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Participation of the conventional calpains in apoptosis
Tao Lu1, Ying Xu, Maura T Mericle
1The Department of Pharmacology and Therapeutics, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA.
Abstract:
The conventional calpains, m- and micro-calpain, are suggested to be involved in apoptosis triggered by many different mechanisms. However, it has not been possible to definitively associate calpain function with apoptosis, largely because of the incomplete selectivity of the cell permeable calpain inhibitors used in previous studies. In the present study, Chinese hamster ovary (CHO) cell lines overexpressing micro-calpain or the highly specific calpain inhibitor protein, calpastatin, have been utilized to explore apoptosis signals that are influenced by calpain content. This approach allows unambiguous alteration of calpain activity in cells. Serum depletion, treatment with the endoplasmic reticulum (ER) calcium ATPase inhibitor thapsigargin, and treatment with calcium ionophore A23187 produced apoptosis in CHO cells, which was increased in calpain overexpressing cells and decreased by induced expression of calpastatin. Inhibition of calpain activity protected beta-spectrin, but not alpha-spectrin, from proteolysis. The calpains seemed not to be involved in apoptosis triggered by a number of other treatments. Calpain protected against TNF-alpha induced apoptosis. In contrast to previous studies, we found no evidence that calpains proteolyze I kappa B-alpha in TNF-alpha-stimulated cells. These studies indicate that the conventional calpains participate in some, but not all, apoptotic signaling mechanisms. In most cases, they contributed to apoptosis, but in at least one case, they were protective.
Insights
Conventional calpains (proteases) influence cell death (apoptosis) through various mechanisms. This study clarifies their specific roles in apoptosis signaling using genetically modified cells, revealing both pro- and anti-apoptotic functions.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Calpains, a family of proteases, are implicated in apoptosis but their precise roles remain unclear due to limitations of existing inhibitors.
- Understanding calpain involvement in apoptosis is crucial for deciphering cell death pathways.
Purpose of the Study:
- To investigate the specific role of conventional calpains (m- and micro-calpain) in apoptosis signaling.
- To elucidate how calpain activity influences apoptosis using genetically modified Chinese hamster ovary (CHO) cells.
Main Methods:
- Utilized CHO cell lines engineered to overexpress micro-calpain or calpastatin (a calpain inhibitor).
- Induced apoptosis via serum depletion, thapsigargin, or calcium ionophore A23187.
- Assessed changes in apoptosis and protein degradation (beta-spectrin, alpha-spectrin, I kappa B-alpha) in response to altered calpain activity.
Main Results:
- Overexpression of micro-calpain enhanced apoptosis, while calpastatin expression reduced it under specific conditions.
- Calpain activity was linked to beta-spectrin proteolysis but not alpha-spectrin.
- Calpains did not appear to be involved in apoptosis triggered by all stimuli and were protective against TNF-alpha induced apoptosis.
- No evidence of calpain-mediated I kappa B-alpha proteolysis in TNF-alpha-stimulated cells was found.
Conclusions:
- Conventional calpains play a role in specific apoptotic signaling pathways, acting as both contributors and protectors.
- Genetically modified cell lines provide a robust model for dissecting calpain function in apoptosis.
- Findings challenge previous assumptions about calpain's universal role in apoptosis and highlight their context-dependent functions.