Malek A M, Zhang J, Jiang J, Alper S L, Izumo S
Department of Neurosurgery, Brigham & Women's Hospital, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
J Mol Cell Cardiol. 1999 Feb;31(2):387-99. doi: 10.1006/jmcc.1998.0873.
Physiological fluid shear stress regulates endothelin-1 (ET-1) gene expression in endothelial cells by inducing an early transient upregulation followed by a sustained suppression, at times greater than 2 h in duration. We evaluated the mechanism of ET-1 mRNA downregulation in confluent monolayers of bovine aortic endothelial (BAE) cells by applying a 6 h steady laminar shear stress of magnitude 20 dyn/cm2. Inhibition of tyrosine kinases using herbimycin A (875 nM) abolished the shear-induced decrease in ET-1 mRNA expression. Similarly, chelation of intracellular calcium ([Ca2+]i) with quin 2-AM (10 microM) blocked the suppression of ET-1 mRNA by shear. To examine the role of the endothelial cytoskeleton in the response to flow, cytochalasin D was used to disrupt F-actin microfilaments. This treatment induced cell retraction and detachment under flow, whereas stabilization of F-actin with phalloidin (1 microM) did not affect shear-induced ET-1 downregulation. In contrast, disruption of the microtubule network with nocodazole (10 micrograms/ml) completely prevented, while microtubule stabilization with taxol (10 microM) did not affect the suppression of ET-1 mRNA by flow. To determine the possible contributions of mechanosensitive channels, barium (1 mM BaCl2), was added to confluent BAE monolayers in a low-sulfate/low-phosphate modified medium and was noted to abrogate the downregulation of ET-1 gene expression and to attenuate the shear-induced increase in cytoplasmic free calcium concentration. Tetraethylammonium (3 mM TEA) partially inhibited the suppression of ET-1 mRNA by shear; in contrast, gadolinium (10 microM GdCl3), an inhibitor of the stretch-activated cation channel ISA, had no effect. Membrane depolarization by elevated extracellular potassium ([K+]o) also attenuated the suppression of ET-1 mRNA by flow at [K+]o = 70 mM and completely inhibited it at [K+]o = 135 mM. In summary, the steady-state downregulation of ET-1 mRNA by physiological levels of fluid shear stress shares signaling features with the morphological and cytoskeletal response to shear stress. These include requirement for intracellular calcium, tyrosine kinase activity, an intact microtubule network, and independence from a Gd(3+)-sensitive ISA. Unlike shear-induced changes in cell morphology and the actin cytoskeleton, the shear-induced decrease in ET-1 mRNA level is blocked by cell depolarization and by Ba2+, a blocker of the shear-activated IKS which also decreases shear-induced cytoplasmic calcium increase.
生理流体剪切应力通过诱导早期短暂上调随后持续抑制来调节内皮细胞中内皮素 -1（ET -1）基因的表达，这种抑制有时持续时间超过2小时。我们通过施加大小为20达因/平方厘米的6小时稳定层流剪切应力，评估了牛主动脉内皮（BAE）细胞汇合单层中ET -1 mRNA下调的机制。使用赫伯霉素A（875 nM）抑制酪氨酸激酶消除了剪切诱导的ET -1 mRNA表达下降。同样，用喹2 - AM（10 microM）螯合细胞内钙（[Ca2 +]i）可阻断剪切对ET -1 mRNA的抑制作用。为了研究内皮细胞骨架在血流反应中的作用，使用细胞松弛素D破坏F -肌动蛋白微丝。这种处理在血流作用下诱导细胞收缩和脱离，而用鬼笔环肽（1 microM）稳定F -肌动蛋白并不影响剪切诱导的ET -1下调。相反，用诺考达唑（10微克/毫升）破坏微管网络可完全阻止，而用紫杉醇（10 microM）稳定微管并不影响血流对ET -1 mRNA的抑制作用。为了确定机械敏感通道的可能作用，在低硫酸盐/低磷酸盐改良培养基中向汇合的BAE单层中加入钡（1 mM BaCl2），发现其可消除ET -1基因表达的下调并减弱剪切诱导的细胞质游离钙浓度增加。四乙铵（3 mM TEA）部分抑制了剪切对ET -1 mRNA的抑制作用；相反，钆（10 microM GdCl3），一种拉伸激活阳离子通道ISA的抑制剂，没有作用。细胞外钾离子浓度升高（[K +]o）引起的膜去极化在[K +]o = 70 mM时也减弱了血流对ET -1 mRNA的抑制作用，在[K +]o = 135 mM时完全抑制了这种作用。总之，生理水平的流体剪切应力对ET -1 mRNA的稳态下调与对剪切应力的形态学和细胞骨架反应具有共同的信号特征。这些特征包括对细胞内钙、酪氨酸激酶活性、完整微管网络的需求以及对Gd(3 +)敏感的ISA的独立性。与剪切诱导的细胞形态和肌动蛋白细胞骨架变化不同，剪切诱导的ET -1 mRNA水平下降被细胞去极化和Ba2 +阻断，Ba2 +是剪切激活的IKS的阻滞剂，它也可减少剪切诱导的细胞质钙增加。
