Abstract
In this work, we analyzed the role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation. We found that inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors.
Highlights
PI3K-p70 S6 kinase (S6K) and p42/p44 MAPK Inhibition Abolish DNA Synthesis in 1G11 Endothelial Cells—In order to determine the importance of PI3K activation in the stimulation of endothelial cell proliferation by growth factors, quiescent 1G11 endothelial cells were stimulated with 20% FCS in the absence or presence of two different inhibitors of PI3K, wortmannin [27] and Ly294002 [28], followed by measurement of DNA synthesis
Given the effect of PI3K inhibitors on DNA synthesis in endothelial cells, we studied the effect of rapamycin on thymidine incorporation stimulated by 20% FCS
In this work we have shown that inhibition of p42/p44 MAPK and PI3K-p70 S6K cascades blocks fetal calf serum-induced DNA synthesis reinitiation in vascular endothelial cells
Summary
Materials—Ly294002 was obtained from Alexis, wortmannin and rapamycin from BioMol, and PD98059 from New England Biolabs. Cells and Culture Conditions—Murine lung endothelial 1G11 cells were obtained from Drs Alberto Mantovani and Annunciata Vecchi (Instituto Ricerche Farmacologiche Mario Negri, Milan, Italy) [20] They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% inactivated FCS, 50 units/ml penicillin, 50 g/ml streptomycin sulfate, 150 g/ml endothelial cell growth supplement (Becton Dickinson), 100 g/ml heparin (Sigma), 1% nonessential amino acids, and 2 mM sodium pyruvate. Retroviral Infection and Generation of Wild Type and Rapamycinresistant p70 S6K-expressing 1G11 Cells—Retroviral supernatants were generated by transient transfection of BOSC23 cells [23] with plasmids pBabe, pBabe-p70 S6K (wild type), and pBabe-p70S6KE389D3E (rapamycin-resistant mutant) [24] Both constructions have an amino-terminal Myc tag. Cells were stimulated with fresh DMEM medium containing 20% FCS and 0.25 Ci/ml [methyl-3H]thymidine (Amersham Pharmacia Biotech) (3 mM final concentration). The p70 S6 kinase and p42/p44 MAPK activities were determined by a mobility shift assay in which, following cell lysis, proteins were separated by SDS-polyacrylamide gel electrophoresis in a 9% gel (acrylamide:bisacrylamide 30:0.3 for p70 S6K) or 12.5% (acrylamide:bisacrylamide 30:0.2 for p42/p44 MAPKs), and Western blotting was performed with anti-S6 kinase antiserum or anti-p42/p44 MAPK antiserum EIB [26]
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