Protein Phosphatase 2A RegulatesbimExpression via the Akt/FKHRL1 Signaling Pathway in Amyloid-β Peptide-Induced Cerebrovascular Endothelial Cell Death

Abstract

Amyloid-␤ peptide (A␤)-induced death in cerebral endothelial cells (CECs)is preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis. Mitochondria-dependent apoptosis engages Bcl-2 family proteins, especially the BH3-only homologues, which play a key role in initiating the apoptotic cascade. Here, we report that the expression of bim, but not other BH3-only members, was selectively increased in cerebral microvessels isolated from 18-month-old APPsw (Tg2576) mice, a model of cerebral amyloid angiopathy (CAA), suggesting a pivotal role for Bim in A␤-induced cerebrovascular degeneration in vivo. A similar expression profile was observed in A␤-treated CECs. Furthermore, A␤ induction of bim expression involved a pro-apoptotic transcription factor, FKHRL1. FKHRL1 bound to a consensus sequence in the bim promoter region and was activated by A␤ before bim expression. FKHRL1 activity was negatively regulated by phosphorylation catalyzed by Akt, an anti-apoptotic kinase. Akt upregulation by adenoviral gene transfer inhibited A␤-induced FKHRL1 activation and bim induction. In addition, A␤ increased the activity of protein phosphatase 2A (PP2A), a ceramide-activated protein phosphatase. Suppression of PP2A activity by RNA interference or a specific inhibitor, okadaic acid, effectively suppressed A␤-induced Akt inactivation and FKHRL1 activation, leading to an attenuation of bim expression and cell death in CECs. Coimmunoprecipitation experiments revealed that A␤ enhanced the binding of the PP2A regulatory subunit PP2AC␣␤ to Akt. These results implicate PP2A as an early regulator of A␤-induced bim expression and CEC apoptosis via the Akt/FKHRL1 signaling pathway. We raise the possibility that this pathway may play a role in cerebrovascular degeneration in CAA.

Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A

“…Moreover, we show that calpain efficiently cleaves B56 alpha and gamma subunits in vitro, and affects B56 alpha stability also in vivo. Notably, B56 subunits have been demonstrated to regulate Akt phosphorylation in many reports (Van Kanegan et al, 2005;Yin et al, 2006;Rocher et al, 2007). On the basis of our findings, we may speculate that calpain could modulate Akt through the regulation of B56 subunits, and hence PP2A recruitment.…”

“…Altogether, the above data suggest that the lack of CAPNS1 is linked to an increase in FoxO activation both in basal conditions and following starvation. To further verify such increase in FoxO activity, electrophoretic mobility shift assay assays were performed with nuclear extracts prepared from CAPNS1 KO and WT cells using an oligonucleotide bearing FoxO-binding sites derived from Fas promoter as a probe (Brunet et al, 1999;Yin et al, 2006). The autoradiography reported in Supplementary Figure 2a shows that CAPNS1 KO MEFs have a higher amount of FoxO-binding activity compared with the WT counterpart.…”

“…Every family accounts for a number of isoforms, in particular the B56 family involves the alpha, beta, gamma, delta and epsilon members, which share high homology. Interestingly, B56 has been implicated in the modulation of Akt phosphorylation (Van Kanegan et al, 2005;Yin et al, 2006;Rocher et al, 2007).…”

“…PTEN is deleted in numerous tumors such as prostate and breast carcinomas (Li et al, 1997;Altomare and Testa, 2005); moreover, FoxO has been implicated in growth factor withdrawal-induced apoptosis (You et al, 2006;Yan et al, 2008) and in amyloid-beta-induced cell death (Yin et al, 2006). PP2A positively regulates FoxO both indirectly, through Akt (Trotman et al, 2006;Yin et al, 2006), and directly, by dephosphorylation both of FoxO1 (Yan et al, 2008) and FoxO3A (Barreyro et al, 2007), leading to FoxO-dependent cell death (Yin et al, 2006;Barreyro et al, 2007;Yan et al, 2008). PP2A is predominantly present as a heterotrimer composed of a scaffolding subunit (A), a catalytic subunit (C) and a variable regulatory subunit (B).…”

Ceramide Induces Apoptosis and Growth Arrest of Human Glioblastoma Cells by Inhibiting Akt Signaling Pathways

“…PP2A controls the activities of several major protein kinase families in the cell, and functions as a positive regulator of apoptosis by dephosphorylating and inactivating antiapoptotic proteins such as Bcl-2 and Akt kinase (Millward et al, 1999;Janssens et al, 2005;Yin et al, 2006). Ceramide is known to stimulate PP2A activity through binding of its catalytic subunit (Law and Rossie, 1995).…”