No abstract

No 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)

G‐protein coupled receptors that activate RhoA can lead to aberrant cell growth and have been linked to human diseases including cancer. We have shown that in 1321N1 astroglioma cells, stimulation of the PAR1 receptor with thrombin activates RhoA and downstream signals required for mitogenesis. One mediator of thrombin induced mitogenesis is the matricellular protein CCN1 which signals through integrins (Walsh, FASEB J, 2008) and another is phospholipase C epsilon (Citro et al PNAS, 2007). Integrin signaling requires the low molecular weight GTPase Rap1, and PLC epsilon serves as an activator or Rap1. Accordingly we asked whether stimulation of the G12/13 and RhoA coupled PAR1 receptor in 1321N1 cells was coupled to activation of Rap1. Using a pull down assay to assess GTP liganded Rap1, we find that thrombin elicits a robust and sustained increase in Rap1 activation, increasing to 3 fold basal levels and maintained for at least 6 hours. Thrombin stimulation also elicits 4–5 fold increases ERK1/2 and PKD activation which are sustained for at least 6 hours. Our results suggest that thrombin mediated Rap1 activation is associated with sustained ERK1/2 and PKD activation. We are currently exploring the possibility of involvement of RhoA and PLC epsilon in thrombin induced Rap1 activation and the role of Rap1 in sustained ERK and PKD activation induced by thrombin in 1321N1 astroglioma cells.Supported by NIH grant, GM 36927