Molecular Mechanism Of Integrin β1 Overexpression-Mediated Up-regulation Of P27～(Kipl) Protein Expression In Human Hepatocellular Carcinoma Cell Line SMMC-7721

Integrins, composed of different α and β subunits, may play great roles in many fundamental cellular events, such as cell proliferation, differentiation and apoptosis. Generally speaking, integrins are always involved in assembly and growth of focal adhesion plaques, and subsequently promote induction of proliferation or inhibition of apoptosis. However, the expression of integrin was often down-regulated in some solid tumors, such as in human hepatocellular carcinoma. Meanwhile, evidence has emerged that integrin can negatively regulate cell growth. We showed previously that overexpression of integrin β1 subunit inhibits cell proliferation. Overexpression of integrin β1 in SMMC-7721 cells regulates the promoter activity of p21^Cip1 and enhances its transcription. Furthermore, overexpression of integrin β1 also up-regulates the protein amount of the other important cyclin-dependent kinase inhibitor p27^Kip1, but not mRNA amount. So in this study, we investigated the molecular mechanism of integrin β1 overexpression up-regulation protein amount of p27 in post-translational level, including protein degradation and protein subcellular distribution.At the beginning, we verified that overexpression of integrin β1 inhibited cell growth in SMMC-7721 and increased the protein amount of p27. Then using semi-quantitative RT-PCR and real-time PCR, we demonstrated overexpression of integrin β1 did not affect the mRNA amount of p27. Furthermore, we found that the p27 protein expression was increased mainly during Gl phase, and the protein amounts of p27 both in cytoplasm and nucleus were up-regulated in integrin β1 overexpressing cells compared with mock-7721 cells. The protein expression of p27 could be inhibited by transfecting p27 SiRNA plasmids in integrin β1 overexpressing cells. The knock-down of p27 protein expression resulted in the block of cell growth inhibition induced by overexpression of integrin β1, indicating the up-regulation of p27 protein expression participated in the cell growth inhibition in integrin β1overexpressing cells.Chx treatment experiment and pulse-chase results showed that the half-life of p27 protein was prolonged in integrin β1 overexpressing cells, indicating that overexpression of integrin β1 inhibited the degradation of p27 protein. The protein amount of p27 in mock cells was increased in the presence of proteasome inhibitors MG132 and LiCl, whereas this up-regulation of p27 protein amount by proteasome inhibitors was resisted in integrin β1 overexpressing cells. Meanwhile, the ratio of phosphorylated p27 at Thr187 and Skp2 protein amount, as well as the amount of p27 polyubiquitination in integrin β1 overexpressing cells were all decreased. These results strongly suggested that overexpression of integrin β1 up-regulated p27 protein amount in nucleus through preventing the Skp2-dependent proteasome degradation.In addition, we found that overexpression of integrin β1 reduced the mRNA expression of Skp2 in SMMC-7721 cells. PI3K signalling pathway could be responsible for the down-regulation of Skp2 expression induced by overexpression of integrin β1. PI3K inhibitor LY294002 also could up-regulate p27 protein expression at post-translational level. Meanwhile, LY294002 inhibited the expression of Skp2 at transcriptional level. These data suggested that overexpression of integrin β1 inhibited Skp2-dependent p27 protein degradation by PI3K pathway. PKB is an important down-stream effector of PI3K. We found that the avtivity of PKB was reduced in integrin β1 overexpressing cells. However, the alteration of PKB activity only change the subcellular distribution of p27 protein in cytoplasm and nucleus, but did not influence the p27 protein amount. Decreased activity of PKB resulted in the accumulation of p27 protein in nucleus, and increased activity of PKB led to the accumulation of p27 protein in cytoplasm. Therefore, at least partially, the accumulation of p27 protein in nucleus was also resulted from reduced activity of PKB induced by integrin β1 overexpression.Both the inhibition of Skp2-dependent p27 protein degradation and reduced PKB activity resulted in the accumulation of p27 protein in nucleus. To further investigate the mechanism of the p27 protein accumulation in cytoplasm in integrin β1 overexpressing cells, we observed the roles of calpain on p27 protein degradation. We found that calpain could mediate the protein dagradation of p27, and overexpression of integrin β1 inhibited the activity of calpain, suggesting that overexpression of integrin β1 increased the p27 protein amount in cytoplasm by inhibition the calpain-mediated degradation. Furthermore, we analyzed the relationship ofproteasome and calpain-mediated p27 protein degradation. We found that these two degradation pathways occurred mainly during G1 phase, and calpain mediated p27 degradation might precede proteasome in SMMC-7721 cells.In summary, overexpression of integrin β1 prevented the Skp2 dependent ubiquitin proteasome degradation of p27 protein through inhibition PI3K pathway, resulting in the accumulation of p27 protein in nucleus. Reduced activity of PKB induced by integrin β1 overexpression also increased the amount of p27 protein in nucleus through regulation the subcellular distribution of p27. Furthermore, overexpression of integrin β1 repressed the calpain-mediated p27 degradation leading to the accumulation of p27 protein in cytoplasm. Taken together, overexpression of integrin β1 subunit inhibited cell growth by up-regulation p27 protein expression at both protein degradation and subcellular distribution levels in SMMC-7721 cells.