Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

Priya Putta; Andrew H Smith; Pinaki Chaudhuri; Rocio Guardia-Wolff; Michael A Rosenbaum; Linda M Graham

doi:10.1016/j.jbc.2021.101180

Activation of the cytosolic calcium-independent phospholipase A₂ β isoform contributes to TRPC6 externalization via release of arachidonic acid

J Biol Chem. 2021 Oct;297(4):101180. doi: 10.1016/j.jbc.2021.101180. Epub 2021 Sep 10.

Authors

Priya Putta¹, Andrew H Smith², Pinaki Chaudhuri³, Rocio Guardia-Wolff³, Michael A Rosenbaum⁴, Linda M Graham⁵

Affiliations

¹ Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio, USA. Electronic address: puttaP@ccf.org.
² Department of Vascular Surgery, Cleveland Clinic, Cleveland, Ohio, USA.
³ Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio, USA.
⁴ Surgical Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA.
⁵ Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio, USA; Department of Vascular Surgery, Cleveland Clinic, Cleveland, Ohio, USA.

Abstract

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A₂ (PLA₂), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA₂ in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA₂ enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA₂ involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA₂α, cPLA₂γ, iPLA₂β, or iPLA₂γ. Downregulation of the β isoform of iPLA₂ blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.

Keywords: calcium channel; canonical transient receptor potential 6 channel (TRPC6 channel); endothelial cell; lysophosphatidylcholine; migration; phospholipase A(2).

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Arachidonic Acid / metabolism*
Calcium Signaling*
Cattle
Cells, Cultured
Endothelial Cells / metabolism*
Enzyme Activation
Lipoproteins, LDL / metabolism
Lysophosphatidylcholines / metabolism
Phospholipases A2 / metabolism*
TRPC6 Cation Channel / metabolism*

Substances

Lipoproteins, LDL
Lysophosphatidylcholines
TRPC6 Cation Channel
oxidized low density lipoprotein
Arachidonic Acid
Phospholipases A2

Abstract

Publication types

MeSH terms

Substances

Grants and funding