Modest Static Pressure Suppresses Columnar Epithelial Cell Growth in Association with Cell Shape and Cytoskeletal Modifications.
Modest Static Pressure Suppresses Columnar Epithelial Cell Growth in Association with Cell Shape and Cytoskeletal Modifications.
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DOI：
10.3389/fphys.2017.00997
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发表时间：
2017
影响因子：
4
通讯作者：
Ito A
中科院分区：
文献类型：
--
作者：
Hagiyama M;Yabuta N;Okuzaki D;Inoue T;Takashima Y;Kimura R;Ri A;Ito A
腔内压力升高可导致退行性疾病，如肠梗阻和肾积水，阈值相当低且恒定，20-30 cm H2O。我们之前设计了一种新的双室培养系统，将培养在半透膜上的细胞置于增加培养介质高度(水压高达60厘米H2O)中。在这里，我们试图确定~30 cm H2O的持续压力负荷如何影响增殖的上皮细胞，特别是与细胞形态的联系。我们利用低静压可加载的双室系统培养了几种不同的细胞系，并检测了细胞生长、细胞周期和细胞形态。Madin-Darby犬肾(MDCK)柱状上皮细胞在静水压2~50cmH2O范围内呈生长抑制，细胞周期在任何特定时相均无停滞。另外两种柱状上皮细胞表现出相似的表型。相反，球状上皮细胞和间充质细胞没有受到生长抑制，即使在50厘米水中也是如此。Phalloidin染色显示，50cmH2O压力负荷使柱状上皮细胞垂直扁平和横向增宽，肌动蛋白纤维分布稀疏，但不影响每细胞的鬼臼蛋白总强度。在50 cm高的培养液中加入100 nM的粘膜保护剂马来酸伊索拉定后，MDCK细胞体积减小，倍增时间缩短。细胞的增殖和形态受Hippo信号通路的调控。50cmH2O的压力负荷增加了丝氨酸-127的磷酸化和胞质保留，提示HIPPO途径参与了压力诱导的细胞生长抑制。对MDCK细胞的RNA测序显示，50 cm的H2O压力负荷使中间丝角蛋白14上调了12倍。免疫荧光在蛋白质水平上证实了这一上调，提示其在细胞骨架增强中起作用。这些结果提供了细胞形态和细胞骨架与细胞生长密切相关的证据。病理性腔内压力升高可能通过直接作用于这一连接和河马途径而导致粘膜退变。
Intraluminal pressure elevation can cause degenerative disorders, such as ileus and hydronephrosis, and the threshold is fairly low and constant, 20–30 cm H2O. We previously devised a novel two-chamber culture system subjecting cells cultured on a semipermeable membrane to increased culture medium height (water pressure up to 60 cm H2O). Here, we sought to determine how a continuous pressure load of ~30 cm H2O affects proliferating epithelial cells with special interest in the link with cell morphology. We cultured several different cell lines using the low static pressure-loadable two-chamber system, and examined cell growth, cell cycle, and cell morphology. Madin–Darby canine kidney (MDCK) columnar epithelial cells were growth-suppressed in a manner dependent on static water pressure ranging from 2 to 50 cm H2O, without cell cycle arrest at any specific phase. Two other types of columnar epithelial cells exhibited similar phenotypes. By contrast, spherical epithelial and mesenchymal cells were not growth-suppressed, even at 50 cm H2O. Phalloidin staining revealed that 50 cm H2O pressure load vertically flattened and laterally widened columnar epithelial cells and made actin fiber distribution sparse, without affecting total phalloidin intensity per cell. When the mucosal protectant irsogladine maleate (100 nM) was added to 50-cm-high culture medium, MDCK cells were reduced in volume and their doubling time shortened. Cell proliferation and morphology are known to be regulated by the Hippo signaling pathway. A pressure load of 50 cm H2O enhanced serine-127 phosphorylation and cytoplasmic retention of YAP, the major constituent of this pathway, suggesting that Hippo pathway was involved in the pressure-induced cell growth suppression. RNA sequencing of MDCK cells showed that a 50 cm H2O pressure load upregulated keratin 14, an intermediate filament, 12-fold. This upregulation was confirmed at the protein level by immunofluorescence, suggesting a role in cytoskeletal reinforcement. These results provide evidence that cell morphology and the cytoskeleton are closely linked to cell growth. Pathological intraluminal pressure elevation may cause mucosal degeneration by acting directly on this linkage and the Hippo pathway.
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