Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway

Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

基质刚度升高与肝细胞癌（hepatocellular carcinoma, HCC）的发生发展密切相关。然而，基质刚度刺激调控HCC进展的分子机制尚未完全阐明。本研究旨在探讨基质刚度介导的骨桥蛋白（osteopontin, OPN）表达调控效应是否存在于HCC细胞中。我们采用此前已报道的可调节基质刚度的体外培养体系，发现随着基质刚度升高，HCC细胞内OPN的表达水平显著上调。此外，糖原合成激酶3β（GSK3β）的磷酸化水平以及核内β-连环蛋白（β-catenin）的表达量亦显著升高，提示GSK3β/β-catenin通路可能参与OPN的表达调控。针对整合素β1（integrin β1）的基因敲低实验结果显示，与对照组相比，在高刚度底物上培养的HCC细胞中，OPN表达水平与磷酸化GSK3β（p-GSK3β）的含量均显著下调。与此同时，抑制GSK-3β可导致β-catenin在细胞质内积累并增强其核转位能力，进而逆转OPN的表达上调，这表明integrin β1/GSK-3β/β-catenin通路被特异性激活，介导了基质刚度诱导的HCC细胞内OPN表达上调。组织微阵列分析证实，OPN的表达与赖氨酰氧化酶（LOX）及I型胶原（COL1）的表达呈正相关。综上，高基质刚度可通过integrin β1/GSK-3β/β-catenin信号通路上调HCC细胞内OPN的表达。本研究为阐明物理机械信号与生化信号分子共同调控HCC细胞OPN表达的通路提供了全新的视角。