烷-N，N，N '，N'-四乙酸-AM（细胞内钙螯合剂）或毒胡萝卜素（其排空细胞内钙库）完全抑制剪切成骨细胞中的应力纤维形成和c-fos/CQX-2产生。新霉素或U-73122抑制磷脂酶C，介导D-肌肌醇1，4，5-三磷酸（IP 3）诱导的细胞内Ca 2+释放，也完全抑制肌动蛋白重组和c-fos/考克斯-2的产生。用U-73122的无活性同种型U-73343预处理MC 3 T3-E1细胞，不抑制这些剪切诱导的反应。这些结果表明，IP 3介导的细胞内Ca 2+释放是调节MC 3 T3-E1细胞中的流动诱导的反应所必需的。
Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca2+ concentration ([Ca2+](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca2+ channel inhibitors or various blockers of intracellular Ca2+ release for 0.5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca2+](i) response, neither gadolinium nor nifedipine, an L-type channel Ca2+ channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-minophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca2+ chelator, or thapsigargin, which empties intracellular Ca2+ stores, completely inhibited stress fiber formation and c-fos/CQX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP3)-induced intracellular Ca2+ release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP3-mediated intracellular Ca2+ release is required for modulating flow-induced responses in MC3T3-E1 cells.