Prohaptoglobin inhibits the transforming growth factor-beta-induced epithelial-to-mesenchymal transition in vitro by increasing Smad1/5 activation and suppressing the Smad2/3 signaling pathway in SK-Hep1 liver cancer cells

Transforming growth factor-beta (TGF-beta) is an important inducer of the epithelial-to-mesenchymal transition (EMT) in various cancers. Our previous study demonstrated that prohaptoglobin (proHp) stimulates Smad1/5 activation via ALK1, a TGF-beta type I receptor, in endothelial cells, suggesting that proHp plays a role in TGF-beta signaling. However, the function of proHp in cellular events downstream of Smads remains unclear. The current study investigated the effects of proHp on TGF-beta-mediated Smad-dependent EMT induction and cell invasion in vitro using proHp-overexpressing SK-Hep1 liver cancer cells. The results of Western blotting, quantitative real-time RT-PCR, and immunocytochemistry indicated that proHp downregulated expression of mesenchymal marker and EMT regulator such as N-cadherin, vimentin, and twist, and upregulated expression of the epithelial marker E-cadherin. Compared with control cells, proHp-overexpressing cells exhibited high levels of ALK1/2/3 receptors and markedly increased Smad1/5 phosphorylation. Interestingly, proHp attenuated TGF-beta-induced expression of mesenchymal markers and Smad2/3 phosphorylation. It also significantly suppressed cell invasion and migration. Knockdown of Smad1/5 abolished the inhibitory effects of proHp on TGF-beta-stimulated Smad2/3 phosphorylation and mesenchymal marker expression. These findings indicate that proHp suppresses the TGF-beta-induced EMT and cell invasion in vitro by enhancing Smad1/5 activation via ALK1/2/3 receptors and thus suppressing the Smad2/3 signaling pathway in SK-Hep1 cells. This study suggests that proHp may prevent a de-differentiation of hepatic cells and induce a cell differentiation by regulating the Smad signaling pathway.