Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells*
Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells*
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DOI：
10.1074/mcp.m113.033423
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发表时间：
2014-04-01
影响因子：
7
通讯作者：
Buettner, Falk F. R.
中科院分区：
文献类型：
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作者：
Konze, Sarah A.;van Diepen, Laura;Buettner, Falk F. R.
人类多能干细胞及其衍生物的预期临床和工业用途已经为建立能够大规模生产细胞的悬浮培养方案提供了主要动力。了解从贴壁培养转移到悬浮培养所伴随的分子变化是至关重要的，因为这些信息可以对优化培养条件的发展产生直接影响。在这项研究中，我们评估了人类胚胎干细胞和诱导多能干细胞生长在表面贴壁培养（二维）与自由浮动悬浮培养球体（三维）的基因表达。我们结合了定量蛋白质组学方法的基础上稳定同位素标记的氨基酸在细胞培养与深度测序为基础的转录。三维培养的细胞显示形成细胞-细胞和细胞-细胞外基质连接的结构组分的蛋白质表达减少。然而，完全出乎意料的是，我们发现了经典Wnt信号传导途径的分泌抑制剂的上调，以及伴随的活性β-连环蛋白水平和Wnt靶基因表达的降低。在Western印迹分析中，半胱氨酸蛋白酶钙蛋白酶显示在三维培养条件下切割E-钙粘蛋白和β-连环蛋白。我们的数据允许开发一种模型，其中E-钙粘蛋白的钙蛋白酶切割诱导局灶性细胞接触的解体，并产生球状体中形成三维细胞-细胞接触所需的100 kDa E-钙粘蛋白片段。β-连环蛋白的平行释放及其通过钙蛋白酶切割的潜在激活被可溶性Wnt途径抑制剂的过表达所抵消。根据该模型，钙蛋白酶在E-钙粘蛋白和β-连环蛋白介导的细胞间粘附与经典Wnt信号通路之间的相互作用中具有关键作用。支持这一模型，我们表明，钙蛋白酶活性的药理学调节，防止球体的形成，并导致解体的预先存在的球体成单细胞，从而提供了新的策略，以改善悬浮培养条件的人多能干细胞在未来。
The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active beta-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and beta-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of beta-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and beta-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that pharmacological modulation of calpain activity prevents spheroid formation and causes disassembly of preexisting spheroids into single cells, thereby providing novel strategies for improving suspension culture conditions for human pluripotent stem cells in the future.