Abstract
The mu- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. Here we compared cell death responses and apoptotic or survival signaling pathways in primary mouse embryonic fibroblasts (MEFs) derived from wild type or capn4 knock-out mice which lack both mu- and m-calpain activities. Capn4(-/-) MEFs displayed resistance to puromycin, camptothecin, etoposide, hydrogen peroxide, ultraviolet light, and serum starvation, which was consistent with pro-apoptotic roles for calpain. In contrast, capn4(-/-) MEFs were more susceptible to staurosporine (STS) and tumor necrosis factor alpha-induced cell death, which provided evidence for anti-apoptotic signaling roles for calpain. Bax activation, release of cytochrome c, and activation of caspase-9 and caspase-3 all correlated with the observed cell death responses of wild type or capn4(-/-) MEFs to the various challenges, suggesting that calpain might play distinct roles in transducing different death signals to the mitochondria. There was no evidence that calpain cleaved Bcl-2 family member proteins that regulate mitochondrial membrane permeability including Bcl-2, Bcl-xl, Bad, Bak, Bid, or Bim. However, activation of the phosphatidylinositol 3 (PI3)-kinase/Akt survival signaling pathway was compromised in capn4(-/-) MEFs under all challenges regardless of the cell death outcome, and blocking Akt activation using the PI3-kinase inhibitor LY294002 abolished the protective effect of calpain to STS challenge. We conclude that the anti-apoptotic function of calpain in tumor necrosis factor alpha- and STS-challenged cells relates to a novel role in activating the PI3-kinase/Akt survival pathway.
Highlights
Biochemical and genetic studies have established that apoptosis can be initiated and executed by caspases, a family of cysteine proteases [2]
These observations support the hypothesis that the capn4-encoded 28-kDa subunit is required for the stability and activity of the -calpain and m-calpain catalytic subunits, -80 and m-80 respectively, and this demonstrates that capn4Ϫ/Ϫ mouse embryonic fibroblasts (MEFs) are devoid of detectable calpain activity
Calpastatin overexpression in human ultraviolet light (UV)(r)-1 fibroblasts protected them against okadaic acid induced apoptosis and reduced c-Jun hyperphosphorylation [52]
Summary
Biochemical and genetic studies have established that apoptosis can be initiated and executed by caspases, a family of cysteine proteases [2]. Many of the studies described above relied on small molecule inhibitors that lack complete calpain specificity on overexpression of calpain or calpastatin or on in vitro proteolysis of the putative calpain substrates. All of these approaches have inherent pitfalls that have confused our understanding of the role of calpain in cell death signaling. Activation of the phosphatidylinositol 3Ј-kinase/AKT survival pathway was correlated with calpain expression in all cell death challenges. This provides novel evidence for an important role for calpain in regulation of a key pro-survival pathway
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