Thrombin stimulated glioblastoma cell proliferation is mediated by RhoA and phospholipase D dependent Rap1 activation

Abstract

We previously reported that stimulation of PAR1 receptors with thrombin activates RhoA and induces mitogenesis in 1321N1 glioblastoma cells. Signaling through RhoA is known to be required for cell transformation by Ras. Another Ras family GTPase, Rap1, plays an important role in cancer and constitutive activation of Rap‐GEFs are associated with aberrant cell growth. Accordingly we asked whether stimulation of PAR1 receptors with thrombin elicits Rap1 activation in 1321N1 cells. We found that thrombin induces a robust and sustained (up to 6 hours) increase in Rap1 activation that is dependent on RhoA signaling. Since RhoA is known to activate phospholipase D (PLD) and diacyclglycerol generated via PLD metabolism can activate GEFs that regulate Rap1 activity, we hypothesized that PLD is a mediator of thrombin‐induced Rap1 activation. Inhibition of the PLD pathway by 1‐butanol and FIPI attenuated thrombin‐induced Rap1 activation and downstream ERK1/2 signaling in 1321N1 cells, indicating that PLD is required for thrombin stimulated Rap1 and ERK1/2 activation. Moreover, blockade of Rap1 activity in these cells significantly suppressed thrombin induced ERK1/2 phosphorylation and cell proliferation. Collectively, our results suggest that PLD is an upstream mediator of thrombin elicited Rap1 activation and that Rap1 signaling is required for mitogenesis in glioblastoma cells.(NIH T32 AG000216‐16 grant)