| Integrins, composed of different α and β subunits, may play a great role 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, integrin expression was often down-regulated in some solid tumors, such as the human hepatocellular carcinoma. Meanwhile, evidence has emerged that integrin can negatively regulate cell growth. So in this study, the molecular mechanisms of integrins affected the cell cycle control will be our focus and were further investigated. At the beginning, we performed the stable transfection assay in the human hepatoma cells SMMC-7721, and observed that S-phase arrest can be induced in the integrin β1-overexpressed cells, such as β1-7721 and α5β1-7721 cells. Next, the western blot was employed and we found that the protein levels of CIP/KIP family of CDK inhibitors, p21 and p27, were increased in the β1-7721 and α5β1-7721 cells. Simultaneously, protein kinase B (PKB) phosphorylation was decreased in these two cell types, although its total protein level was not changed. The total protein and its phosphorylation levels of focal adhesion kinase (FAK) were not affected in the integrin β1-overexpressed cells. Overexpression of β1 integrin in SMMC-7721 cells did not interfere with the mRNA level of p27 gene. The p27 protein level was up-regulated when activities of PI3K and its downstream target PKB were inhibited with wortmannin; but its protein level was decreased when the integrin β1-overexpressed cells were grown on the FN- or LN- coated culture dishes. These results suggested that PKB maybe plays a great role in the S-phase arrest observed in this research. Except for the increased level of p21 protein detected by western blot assay, we also found the mRNA level of p21 was elevated in the integrin β1-overexpressed cells. Subsequently, the p21 promoter activity was detected using the luciferase reporter gene assay system. We found that the luciferase activity controlled by the proximalsequence (-189―+28) of p21 5'-flanking region was enhanced when integrin β1 subunit was overexpressed in the SMMC-7721 cells. At the mean time, disruption of cytoskeleton with cytochalasin B can also up-regulated the p21 promoter activity. These findings told that transcription of p21 gene can be exactly controlled by the β1 integrins and the mechanisms underlying this phenomenon may involve the dynamic assembly of microfilament cytoskeleton.As mentioned above, PKB phosphorylation was greatly decreased in the integrin β1-overexpressed cells, β1-7721 and α5β1-7721 when these cells were grown on the flasks without the coating of fibronectin (FN) or laminin (LN). But why the overexpression of β1 integrins can down-regulated the phosphorylation of PKB? In the following studies, we found that β1-7721 or α5β1-7721 cells were prone to spread compared with the mock and α5 -7721 when these cells were plated and grown on the FN- or LN-coated culture dishes. Under the same conditions, the increasing levels of PKB or FAK phosphorylation were also observed in the β1-7721 or α5β1-7721 cells. These results showed that the extracellular matrix might be of great importance to the PKB phosphorylation control in the integrin β1-transfected cells. Consequently, we hypothesized that the relative lack of adhesion matrix may be contributed to the decrease of PKB phosphorylation and the following S-phase arrest in theβ1-7721 or α5β1-7721 cells. Based on this hypothesis, the similar cell cycle pattern and PKB activity would take place if the attachment of the parental SMMC-7721 cells on the culture dishes were blocked by the attachment agonist Poly-HEME. So is the case. The S-phase arrest and the decreased PKB phosphorylation level occurred at 72 h after the parental cells were plated on the Poly-HEME coated Petri dishes.Furthermore, we also observed that the PKB ph...
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