Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-b is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-b1 treatment enhanced the amount of a5b1 integrin on cell surface, the mRNA level of a5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-b1 could also promote cell migration. Furthermore, our results showed that TGF-b1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-b1 could down-regulate PTEN at protein level suggested that TGF-b1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-b1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.

N expression at both mRNA and protein levels compared with immortalized L02 hepatic cells. PTEN mRNA in SMMC-7721 hepatoma cells could be reduced by TGF-βI treatment. We also found that the phosphorylation levels of FAK in both of the hepatoma cell lines were higher than that in L02 hepatic cells. Transient expression of the PTEN gene in SMMC-7721 and HepG2 hepatoma cells resulted in decreased FAK phosphorylation. The level of FAK tyrosine phosphorylation appeared to be inversely correlated with the level of the PTEN protein. In summary, our results indicated that the function of the PTEN gene in hepatocarcinomas may be impaired mainly through point mutations and expression deficiency and that the defect of PTEN in tumor cells could alter the phosphorylation of FAK. (Mol Cell Biochem 262: 25–33, 2004)

PTEN is a major tumor suppressor gene that has been shown to inhibit cell invasion. Its mutation has been found in 20-40% of malignant gliomas. Meanwhile, the type III EGFR mutation (EGFRvIII), which was frequently found in gliomas, promoted cell invasion. In the present study, the effects of PTEN on cell invasion were investigated in U87DEGFR glioblastoma cells with EGFRvIII expression but missing PTEN. The cell invasion was downregulated by transfection of phosphatase-active forms of PTEN (wild-type and G129E) but not by PTEN (C124A) with an inactive phosphatase domain; the effects were correlated with decreased tyrosine phosphatase levels of FAK at Tyr397, which was increased by EGFRvIII. Overexpression of FAK mutant (Y397F) could partially mimic the effect of PTEN on cell invasion. Although EGFRvIII increased the levels of P-Akt and PTEN eliminated it, PI-3K inhibitors, wortmannin or Ly294002, could not decrease the cell invasion. In conclusion, PTEN could inhibit cell invasion even in the presence of the constitutively active EGFR; this inhibition depended on its protein phosphatase activity, partially by dephosphorylating FAK, but not depended on its lipid phosphatase activity.

Integrin a 5β1 and a 2β1 are the major integrin receptors in human hepatocytes. However, in human hepatocellular carcinoma cells it was found that the expression of integrin a 5β1 was decreased and another integrin a 6 β1 increased. In this study, the SMMC7721 human hepatocellular carcinoma cells cotransfected or singlely transfected with integrin a 5 and/or β 1 cDNAs were established, and designated a 5β 1.6-7721, a 5.3-7721, and β 1.6-7721 cell lines, respectively. Transfection with cDNAs of integrin a 5 and β 1 subunits resulted in the over expression of each integrin and modified biological properties, including a slowed growth rate, changes in the cell cycle from 15.5% of control cells in the G2/M phase to 12.1%, 9.6% and 9.4% in a 5.3-7721, β 1.6-7721, a 5 β 1.6-7721, respectively, and a decrease in the Cell Mitosis Index from 1.6 in controls to 0.96, 0.95, and 0.72, and 34%, 28% and 52% derived from colony forming ability, respectively. Tumorigenicity was also tested in nude mice with inoculation of cells subcutaneously. Tumor masses growing in nude mice following inoculation with b 1.6-7721, and a 5 β 1.6-7721 cells weighed only 52% or 31% those of control cells. These results indicated that deletion or low expression of integrin a 5 β 1 may play an important role in the development of hepatocellular carcinoma. Therefore, induction of expression of the integrin a 5 β 1 in malignant cells could be a potential means of treating hepatocellular carcinoma. (Mol Cel Biochem 207: 49-55, 2000)

How cells couple and uncouple regulation of cellular processes such as shape change and survival is an important question in molecular cell biology. PINCH-1, a widely expressed protein consisting of five LIM domains and a C-terminal tail, is an essential focal adhesion protein with multiple functions including regulation of the integrin-linked kinase (ILK) level, cell shape, and survival signaling. We show here that the LIM1-mediated interaction with ILK regulates all these three processes. By contrast, the LIM4-mediated interaction with Nck-2, which regulates cell morphology and migration, is not required for the control of the ILK level and survival. Remarkably, a short 15-residue tail C-terminal to LIM5 is required for both cell shape modulation and survival, albeit it is not required for the control of the ILK level. The C-terminal tail not only regulates PINCH-1 localization to focal adhesions but also functions after it localizes there. These findings suggest that PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but yet distinct domain interactions.