Flow-induced DNA synthesis requires signaling to a translational control pathway.
BACKGROUND
The mTOR translational control pathway that signals to the P70/P85 S6 kinase (pp70(S6k)) is essential for mitogenesis. We have previously shown that pp70(S6k) is activated by fluid flow. We hypothesized that oscillatory fluid flow in the absence of exogenous mitogens would induce endothelial cells to synthesize DNA via activation of the mTOR pathway. For comparison, we also studied the ERK1/2 transcriptional signaling pathway.


METHODS
Confluent human umbilical vein endothelial cells (HUVECs) were exposed to oscillatory flow (12 dyn/cm(2) peak shear stress; 3.3 Hz) or kept static in serum-deprived culture medium. Rapamycin or PD98059 was used to inhibit pp70(S6k) or ERK1/2 activation, respectively.


RESULTS
Oscillatory flow activated both the pp70(S6k) and ERK1/2 signaling pathways. Rapamycin blocked activation of pp70(S6k) but not ERK1/2, while PD98059 blocked ERK1/2 but not pp70(S6k). DNA synthesis, as measured by [3H]thymidine uptake, increased by approximately twofold (P < 0.01) in HUVEC cultures exposed to oscillatory flow compared with those kept static. Rapamycin completely abolished the flow-induced increase in DNA synthesis while PD98059 did not. Oscillatory flow upregulated expression of cyclin-dependent kinases 1 and 4 mRNA in a temporal pattern consistent with cell cycle entry; rapamycin also inhibited these changes.


CONCLUSIONS
Oscillatory flow activates both the ERK 1/2 and pp70(S6k) signaling pathways in HUVECs and induces DNA synthesis in the absence of other exogenous mitogens. Complete blockade of [3H]thymidine uptake by the mTOR pathway inhibitor rapamycin indicates that separate and distinct signaling to a translational control pathway is necessary to mediate flow-induced DNA synthesis by endothelial cells. Oscillatory flow-induced endothelial proliferation may contribute to atherogenesis.
Kraiss
L W
LW
Division of Vascular Surgery, University of Utah School of Medicine, Salt Lake City, Utah 84112-5330, USA. larry.kraiss@hmbg.utah.edu
Ennis
T M
TM
Alto
N M
NM
D016203:CDC2 Protein Kinase / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D014176:Protein Biosynthesis; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D013312:Stress, Physiological; D058570:TOR Serine-Threonine Kinases; D015533:Transcriptional Activation
D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D004247:DNA; D011494:Protein Kinases; C546842:MTOR protein, human; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020123:Sirolimus