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Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells
1Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Pettenkoferstrasse 9, 80336 München, Germany. messler@klp.med.uni-muenchen.de
Abstract:
The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.
Insights
Thrombin activates the Rho/Rho kinase pathway, which inactivates myosin light chain phosphatase, leading to endothelial cell contraction. This pathway controls cell contraction by regulating myosin light chain phosphorylation.
Area of Science:
- Cell Biology
- Biochemistry
- Physiology
Background:
- Endothelial cell contraction is crucial for vascular function.
- Rho GTPase signaling pathways regulate cellular processes, including contractility.
Purpose of the Study:
- To investigate the role of Rho GTPase, Rho kinase, and myosin light chain phosphatase in thrombin-induced endothelial cell contraction.
- To elucidate the signaling mechanisms linking thrombin stimulation to endothelial cell contraction.
Main Methods:
- Utilized C3-transferase to inhibit Rho.
- Employed microinjection of Rho kinase domains and myosin light chain phosphatase.
- Assessed effects of phosphatase inhibitors on cell contraction.
Main Results:
- Inhibition of Rho or Rho kinase abolished thrombin-induced contraction.
- Activation of Rho or Rho kinase promoted contraction.
- Thrombin inactivated myosin light chain phosphatase, increasing myosin light chain phosphorylation and causing contraction.
Conclusions:
- Thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase.
- This pathway is essential for regulating myosin light chain phosphorylation and endothelial cell contraction.
- Findings elucidate a key signaling network controlling vascular cell contractility.