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P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

Sathanoori, Ramasri LU ; Bryl-Gorecka, Paulina LU ; Müller, Christa E.; Erb, Laurie; Weisman, Gary A.; Olde, Björn LU and Erlinge, David LU (2017) In Cellular and Molecular Life Sciences1997-01-01+01:00 74(4). p.731-746
Abstract

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin... (More)

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
FAK, Integrins, P2Y receptors, Shear stress, Wound repair
in
Cellular and Molecular Life Sciences1997-01-01+01:00
volume
74
issue
4
pages
731 - 746
publisher
Birkhaüser
external identifiers
  • scopus:84988644296
  • wos:000393694900011
ISSN
1420-682X
DOI
10.1007/s00018-016-2365-0
language
English
LU publication?
yes
id
feebd9c2-419e-45bc-bc33-32e551288cac
date added to LUP
2016-11-02 14:46:55
date last changed
2018-01-07 11:33:07
@article{feebd9c2-419e-45bc-bc33-32e551288cac,
  abstract     = {<p>Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y<sub>2</sub> receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y<sub>2</sub> receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y<sub>2</sub> Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y<sub>2</sub> Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y<sub>2</sub> receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y<sub>2</sub> RGE mutant receptors. Consistent with the role of P2Y<sub>2</sub> receptors in vasodilation, receptor knockdown and overexpression of P2Y<sub>2</sub> RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y<sub>2</sub> receptor in shear stress-induced cytoskeletal alterations in HUVECs.</p>},
  author       = {Sathanoori, Ramasri and Bryl-Gorecka, Paulina and Müller, Christa E. and Erb, Laurie and Weisman, Gary A. and Olde, Björn and Erlinge, David},
  issn         = {1420-682X},
  keyword      = {FAK,Integrins,P2Y receptors,Shear stress,Wound repair},
  language     = {eng},
  number       = {4},
  pages        = {731--746},
  publisher    = {Birkhaüser},
  series       = {Cellular and Molecular Life Sciences1997-01-01+01:00},
  title        = {P2Y<sub>2</sub> receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells},
  url          = {http://dx.doi.org/10.1007/s00018-016-2365-0},
  volume       = {74},
  year         = {2017},
}