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

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.

SUBMITTER: Putta P

PROVIDER: S-EPMC8498464 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H(2)O(2) stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H(2)O(2) and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H(2)O(2), sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H(2)O(2) and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H(2)O(2) and sorbitol increased phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA(2) by H(2)O(2) occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA(2) activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.

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

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