Glutaminolysis Was Induced By TGF-β1 Through PP2Ac Regulated Raf-MEK-ERK Signaling Pathway In Endothelial Cell

Objective To explore the glutaminolysis changes and possible existence molecular regulatory mechanisms in the pathological and physiological changes of endothelial cell induced by transforming growth factor-β1(TGF-β1).Methods We taked human umbilical vein endothelial cell(HUVECs) as the research object and stimulated endothelial cells for 48 h with TGF-β1(10 ng/ml). Then we detected the key enzyme, kidney-type glutaminase(KGA), expression with western blot and tested intracellular glutamate content with the Glutamic Acid/Glutamate Oxidase Assay Kit. Meanwhile we detected the expression of p-c-Raf(Ser 259), T-c-Raf, p-ERK1/2, T-ERK1/2 and protein phosphatase 2Ac(PP2Ac) at different time points with western blot method, and detected the activity of protein phosphatase 2A(PP2A) with Serine/Threonine Protein Phosphatase Activity Kit. With MEK1/2 specific inhibitor U0126 pretreatment cells for 30 min, we detected the KGA expression in endothelial cells stimulated by TGF-β1 for 15 min or 60 min with western blot. Then we inhibited the activity of PP2 A with PP2 A specific inhibitor OA or transfection cells with small interfering RNA(PP2Ac si RNA) and detected p-c-Raf(Ser 259), T-c-Raf, p-ERK1/2 and T-ERK1/2 expression in endothelial cells stimulated by TGF-β1 for 15 min or 60 min with western blot method.Results The expression of KGA and intracellular glutamate were significantly increase in endothelial cells with the stimulation of TGF-β1. The expression of p-c-Raf(Ser 259), T-c-Raf and p-ERK1/2 were increase in a short period of time(90min), while the rate of p-c-Raf(Ser 259) to total c-Raf decreased. And PP2 Ac expression has no obvious change but its activity was increase from 15 min and reached the peak at 60 min. Compared with the model group(TGF-β1) at 15 min and 60 min, KGA expression of the inhibitor intervention group(TGF-β1+U0126) was down regulation(P﹤0.05). P-c-Raf(Ser 259), T-c-Raf and p-ERK1/2 protein expression were decrease when the activity of PP2 A was inhibited by OA or by depletion of its catalytic subunit(PP2Ac), but in total c-Raf protein, the ratio of p-c-Raf(Ser 259) was increase(P﹤0.05).Conclusion TGF-β1 promotes glutaminolysis metabolize in endothelial cell. And the key enzyme catalyzing the action of glutaminolysis, KGA, is regulated by Raf-MEK-ERK signaling pathway relying on PP2 A positive regulation.