Flow-Induced DNA Synthesis Requires Signaling to a Translational Control Pathway

Abstract

Background. The mTOR translational control pathway that signals to the P70/P85 S6 kinase (pp70S6k) is essential for mitogenesis. We have previously shown that pp70S6k 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/cm2 peak shear stress; 3.3 Hz) or kept static in serum-deprived culture medium. Rapamycin or PD98059 was used to inhibit pp70S6k or ERK1/2 activation, respectively.Results. Oscillatory flow activated both the pp70S6k and ERK1/2 signaling pathways. Rapamycin blocked activation of pp70S6k but not ERK1/2, while PD98059 blocked ERK1/2 but not pp70S6k. 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 pp70S6k 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.