Abstract
Cell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2+]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells. Akt was down-regulated when [Ca2+]i was elevated by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, but was up-regulated when it was suppressed by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), a cell permeable Ca2+ chelator. The inactivation of Akt was well correlated with the susceptibility to oxidant-induced apoptosis in H9c2 cells. To investigate the mechanism of the Ca(2+)-dependent regulation of Akt via the regulation of PP2A, we examined the transcriptional regulation of PP2Acalpha in H9c2 cells with Ca2+ modulators. Transcription of the PP2Acalpha gene was increased by thapsigargin but decreased by BAPTA-AM. The promoter activity was examined and the cAMP response element (CRE) was found responsible for the Ca(2+)-dependent regulation of PP2Acalpha. Furthermore, phosphorylation of CRE-binding protein increased with thapsigargin but decreased with BAPTA-AM. A long term change of [Ca2+]i regulates PP2Acalpha gene transcription via CRE, resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis.
Highlights
Cell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2؉]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells
To investigate the influence of the long term change of [Ca2ϩ]i on susceptibility to oxidative stress-induced cell injury, cells were treated with Ca2ϩ modulators for 4 h exposed to 75 M hydrogen peroxide (H2O2) for 0 –120 min, and cell damage was examined at predetermined times using the Lactate Dehydrogenase (LDH)
To investigate whether apoptosis is involved in the mechanism of thapsigargin-induced cell damage, cells were treated with Ca2ϩ modulators for 4 h exposed to H2O2 (75 M) for 2 h, and apoptosis was examined
Summary
Cell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2؉]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells. A long term change of [Ca2؉]i regulates PP2Ac␣ gene transcription via CRE, resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis. A cellular Ca2ϩ overload or the perturbation of intracellular Ca2ϩ compartmentalization can cause cytotoxicity and trigger apoptosis or necrosis [3, 4] Under such circumstances, various Ca2ϩ-dependent signaling cascades with kinases and phosphatases directly or indirectly influence cellular signaling. Calcineurin/PP2B, a Ca2ϩ-dependent Ser/Thr phosphatase [6], appears to be involved in apoptosis [7] Together, these findings show that Ca2ϩ has a pivotal role in the regulatory mechanism of signaling pathways in cell survival and death, the precise mechanism of Ca2ϩ-dependent cross-talk has not been fully clarified
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