Chen N X, Ryder K D, Pavalko F M, Turner C H, Burr D B, Qiu J, Duncan R L
Department of Anatomy, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Am J Physiol Cell Physiol. 2000 May;278(5):C989-97. doi: 10.1152/ajpcell.2000.278.5.C989.
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 Ca(2+) concentration (Ca(2+)) 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 Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the Ca(2+) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) 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-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) 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 IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.
在体内，受到流体剪切力作用的成骨细胞会增加早期反应基因c-fos以及环氧化酶诱导型同工酶COX-2的表达，这两种蛋白质与骨骼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力诱导的肌动蛋白应力纤维的形成。在此，我们证明MC3T3-E1成骨样细胞对剪切力的反应是细胞内Ca(2+)浓度（[Ca(2+)]i）迅速增加，我们推测这对随后细胞对剪切力的反应很重要。为了验证这一假设，将MC3T3-E1细胞接种在涂有纤连蛋白的载玻片上，并使其受到层流（12达因/平方厘米）作用。在施加剪切力之前，用两种Ca(2+)通道抑制剂或各种细胞内Ca(2+)释放阻滞剂处理细胞0.5 - 1小时。尽管机械敏感通道阻滞剂钆显著降低了[Ca(2+)]i反应，但钆和L型通道Ca(2+)通道阻滞剂硝苯地平都不能阻断MC3T3-E1细胞中剪切力诱导的应力纤维形成以及c-fos和COX-2的增加。然而，细胞内Ca(2+)螯合剂1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸-AM或耗尽细胞内Ca(2+)储存的毒胡萝卜素，完全抑制了剪切力作用下成骨细胞中的应力纤维形成以及c-fos/COX-2的产生。新霉素或U-73122对磷脂酶C的抑制作用，磷脂酶C介导D-肌醇1,4,5-三磷酸（IP(3)）诱导的细胞内Ca(2+)释放，也完全抑制了肌动蛋白重组以及c-fos/COX-2的产生。用U-73122的无活性同工型U-73343预处理MC3T3-E1细胞，并未抑制这些剪切力诱导的反应。这些结果表明，IP(3)介导的细胞内Ca(2+)释放是调节MC3T3-E1细胞中流体诱导反应所必需的。
