Molecular Mechanism Of Integrin β1 Overexpression-Mediated Growth Inhibion And Of P21Cip1 Upregulation In Human Hepatocellular Carcinoma Cell Line SMMC-7721

Integrins,composed of differentαandβsubunits,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 integrinβ1 subunit inhibits cell proliferation.Overexpression of integrinβ1 also up-regulates the protein amount of the other important cyclin-dependent kinase inhibitor p27^Kipl,but not mRNA amount.Furthermore, overexpression of integrinβ1 in SMMC-7721 cells regulates the promoter activity of p21^Cip1 and enhances its transcription.So in this study,we investigated the molecular mechanism of integrinβ1 overexpression-mediated up-regulation of p21 transcription.The cell line used in this study includes the integrinβ1 subunit stably overexpressed SMMC-7721 cells,β1-7721cells,and Mock-cells.We found overexpression of integrinβ1 significantly increases protein and mRNA level of p21. The protein amounts of p21 in both cytoplasm and nucleus were increased inβ1-7721 cells compared with mock-7721 cells.We found knock-down of p21 protein expression using SiRNA could block the cell growth inhibition and S-phase delay induced by integrinβ1 overexpression in SMMC-7721 cells.This indicated the significant role of p21 in integrinβ1 overexpression-mediated growth arrest.Since the stability of p21 was not affected by integrinβ1 overexpression,the high p21 protein level was mainly due to the transcriptional activation of p21 gene.Aftrer 5' deletion of p21 promoter analysis,we found integrinβ1 subunit overexpression activates p21 transcription mainly through the proximal region of p21 promoter which does not contain p53 binding sites.And since the p53 protein level was also not influenced by integrinβ1 overexpression,integrinβ1 overexpression upregulates p21 expression though a p53-independent pathway.After the mutation analysis of p21 promoter,we found one of the Sp1 sites(-82～-77) on the proximal region of p21 promoter plays an important role in integrinβ1 overexpression-meidated p21 transcription.Result of chromatin immunoprecipitation also showed more transcription factor Sp1 was recruited into the p21 promoter in theβ1 subunit-overexpressing cells.Since the protein level of Sp1 was not influenced by integrinβ1 ovrexpression,we wonder integrinβ1 overexpression facilitates the binding of the transcription factor to the p21 promter.Using chromatin immunoprecipitation assay,we found the acetylation value of histone proteins across the p21WAF1/Cipl gene was increased after the integrinβ1 subunit was over-expressed.And the co-activator p300/CBP which pocess potential histone acetyltransferase acitvity was found to be invloved in this integrin-mediated histone modification.Then we set out to find the signaling pathway that involved in this integrinβ1 overexpression-induced p21 expression.Inhibited PI3K/PKB pathway and deceased expression of c-Jun protein was observed in integrinβ1 overexpressing cells while the level of c-Jun phosphorylation was inceased.We found PI3k/Akt pathway was not involved in the integrin-mediated p21 regualtion.Both the decreased expression of c-Jun protein level and the increased phosphorylation of c-Jun play important roles in integrinβ1-mediated p21 transcriptional regulation.Furthermore,we found the phosphorylated form of c-Jun could directly bind to Sp1 and enhances the transcription of p21 gene.Integrin are not monomers,they mediate their biological function through heterodimer formation and ligand binding.In this study we found integrinβ1 subunit overexpression could induce diverseαsubunit in different tissue-origined cancer cells. Integrinβ1 subunit overexpression could neither induce expression of p21 and p27 nor influence the cell proliferation in the integrinα5-deficient cancer cell line. Furthermore,integrinβ1 overexpression-meidated growth arrest could be reversed by downregulation of integrinα5 expression.These indicated the significant role of integrinα5 subunit in integrinβ1 overexperssion-mediated growth arrest.We also showed inadequate cell-matrix interactions due to the relative lack of integrin ligands might be a reason for the integrinβ1 overexpression-mediated growth arrest.