Abstract
Cell proliferation, an event associated with angiogenesis, involves coordinated activities of a number of proteins. The role of plasminogen activator inhibitor-1 (PAI-1) in angiogenesis remains controversial. Utilizing proliferating PAI-1-/- endothelial cells (EC), the impact of a host PAI-1 deficiency on Akt activation was evaluated. Hyperactivation of Akt(Ser(P)473) was observed in PAI-1-/- EC, and this was probably due to enhanced inactivation of tumor suppressor PTEN, thus rendering the cells resistant to apoptotic signals. Higher levels of inactivated caspase-9 in PAI-1-/- EC led to lower levels of procaspase-3 and cleaved caspase-3, thereby promoting survival. These effects were reversed when recombinant PAI-1 was added to PAI-1-/- EC. Additional studies demonstrated that regulation of proliferation is dependent on its interaction with low density lipoprotein receptor-related protein. Thus, PAI-1 is a negative regulator of cell growth, exerting its effect on the phosphatidylinositol 3-kinase/Akt pathway and allowing controlled cell proliferation.
Highlights
Lular matrix, either directly or indirectly through activation of matrix metalloproteases
To determine whether Akt activation is transduced by PI3K, proliferating WT and PAI-1Ϫ/Ϫ endothelial cells (EC) were treated for 1 h with wortmannin, a selective inhibitor of PI3K
Immunoblot analysis using anti-Akt(Ser(P)473) and anti-Akt antibodies, demonstrated that at doses of 150 nM or above, wortmannin inhibited phosphorylation of Akt at Ser473 in both cell types, whereas total levels of Akt were not altered (Fig. 1A). These results suggest that, as in WT cells, Akt activation in PAI-1Ϫ/Ϫ cells is mediated by PI3K
Summary
The ␣-tubulin antibody was obtained from Sigma and from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The ␣-tubulin antibody was obtained from Sigma and from Santa Cruz Biotechnology, Inc. Rabbit antibodies against mouse Akt, mouse Akt(Ser(P)473), human PTEN, human PTEN(Ser(P)380), mouse caspase-9, human caspase-9(Thr(P)125), and human caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). The secondary antibody used was horseradish peroxidase-conjugated polyclonal goat anti-rabbit IgG (Bio-Rad) and horseradish peroxidase-conjugated goat anti-mouse IgM antibody (Santa Cruz Biotechnology Inc.). Final endotoxin levels of the PAI-1 protein preparations used were less than 0.01 enzyme units/ml. The characteristics of PAI-1Ϫ/Ϫ mice have been previously described [29, 30]. PAI-1Ϫ/Ϫ mice were back-crossed into the C57BL/6J strain (ϾF8). WT C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were used as controls.
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