The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability of E-cadherin junctions in response to DPAGT1 inhibition. We show the impact of pathway dysregulation through measurements of cell migration in scratch-wound assays. Collectively, our results highlight the importance of numerical analyses of cellular networks dynamics to gain insights into physiological processes and potential design of therapeutic strategies to prevent epithelial cell invasion in cancer.

由蛋白质N-糖基化（protein N-glycosylation）代谢通路、Wnt/β-连环蛋白信号通路（Wnt/β-catenin signaling pathway）以及E-钙粘蛋白介导的细胞间黏附通路这三类进化保守的通路所构成的细胞网络，在发育过程中调控细胞增殖与细胞间黏附的平衡，并在分化组织的稳态维持中发挥关键作用。上述三类通路共享一个高度保守的调控分子——β-连环蛋白，其既可作为E-钙粘蛋白黏着连接的结构组分，又可作为Wnt/β-连环蛋白信号通路的共转录激活因子；而该信号通路的靶基因正是N-糖基化调控基因DPAGT1。尽管上述通路已被独立研究，但学界对其相互作用的动态机制仍知之甚少。本研究首次构建了该细胞网络在MDCK细胞中的数值模型。由于该网络包含大量分子，且其表达水平随细胞微环境与时间动态变化，因此可产生诸多难以追踪的潜在细胞状态。我们借助各组分通路中各反应的已知动力学参数，建立了理论分析框架，并对该网络的细胞调控机制获得了全新认识。具体而言，我们开发了数学模型，可量化在不同条件下，以Wnt3a模拟激活Wnt/β-连环蛋白信号通路时，该网络数学表征中任一分子的浓度倍数变化。我们定量分析了蛋白质N-糖基化以及DPAGT1编码的酶GPT的合成，在决定细胞质β-连环蛋白丰度中的重要性。我们验证了轴蛋白（axin）在β-连环蛋白降解过程中的作用。最后，我们的数据表明，细胞间黏附对细胞内E-钙粘蛋白的循环再利用不敏感。我们通过抑制β-连环蛋白介导的DPAGT1表达激活，并预测DPAGT1抑制后细胞质β-连环蛋白浓度与E-钙粘蛋白黏着连接稳定性的变化，从而验证了该模型。我们通过划痕愈合实验检测细胞迁移，展示了通路失调所产生的影响。综上，我们的研究结果凸显了对细胞网络动态进行数值分析的重要性，这不仅有助于深入理解生理过程，还可为预防癌症中上皮细胞侵袭的治疗策略设计提供新思路。