PAR-1 activation rescues astrocytes through the PI3K/Akt signaling pathway from chemically induced apoptosis that is exacerbated by gene silencing of β-arrestin 1

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Protease-activated receptors (PARs) are seven transmembrane-domains G-protein-coupled receptors with four members: PAR-1, PAR-2, PAR-3, and PAR-4. The scaffold proteins β-arrestin 1 and β-arrestin 2 have been shown mediating responses to various receptor agonists, including PAR-1 and PAR-2. The aim of this study was to investigate whether the signaling adaptor β-arrestin 1 is (i) associated with apoptosis of astrocytes and (ii) involved in thrombin-induced PAR-1-mediated cytoprotection. Here, we found firstly that staurosporine-induced apoptosis, detected as cleavage of caspase 3 is more than 3-times higher in β-arrestin 1-lacking astrocytes than in control cells. This indicates that β-arrestin 1 is important to protect astrocytes from apoptosis. Secondly, PAR-1 activation by thrombin protects non-silenced and β-arrestin 1-deficient astrocytes from staurosporine-induced chemical toxicity. Furthermore, application of thrombin rescues β-arrestin 1-lacking astrocytes from apoptosis by enhanced Akt (Ser 473) phosphorylation. Rescue from cell death was measured by quantification of the cleavage of caspase 3. Thus, we conclude that the thrombin-activated PI3K/Akt signaling cascades play pivotal roles in survival of β-arrestin 1-deficient astrocytes. Our most striking novel finding is that β-arrestin 1 inhibits long-term thrombin-stimulated phosphorylation of Akt (Ser 473). This has been demonstrated by enhanced Akt (Ser 473) phosphorylation in astrocytes with knockdown of β-arrestin 1. Blockade of the PI3K/Akt signaling pathway by LY294002 abrogates the protection caused by thrombin treatment. In addition, we also found that thrombin-induced phosphorylation of Akt (Ser 473) is increased by transactivation of the EGF and PDGF receptors in β-arrestin 1-silenced astrocytes.