Abstract
We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-alpha markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-alpha or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury.
Highlights
From the Departments of ‡Medicine and ¶Biochemistry, University of Arkansas for Medical Sciences and §Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205
We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells
Translocation of Mitochondrial AIF to Nuclei—We have examined the role of AIF and its translocation during cisplatin injury to renal tubular epithelial cells (RTEC)
Summary
From the Departments of ‡Medicine and ¶Biochemistry, University of Arkansas for Medical Sciences and §Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205. Caspase-2 activation was dependent on the p53responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury. These studies were supported by the evidence that caspase inhibitors [10, 11] and Apaf-1- or caspase-3-deficient neuronal and mouse embryonic fibroblasts were unable to prevent mitochondrial release of AIF in response to death signals [7, 12] These studies suggest that AIF translocation is independent of caspase-9 and caspase-3 activation in these cells. Nephrotoxicity is one of the major side effects when the domain; Smac, second mitochondria-derived activator of caspase; Z, benzyloxycarbonyl; fmk, fluoromethylketone; DAPI, 4Ј, 6Ј-diamidino-2phenylindole; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Diablo, direct IAP-binding protein with low pl; HtrA2, high temperature requirement A2; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,3-diphenyltetrazolium bromide; siRNA, small interfering RNA; AMC, amino-4-methylcoumarin; IAP, hihibitor of apoptosis protein
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