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A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension

Abstract

Nitric oxide (NO) production by endothelial cell nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. We sought to understand the mechanism underlying defective eNOS function. Phosphorylation of the serine-threonine kinase Akt, which activates eNOS, was substantially reduced in sinusoidal endothelial cells from injured livers. Overexpression of Akt in vivo restored phosphorylation of Akt and production of NO and reduced portal pressure in portal hypertensive rats. We found that Akt physically interacts with G-protein-coupled receptor kinase-2 (GRK2), and that this interaction inhibits Akt activity. Furthermore, GRK2 expression increased in sinusoidal endothelial cells from portal hypertensive rats and knockdown of GRK2 restored Akt phosphorylation and NO production, and normalized portal pressure. Finally, after liver injury, GRK2-deficient mice developed less severe portal hypertension than control mice. Thus, an important mechanism underlying impaired activity of eNOS in injured sinusoidal endothelial cells is defective phosphorylation of Akt caused by overexpression of GRK2 after injury.

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Figure 1: Phosphorylation of eNOS and Akt and NO production are significantly reduced in injured sinusoidal endothelial cells.
Figure 2: Constitutively active Akt restores phosphorylation of eNOS and production of NO in injured endothelial cells.
Figure 3: Characterization of GRK2 expression in sinusoidal endothelial cells.
Figure 4: Interaction between GRK2 and Akt.
Figure 5: Inhibition of GRK2 expression increases Akt phosphorylation and NO production in injured endothelial cells.
Figure 6: A model for the effect of GRK2-mediated inhibition of Akt activation in injured sinusoidal endothelial cells.

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Acknowledgements

This work was supported by the US National Institutes of Health (grants R01 DK 50574 and R01 DK 57830 to D.C.R., HL 03557 to C.D.K. and R01 GM 59989 and DA 16347 to R.T.P.), the Burroughs Welcome Fund (D.C.R. is the recipient of a Burroughs Welcome Fund Translational Scientist Award) and the American Heart Association (Grant-in-Aid award to R.T.P. and Mid-Atlantic Affiliate of the American Heart Association award 0051276U to C.D.K.). We thank J. Huang and X.-L. Niu for assistance with adenovirus, G. Liang for assistance with microscopy, J. Walker and M. Caron for GRK2 mice and D. Capel and R. Lefkowitz for purified GRK2 (all from Duke University Medical Center).

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Correspondence to Don C Rockey.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

GRK5 and GRK6 expression in normal and injured sinusoidal endothelial cells. (PDF 915 kb)

Supplementary Fig. 2

Overexpression of GRK2 in sinusoidal endothelial cells does not affect the activation of the MAP kinase pathway. (PDF 2069 kb)

Supplementary Fig. 3

Phosphorylation of GSK-3 by active Akt is decreased with increasing concentrations of GRK2. (PDF 1101 kb)

Supplementary Fig. 4

Expression and purification of GRK2 fragments. (PDF 971 kb)

Supplementary Fig. 5

Agonist stimulation leads to Akt/GRK2 disassociation in injured sinusoidal endothelial cells. (PDF 1098 kb)

Supplementary Fig. 6

GRK2 siRNA does not affect GRK5 and GRK6 expression. (PDF 2148 kb)

Supplementary Methods (PDF 99 kb)

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Liu, S., Premont, R., Kontos, C. et al. A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension. Nat Med 11, 952–958 (2005). https://doi.org/10.1038/nm1289

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