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

Abstract

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.

Fig 1. Higher matrix stiffness upregulates OPN expression in HCC cells and activates the GSK3β/β-catenin signaling pathway.

(A) Morphology of Hep3B and Huh7 cells cultured on low stiffness substrate (6 kPa), medium-stiffness substrate (10 kPa) and high-stiffness substrate (16 kPa). (B) mRNA expression of OPN in Hep3B and Huh7 cells cultured on different stiffness substrates. (C) Increasing matrix stiffness upregulates OPN expression and activates the GSK3β/β-catenin signaling pathway in both Hep3B and Huh7 cells. (D) Osteopontin concentrations in supernatant of Huh7 and Hep3B cells cultured on different stiffness substrates and HCC cells with LV-INTGB1-RNAi cultured on high stiffness substrate. In each case, error bars represent SD, *p < 0.05, **p < 0.01, ***p<0.0001.

Fig 2. Higher matrix stiffness activates GSK3β/β-catenin signaling pathway mediated by integrin-β1 to modulate OPN expression.

(A) Levels of P-GSK3β and β-catenin were attenuated in HCC cells infected with LV-INTGB1-RNAi on high stiffness substrate. Nuclear expression of β-catenin and TCF dropped sharply, and the expression of OPN also decreased significantly. (B) OPN expression Huh7 and Hep3B cells infected with LV-ITGB1-RNAi on high stiffness substrate. In each case, error bars represent SD, *p < 0.05, **p < 0.01, ***p<0.0001.

Fig 3. GSK-3β inhibitor rescues OPN expression in HCC cells infected with LV-INTGB1- RNAi on high stiffness substrate.

In the infected HCC cells infected with LV-INTGB1-RNAi grown on high stiffness substrate, inhibition of GSK-3β reversed the previously described increase in OPN expression and β-catenin expression compared to the control cells.

Fig 4. Expressions of COL1, LOX, and OPN in HCC tissue with different matrix stiffness backgrounds.

(A) Representative HCC tumor samples show the expression levels of COL1, LOX and OPN. HCC tissue in Groups 1, 2, and 3 are defined as normal liver stiffness background tissue, medium stiffness and high stiffness liver background tissue, respectively[22]. (B) Graphs showing the average IOD value of COL1, LOX and OPN expression in the three groups analyzed by image pro-plus 6.0 software. In each case, error bars represent SD, *p < 0.05, **p < 0.01, ***p<0.0001. (C) Multiple linear regression indicates that the levels of OPN are positively correlated with the expression levels of LOX and COL1.

Fig 5. Schematic of the proposed mechanism by which matrix stiffness triggers the integrinβ1/GSK3β/β-catenin pathway to modulate OPN expression.