Neuroprotective Mechanism of Taurine due to Up-regulating Calpastatin and Down-regulating Calpain and Caspase-3 during Focal Cerebral Ischemia

Aims Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death. Methods Male Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose–responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and αII-spectrin, and the apoptotic cell death in penumbra were evaluated. Results Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on μ-calpain activity. Moreover, taurine significantly increased the MAP-2 and αII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo. Conclusions Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."     

Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina subject to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mmHg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin (an endogenous protein inhibitor of calpain) in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 microl of 100 microM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI.     

Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina was subjected to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mm Hg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain, as well as calpastatin (an endogenous protein inhibitor of calpain), in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 μl of 100 μM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24 h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 258–264. The text was submitted by the authors in English.     

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.     

Evidence for apoptosis in the selenite rat model of cataract

The purposes of this experiment were (1) to determine if apoptosis was accelerated during formation of selenite cataract, and (2) to determine the role of calpains and caspases in lens apoptosis. Evidence for apoptosis in selenite-injected rats included: approximately 7-8% of epithelial cells in germinative zone were positive, disappearance of the nuclear membrane, condensation of the chromatin, and breakdown of PARP. Activation of calpains was indicated by characteristic limited proteolysis of crystallins, breakdown of alpha-spectrin to 150/145 kDa fragments, hydrolysis of vimentin, and autolytic breakdown of m-calpain. Selenite cataract did not have an appreciable effect on the mRNA levels for caspase-3, calpains, and calpastatin. This indicated the increased enzyme activity of m-calpain and caspase-3 in selenite cataract occurred at the enzyme level rather than by upregulation of mRNAs. Increased calpain and caspase activity may be linked to the selenite-induced apoptosis. Such data are important because they indicate that apoptosis may be a fairly early event in selenite cataract.     

Calpain,calpastatin activities and ratios during myocardial ischemia-reperfusion

The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a calpain burst impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). As expected no detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without affect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain–calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downegulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of I/R changes, although the role of calpastatin downregulation remains to be elucidated.     

Caspase-1-induced calpastatin degradation in myoblast differentiation and fusion: cross-talk between the caspase and calpain systems

Previously, we found that calpastatin diminished transiently prior to myoblast fusion (rat L8 myoblasts), allowing calpain-induced protein degradation, required for fusion. Here we show that the transient diminution in calpastatin is due to its degradation by caspase-1. Inhibition of caspase-1 prevents calpastatin diminution and prevents myoblast fusion. Caspase-1 activity is transiently increased during myoblast differentiation. Both calpain and caspase appear to be responsible for the fusion-associated membrane protein degradation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in L8 myoblast fusion represents a novel function for this caspase in a non-apoptotic differentiation process, and points to cross-talk between the calpain and caspase systems in some differentiation processes.     

Ucf-101对大鼠局灶性脑缺血再灌注后Caspase-3蛋白表达的影响

目的观察Ucf—101对大鼠脑缺血再灌注后神经元caspase-3蛋白表达及细胞凋亡的影响,研究其对缺血性脑损伤是否具有保护作用。方法将36只雄性WiStar大鼠随机分为3组：假手术组、缺血组及Ucf—101组,采用线栓法建立大鼠右侧大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）2h再灌注模型,于再灌注后6h和24h断头取脑,采用TTC法测梗死体积,TUNEL法原位标记DNA片段,检测TUNEL阳性细胞的变化,免疫组化法观察脑皮质神经元caspase-3的表达。结果脑缺血再灌注后不同时间点（6h、24h）,Ucf-101组与缺血组相比梗死体积明显缩小,有显著性差异（P〈0．05）;假手术组未见梗死现象。缺血组TUNEL阳性细胞数较假手术组明显增多（P〈0．05）,脑皮质caspase-3的表达较假手术组亦显著增强（P〈0．05）,给予Ucf-101处理后,TUNEL阳性细胞数较缺血组明显减少（P〈0．05）,caspase-3的表达较缺血组亦明显减弱（P〈0．05）。结论Ucf-101能有效地抑制脑缺血再灌注损伤,下调脑皮质神经元Caspase-3蛋白的表达,抑制神经元的凋亡,发挥神经保护作用。     

Effect of Ca2+ on binding of the calpains to calpastatin

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.     

Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells

Abstract

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H₂O₂-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H₂O₂ induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H₂O₂-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.     

Postischemic Reperfusion Induces α-Fodrin Proteolysis by m-Calpain in the Synaptosome and Nucleus in Rat Brain

Abstract: A membrane cytoskeletal protein, fodrin, is a substrate for a Ca²⁺-dependent protease, calpain. It remains unknown whether μ-calpain or m-calpain is involved in the proteolysis of either α- or β-fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of μ-calpain or a significant change of m-calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca²⁺-dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE-cellulose column. α- and β-fodrins and m-calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of α-fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 µ M , 1 ml, i.v.). The μ-calpain-specific fragment of β-fodrin was not generated during ischemia-reperfusion, supporting the possibility of the involvement of m-calpain rather than μ-calpain in the α-fodrin proteolysis.     

Degradation of Microtubule-Associated Protein 2 and Brain Spectrin by Calpain: A Comparative Study

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and spectrin by the calcium-dependent neutral protease calpain was studied. Five major results are reported. First, MAP-2 isolated from twice-cycled microtubules (2 X MT MAP-2) was extremely sensitive to calpain-induced hydrolysis. Even at an enzyme-to-substrate ratio (wt/wt) of 1:200, 2 X MT MAP-2 was significantly degraded by calpain. Second, MAP-2 purified from the total brain heat-stable fraction (total MAP-2) was significantly more resistant to calpain-induced hydrolysis compared with 2 X MT MAP-2. Third, MAP-2a and MAP-2b were proteolyzed similarly by calpain, although some relative resistance of MAP-2b was observed. Fourth, the presence of calmodulin significantly increased the extent of calpain-induced hydrolysis of the alpha-subunit of spectrin. Fifth, the two neuronal isoforms of brain spectrin (240/235 and 240/235E, referred to as alpha/beta N and alpha/beta E, respectively) showed different sensitivities to calpain. alpha N-spectrin was significantly more sensitive to calpain-induced degradation compared to alpha E-spectrin. Among other things, these results suggest a role for the calpain-induced degradation of MAP-2, as well as spectrin, in such physiological processes as alterations in synaptic efficacy, dendritic remodeling, and in pathological processes associated with neurodegeneration.     

Microinjection of calpastatin inhibits fusion in myoblasts

Rat satellite cells (RSC) were microinjected with purified calpastatin or m-calpain, and myoblasts from a C2C12 mouse line were microinjected with purified calpastatin. Microinjection with calpastatin completely prevented fusion of myoblasts from both sources, whereas microinjection with m-calpain significantly increased the rate of fusion of cultured RSC; 44% of the nuclei of RSC cultures were in multinucleated myotubes within 48 h after microinjection with m-calpain plus labeled dextran, whereas only 15% of the nuclei were in multinucleated myotubes after microinjection with dextran alone. Western analyses indicated that neither RSC nor C2C12 myoblasts contained detectable amounts of mu-calpain before fusion. The levels of calpastatin in C2C12 myoblasts increased as cells passed from the proliferative stage to the onset of fusion, and these levels increased substantially in both the C2C12 and the RSC cells as they progressed to the late or postfusion stage. Both RSC and C2C12 myoblasts contained an 80-kDa polypeptide that was labeled with an anti-m-calpain antibody in Western blots. The results are consistent with a role of the calpain system (m-calpain in these myoblast lines) in remodeling of the cytoskeletal/plasma membrane interactions during cell fusion.     

Inflammatory cytokines induced down-regulation of m-calpain mRNA expression in fibroblastic synoviocytes from patients with osteoarthritis and rheumatoid arthritis

Our previous reports revealed that calpain has proteoglycanase activity and exists in synovial fluid in osteoarthritis and rheumatoid arthritis. We examined the effects of cytokines on expression of the calpain-calpastatin system in fibroblastic synoviocytes (FLS). Primary cultures of human FLS from osteoarthritis (OA) and rheumatoid arthritis (RA) patients were stimulated with inflammatory cytokines and the amounts of m-calpain and calpastatin mRNAs expressed were determined by Northern blotting. Northern blots were subjected to computerized densitometer and band intensities were determined. Interleukin-1 (IL-1) down-regulated m-calpain and tissue-type calpastatin mRNA expression in OA and RA FLS. In RA FLS, although IL-6 did not alter m-calpain mRNA expression, IL-1 + tumor necrosis factor (TNF) and IL-1 + transforming growth factor (TGF) down-regulated m-calpain mRNA expression. These results provide new information about the effects of inflammatory cytokines on calpain and calpastatin system in OA and RA pathology.     

Protective functions of taurine against experimental stroke through depressing mitochondria-mediated cell death in rats

Taurine, an abundant amino acid in the nervous system, is reported to reduce ischemic brain injury in a dose-dependent manner. This study was designed to investigate whether taurine protected brain against experimental stroke through affecting mitochondria-mediated cell death pathway. Rats were subjected to 2-h ischemia by intraluminal filament, and then reperfused for 22 h. It was confirmed again that taurine (50 mg/kg) administered intravenously 1 h after ischemia markedly improved neurological function and decreased infarct volume at 22 h after reperfusion. In vehicle-treated rats, the levels of intracellular ATP and the levels of cytosolic and mitochondrial Bcl-xL in the penumbra and core were markedly reduced, while the levels of cytosolic Bax in the core and mitochondrial Bax in the penumbra and core were enhanced significantly. There was a decrease in cytochrome C in mitochondria and an increase in cytochrome C in the cytosol of the penumbra and core. These changes were reversed by taurine. Furthermore, taurine inhibited the activation of calpain and caspase-3, reduced the degradation of αII-spectrin, and attenuated the necrotic and apoptotic cell death in the penumbra and core. These data demonstrated that preserving the mitochondrial function and blocking the mitochondria-mediated cell death pathway may be one mechanism of taurine’s action against brain ischemia.     

Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis

Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.     

Calpain activates caspase-8 in neuron-like differentiated PC12 cells via the amyloid-β-peptide and CD95 pathways

The neurotoxic amyloid-β-peptide (Aβ) is important in the pathogenesis of Alzheimer's disease (AD). Calpain (Ca²⁺-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in AD/Aβ toxicity. We previously found that Aβ promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. We now report on the previously unrecognized caspase-8 activation by calpain. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the Aβ and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIP_S). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIP_S degradation in the Aβ-treated and CD95-triggered cells. Increased cellular Ca²⁺ per se results in calpain activation but does not lead to caspase-8 activation or FLIP_S degradation. The results suggest that procaspase-8 and FLIP_S association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase-8, and CD95 pathway in AD/Aβ toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.     

Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.