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.     

Effector CD4+ T cells generate intermediate caspase activity and cleavage of caspase-8 substrates

Caspase-8 activation promotes cell apoptosis but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4(+) T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIP(L), receptor interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4(+) T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling.     

TNF-alpha-mediated cardiomyocyte apoptosis involves caspase-12 and calpain

Following ischemia-reperfusion, there is a sustained increase of TNF-alpha both locally in the heart as well as in circulating levels in blood. While TNF-alpha has been implicated in cardiomyocyte apoptosis which occurs in several cardiomyopathies, the molecular pathways by which TNF-alpha induces apoptosis in these cells are not fully elucidated. We investigated the role of the two families of cysteine proteases, caspases and calpains, which are known to participate in apoptotic cell death. The effect of the highly specific calpain inhibitor, Z-LLY-fmk, and the caspase pathways involved in TNF-alpha-mediated apoptosis of the HL-1 cardiomyocyte cell line were examined. Activation of the downstream caspase-3, and the cleavage of poly ADP-ribose polymerase (PARP) were observed in a time-dependent manner upon treatment with TNF-alpha. Caspase-12, but not caspase-9, was activated in response to TNF-stimulation, indicating that an endoplasmic reticulum (ER)/calcium-dependent pathway may be involved. In HL-1 cardiomyocytes, TNF-alpha-induced apoptosis appears to be mediated by calpain as apoptotic changes were abrogated in the presence of the highly specific calpain inhibitor, Z-LLY-fmk. In conclusion, our results suggest that TNF-alpha-mediated apoptosis in HL-1 cardiomyocytes follows the caspase-12 apoptotic pathway that involves calpain.     

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.     

Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.     

Calpain and calpastatin in myoblast differentiation and fusion: Effects of inhibitors

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca²⁺-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-β, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-β and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of μ-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.     

Caspases Mediate 6-Hydroxydopamine-Induced Apoptosis but Not Necrosis in PC12 Cells

Abstract: The neurotoxin 6-hydroxydopamine (6-OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6-OHDA-induced apoptosis is morphologically indistinguishable from serum deprivation-induced apoptosis. Exposure of PC12 cells to a low concentration of 6-OHDA (25 µ M ) results in apoptosis, whereas an increased concentration (50 µ M ) results in a mixture of apoptosis and necrosis. We investigated the involvement of caspases in the apoptotic death of PC12 cells induced by 6-OHDA, using a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), and compared this with serum deprivation-induced apoptosis, which is known to involve caspases. We show that zVAD-fmk (100 µ M ) completely prevented the apoptotic morphology of chromatin condensation induced by exposure to either 6-OHDA (25 and 50 µ M ) or serum deprivation. Furthermore, cell lysates from 6-OHDA-treated cultures showed cleavage of a fluorogenic substrate for caspase-3-like proteases (caspase-2, 3, and 7), acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin, and this was inhibited by zVAD-fmk. However, although zVAD-fmk restored total cell viability to serum-deprived cells or cells exposed to 25 µ M 6-OHDA, the inhibitor did not restore viability to cells exposed to 50 µ M 6-OHDA. These data show the involvement of a caspase-3-like protease in 6-OHDA-induced apoptosis and that caspase inhibition is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6-OHDA neurotoxicity.     

Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells

Neuronal cell death after traumatic brain injury, Alzheimer’s disease and ischemic stroke may in part be mediated through endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR results in induction of molecular chaperone GRP78 and the ER-resident caspase-12, whose activation has been proposed to be mediated by calpain and caspase processing, although their relative contribution remains unclear. In this study we induced ER stress with thapsigargin (TG), and determined the activation profile of calpain-2, caspase-3, caspase-7, and caspase-12 by analyses of protein levels, corresponding substrates and breakdown products (BDP). Specific calpain and caspase activity was assessed by analysis of αII-spectrin BDP of 145 kDa (SBDP145), BDP of 150 kDa (SBDP150) and BDP of 120 kDa (SBDP120). Decrease in pro-calpain-2 protein and increased SBDP145 levels by 3 h after TG treatment indicated early calpain activity. Active caspase-7 (p20) increase occurred after 8 h, followed by concomitant up-regulation of active caspase-3 and SBDP120 after 24 h. In vitro digestion experiments supported that SBDP120 was exclusively generated by active caspase-3 and validated that kinectin and co-chaperone p23 were calpain and caspase-7 substrates, respectively. Pro-caspase-12 protein processing by the specific action of calpain and caspase-3/7 was observed in a time-dependent manner. N-terminal pro-domain processing of pro-caspase-12 by calpain generated a 38 kDa fragment, while caspase-3/7 generated a 35 kDa fragment. Antibody developed specifically against the caspase-3/7 C-terminal cleavage site D³⁴¹ detected the presence of large subunit (p20) containing 23 kDa fragment that increased after 24 h of TG treatment. Significant caspase-12 enzyme activity was only detected after 24 h of TG treatment and was completely inhibited by caspase 3/7 inhibitor DEVD-fmk and partially by calpain inhibitor SNJ-1945. ER-stress-induced cell death pathway in TG-treated PC12 cells was characterized by up-regulation of GRP-78 and processing and activation of caspase-12 by the orchestrated proteolytic activity of calpain-2 and caspase-3/7.     

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.     

Expression and characterization of constitutively active human caspase-14

Caspase-14 is a cysteine endoproteinase that is expressed in the epidermis and a limited number of other tissues. It is activated during keratinocyte differentiation by zymogen processing, but its precise function is unknown. To obtain caspase-14 for functional studies, we engineered and expressed a constitutively active form of human caspase-14 (Rev-hC14) in Escherichia coli and cultured mammalian cells. Rev-hC14 required no proteolytic processing for activity, showed strong activity against the caspase substrate WEHD, and was inhibited by the pan-caspase inhibitor zVAD-fmk. Mammalian cells that expressed active caspase-14 showed normal cell adherence and morphology. Using positional scanning of synthetic tetrapeptide libraries, we determined the substrate preference of human caspase-14 to be W (or Y)-X-X-D. These studies affirm that caspase-14 has a substrate specificity similar to the group I caspases, and demonstrate that it functions in a distinct manner from executioner caspases to carry out specific proteolytic events during keratinocyte differentiation.     

Active caspase-1-mediated secretion of retinoic acid inducible gene-I

Caspase-1 is an inflammatory caspase that controls the activation and secretion of the inflammatory cytokines, IL-1beta and IL-18. We observed that cellular levels of retinoic acid-inducible gene-I (RIG-I) were enhanced when the pan-caspase inhibitor Z-VAD-fmk or caspase-1-specific inhibitor Z-WEHD-fmk blocked caspase activity. Overexpression of caspase-1 reduced cellular levels of RIG-I and inhibited RIG-I-mediated signaling activity. Enzymatic activity of caspase-1 was necessary to control RIG-I, although it was not a substrate of proteolytic cleavage by caspase-1. Caspase-1 physically interacted with full length RIG-I, but not with mutant forms lacking either the amino- or carboxyl-terminal domains. RIG-I was present in the supernatant of cells transfected with active caspase-1 but not with caspase-4. Stimulating cells with LPS and ATP also induced secretion of endogenous RIG-I in macrophages. Our data suggest a novel mechanism that negatively regulates RIG-I-mediated signaling activity via caspase-1-dependent secretion of RIG-I protein.     

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.     

Calpastatin in rat myoblasts: transient diminution and decreased phosphorylation depend on myogenin-directed myoblast differentiation

The formation of skeletal muscle fibers involves cessation of myoblast division, followed by myoblast differentiation and fusion to multinucleated myofibers. The myogenic regulatory factor myogenin appears at the onset of differentiation; it is required for muscle fiber formation, and cannot be replaced by other factors. The myogenin-dependent pathways and targets are not fully known. Previous studies, indicating an involvement of calpain-calpastatin and caspase in myoblast fusion, were based on the use of various inhibitors. The availability of myogenin deficient cell lines that are incapable of fusion, but regain the ability to differentiate when transfected with myogenin, provide a convenient means to study calpain-calpastatin and caspase in fusing and non-fusing myoblasts without the use of inhibitors. The differentiating wild type myoblasts exhibit decreased calpastatin phosphorylation, transient diminution in calpastatin mRNA, caspase-1 dependent diminution in calpastatin protein, and calpain-promoted proteolysis. In the myogenin-deficient myoblasts, calpastatin phosphorylation is not diminished, caspase-1 is not activated, calpastatin mRNA and protein are not diminished, and protein degradation does not occur. The myogenin-deficient myoblasts transfected with myogenin gene regain the ability to fuse, and exhibit the alterations in calpastatin and proteolysis observed in the wild type cells. Overall, the results demonstrate that the regulation of calpain in these myoblasts is independent of myogenin. In contrast, the regulation of calpastatin depends on myogenin function. The temporary diminution of calpastatin during myogenin-directed differentiation of myoblasts allows calpain activation and calpain-induced protein degradation, required for myoblast differentiation and fusion.     

Involvement of endoplasmic reticulum stress and activation of MAP kinases in beta-lapachone-induced human prostate cancer cell apoptosis

Beta-lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the beta-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of beta-lapachone. Beta-Lapachone-induced DU145 cell apoptosis was dose-dependent and accompanied by cleavage of procaspase-12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent beta-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of beta-lapachone-treated cells. These findings show that beta-lapachone-induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of beta-lapachone.     

Caspase-3在roscovitine诱发PC12细胞凋亡中发挥重要作用

我们已证实周期蛋白激酶（cyclin-dependent kinases）cdk2、cdc2和cdk5抑制剂roscovitine诱导PC12细胞凋亡。本实验应用caspase-3免疫细胞化学与hoechst 33342荧光化学双标、MTT比色法细胞活性测定和Western blot方法,研究了caspase-3在roscovitine所致PC12细胞凋亡中的作用。结果显示,roscovitine（50μmol／L）处理PC12细胞12h,细胞核染色质凝缩及核碎片形成,同时胞浆中出现caspase-3阳性标志,caspase-3阳性细胞占细胞总数的42％。非特异性caspases抑制剂Z-VAD-FMK（50μmol／L）和caspase-3特异性抑制剂Z-DEVD-FMK（100μmol／L）可部分降低roscovitine所致的细胞死亡,使细胞存活率分别由29．03％（roscovitine）增至58．06％（Z-VAD-FMK＋roscovitine）和45．16％（Z-DEVD-FMK＋roscovitine）：用单克隆non-erythroid α-spectrin抗体检测roscovitine处理组细胞匀浆提取液,表明caspase-3裂解的特异性spectfin 120kDa蛋白产物较对照组显著增加。提示细胞凋亡成分caspases参与roscovitine所敛的细胞凋亡,其中caspase-3发挥重要作用。     

Characterization of the calcium-dependent proteolytic system in a mouse muscle cell line

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C₂C₁₂) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.     

M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase- and MAPK/ERK-independent pathways

When PC12 cells are deprived of trophic support they undergo apoptosis. We have previously shown that survival of trophic factor-deprived PC12M1 cells can be promoted by activation of the G protein-coupled muscarinic receptors. The mechanism whereby muscarinic receptors inhibit apoptosis is poorly understood. In the present study we investigated this mechanism by examining the effect of muscarinic receptor activation on the serum deprivation-induced activity of key players in apoptosis, the caspases, in PC12M1 cells. The results showed that m1 muscarinic activation inhibits caspase activity induced by serum deprivation. This effect appeared to be caused by the prevention of activation of caspases such as caspase-2 and caspase-3, and not by the inhibition of existing activity. Muscarinic receptor activation also stimulated the mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/ERK) and phosphoinositide (PI) 3-kinase signaling pathways. The PI 3-kinase pathway inhibitors wortmannin and LY294002, as well as the MAPK/ERK pathway PD98059 inhibitor, did not however suppress the inhibitory effect of the muscarinic receptors on caspase activity. The results therefore suggested that the muscarinic survival effect is mediated by a pathway that leads to caspase inhibition by MAPK/ERK- and PI 3-kinase-independent signaling cascades.     

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways

Anandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca(2+), nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by alpha-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.     

Cell death induced by novel procaspase-3 activators can be reduced by growth factors

Caspase-3 is known as the key executioner caspase, activated in both the intrinsic and extrinsic apoptotic pathway, and an effector far downstream in the apoptotic cascade. Procaspase-activating compound-1 (PAC-1) and 1541 were launched as direct activators of procaspase-3 to caspase-3, and anticipated to be promising therapeutic agents for the treatment of cancer. PAC-1 has recently been evaluated in a phase I preclinical trial. However, little is known about the effect of these substances in cells. Activation of caspase-3 in whole cells may be more complicated than thought, as it is likely that this key protease is tightly regulated both in development and apoptosis. In this study, we investigated the effect of epidermal growth factor (EGF) on PAC-1-induced caspase-3 activity and cell death. We show that EGF can block caspase-3 activity generated by PAC-1, and protect both PC12 cells and primary cerebellar granule neurons against PAC-1-induced death. Similar results were obtained with 1541. Both substances reduced cellular p-ERK levels. Crosstalk between caspase-3 and growth factor signaling pathways may present a challenge for the use of such caspase-3-activating substances in cancer therapy, since aberrant growth factor signaling is frequently seen in malignant cells. This study adds important knowledge about cellular effects of procaspase-3 activators like PAC-1 and 1541. Effects mediated by these substances may also contribute to the understanding of caspase signaling in cells.     

Myoblast migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization

Cell migration is a fundamental cellular function particularly during skeletal muscle development. Ubiquitous calpains are well known to play a pivotal role during muscle differentiation, especially at the onset of fusion. In this study, the possible positive regulation of myoblast migration by calpains, a crucial step required to align myoblasts to permit them to fuse, was investigated. Inhibition of calpain activity by different pharmacological inhibitors argues for the involvement of these proteinases during the migration of myoblasts. Moreover, a clonal cell line that fourfold overexpresses calpastatin, the endogenous inhibitor of calpains, and that exhibits deficient calpain activities was obtained. The results showed that the migratory capacity of C2C12 and fusion into multinucleated myotubes were completely prevented in these clonal cells. Calpastatin-overexpressing myoblasts unable to migrate were characterized by rounded morphology, the loss of membrane extensions, the disorganization of stress fibers and exhibited a major defect in new adhesion formation. Surprisingly, the proteolytic patterns of desmin, talin, vinculin, focal adhesion kinase (FAK) and ezrin, radixin, moesin (ERM) proteins are the same in calpastatin-overexpressing myoblasts as compared to control cells. However, an important accumulation of myristoylated alanine-rich C kinase substrate (MARCKS) was observed in cells showing a reduced calpain activity, suggesting that the proteolysis of this actin-binding protein is calpain-dependent and could be involved in both myoblast adhesion and migration.