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Calcineurin regulates endothelial barrier function by interaction with and dephosphorylation of myosin phosphatase

Bernadett Kolozsvári1, Éva Bakó, Bálint Bécsi

  • 1Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen, Nagyerdei krt 98, Debrecen H-4032, Hungary.

Cardiovascular Research
|August 8, 2012
PubMed
Abstract

Insights

Calcineurin (CN) dephosphorylates MYPT1 at Thr696, enhancing endothelial barrier function. This mechanism involves activating myosin phosphatase (MP) and reducing myosin phosphorylation, crucial for cell integrity.

Area of Science:

  • Cell biology
  • Molecular biology
  • Biochemistry

Background:

  • Calcineurin (CN) is known to influence endothelial barrier function, but the precise molecular mechanisms remain unclear.
  • CN's role in myosin phosphorylation, a key regulator of endothelial cell permeability, is under investigation.

Purpose of the Study:

  • To investigate whether calcineurin (CN) controls myosin phosphorylation by regulating the phosphorylation state of MYPT1 (myosin phosphatase target subunit 1) at Thr696.
  • To elucidate the molecular mechanism by which CN impacts endothelial barrier function.

Main Methods:

  • Utilized bovine and human pulmonary artery endothelial cells (BPAECs and HPAECs).
  • Employed calcineurin inhibitor cyclosporin A (CsA), intracellular Ca(2+) measurements, and Rho-kinase activity assays.
  • Performed overexpression of CN fragments, in vitro dephosphorylation assays, pull-down experiments, co-localization studies, and surface plasmon resonance (SPR).

Main Results:

  • Cyclosporin A (CsA) increased MYPT1(Thr696) phosphorylation in a Ca(2+)- and Rho-kinase-dependent manner.
  • Active calcineurin (CN) decreased endogenous MYPT1(pThr696) levels and directly dephosphorylated MYPT1 in vitro.
  • CN interacts with MYPT1 via a specific motif (PLIEST), stabilizing the complex and potentially activating myosin phosphatase (MP).
  • CsA treatment maintained MYPT1 and myosin phosphorylation levels, correlating with impaired recovery of endothelial barrier function after thrombin stimulation.

Conclusions:

  • Calcineurin (CN) likely improves endothelial barrier function by dephosphorylating cofilin(pSer3) and MYPT1(Thr696).
  • This dephosphorylation activates myosin phosphatase (MP), leading to decreased myosin phosphorylation and altered actin polymerization.
  • CN's interaction with MYPT1 is critical for regulating endothelial permeability and maintaining barrier integrity.

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