calpastatin and Neuroblastoma

Previously, we showed that contamination of SH-SY5Y neuroblastoma cells by Mycoplasma hyorhinis strains NDMh and MCLD leads to increased levels of calpastatin (the endogenous, specific inhibitor of the Ca2+-dependent protease calpain), resulting in inhibition of calpain activation. We have found that the increased calpastatin level is promoted by the lipoprotein fraction (MhLpp) of the mycoplasmal membrane. Here, we present MhLpp-based novel synthetic lipopeptides that induce upregulation of calpastatin in SH-SY5Y neuroblastoma cells, leading to protection of the treated cells against Ca2+/amyloid-β-peptide toxicity. These lipopeptides present a new class of promising agents against calpain-induced cell toxicity.

Topics: Amyloid beta-Peptides; Calcium-Binding Proteins; Cell Line, Tumor; Drug-Related Side Effects and Adverse Reactions; Humans; Lipopeptides; Mycoplasma hyorhinis; Neuroblastoma; Neuroprotective Agents; Up-Regulation

Methamphetamine (METH) is an addictive stimulant drug that has many negative consequences, including toxic effects to the brain. Recently, the induction of inflammatory processes has been identified as a potential contributing factor to induce neuronal cell degeneration. It has been demonstrated that the expression of inflammatory agents, such as cyclooxygenase 2 (COX-2), depends on the activation of calcineurin (CaN) and nuclear factor of activated T-cells (NFAT). Moreover, the excessive elevation in cytosolic Ca

Topics: Calcineurin; Calcium-Binding Proteins; Cell Line, Tumor; Cell Survival; Central Nervous System Stimulants; Cyclooxygenase 2; Dose-Response Relationship, Drug; Gene Expression; Humans; Methamphetamine; Neuroblastoma; NFATC Transcription Factors

Calpain (intracellular Ca(2+)-dependent protease) and calpastatin (calpain specific endogenous inhibitor) are widely distributed in biological systems, and have been implicated in many cellular physiological and pathological processes. Calpastatin level is of central importance to the control of calpain activity. We demonstrated for the first time that calpastatin is overexpressed in mycoplasma-contaminated cultured cells (SH-SY5Y cells that are infected by a strain of Mycoplasma hyorhinis (NDMh)). We have found that the calpastatin-upregulating activity resides in the mycoplasmal membrane lipoproteins, and is associated with NF-κB activation. Calpain-promoted proteolysis is attenuated in the NDMh lipoprotein-treated cells. Here we show that the NDMh lipoproteins promoted an increase in calpastatin in SH-SY5Y cells via the TLR2/TAK1/NF-κB pathway. The synthetic mycoplasmal lipopeptide MALP-2 and the bacterial lipopeptide PAM3CSK4 (TLR2 agonists) also promoted calpastatin upregulation. LPS (TLR4 agonist) activated NF-κB without calpastatin increase in the cell. In contrast, lipoteichoic acid (TLR2 agonist) upregulated calpastatin not via NF-κB activation, but via the MEK1/ELK1 pathway. Zymosan and peptidoglycan, TLR2 agonists that lack lipids, did not induce calpastatin upregulation. Cell treatment with a calpastatin-upregulating agonist (lipoteichoic acid) led to the attenuation of Ca(2+)-promoted calpain activity, whereas agonists that do not upregulate calpastatin (LPS, Zymosan) were ineffective. Overall, the results indicate that in these non-immune cells, calpastatin is upregulated by TLR2-agonists containing lipids, with more than one downstream pathway involved. Such agonists may be useful for studying mechanisms and factors involved in calpastatin regulation. In addition, suitable TLR2 agonists may be of interest in devising treatments for pathological processes involving excessive calpain activation.

Topics: Calcium-Binding Proteins; Calpain; ets-Domain Protein Elk-1; Humans; Immunoblotting; Lipopeptides; Lipopolysaccharides; Lipoproteins; MAP Kinase Kinase 1; Mycoplasma hyorhinis; Neuroblastoma; NF-kappa B; Proteolysis; Teichoic Acids; Toll-Like Receptor 2; Tumor Cells, Cultured; Up-Regulation

Calpains are involved in calcium-induced neuronal cell toxicity, which is associated with the pathophysiology of Alzheimer's disease (AD). The activity of calpains is regulated by the inhibitor calpastatin, and increased activity of calpains and decreased calpastastin are often found in AD. Histone deacetylase (HDAC) inhibitors are implicated in AD treatment through the improvement of learning and memory but the underlying mechanism is yet to be understood. Here, using SH-SY5Y neuroblastoma cells and a calcium ionophore ionomycin, we examined whether and how HDAC inhibitor trichostatin A (TSA) inhibits calcium-induced neuronal cell death. TSA increased both the mRNA and protein levels of calpastatin, with no alterations in those of calpain 1 and calpain 2. Furthermore, TSA-stimulated increase of calpastatin was accompanied by a significant attenuation of ionomycin-induced autolysis of calpain 1, but not of calpain 2, and calpain-dependent 150 kDa αII spectrin cleavage. Under these conditions, however, caspase activity was unaltered. Moreover, ectopic expression of small interfering RNA of calpastatin reversed the inhibitory effect of TSA on ionomycin-induced calpain 1 autolysis and αII spectrin cleavage. Chromatin immunoprecipitation assay revealed the increased levels of acetylation at lysine 5 of histone H4 (H4K5-Ac), H3K9-Ac and H3K14-Ac within the calpastatin promoter region in TSA-treated cells relative to control cells. Finally, TSA significantly decreased ionomycin-induced cell toxicity. This study demonstrates that TSA attenuates calcium-induced neuronal cell death by the inhibition of calpain activity which is mediated in part by increased calpastatin expression via histone hyperacetylation within the calpastatin promoter region. Our study provides a novel mechanism for the neuroprotective effect of HDAC inhibitors on AD.

Topics: Acetylation; Apoptosis; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Proliferation; Chromatin Immunoprecipitation; Epigenomics; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Ionomycin; Luciferases; Neuroblastoma; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

Methamphetamine (METH) is one of the most commonly abused substances in today's society. Many studies have shown that the process of cell death induced by METH involves with the reception of death signals, an increase in pro-apoptotic proteins (Bax) and an activation of cysteine protease death pathway. The objective of this study was to investigate the neuroprotective effects of calpastatin against METH-induced toxicity in SH-SY5Y neuroblastoma cells by observing cell viability, phosphorylation of transcription factor, c-Jun (phospho-c-Jun) and levels of Bax and Bcl-2. The results showed that METH significantly decreased cell viability in SH-SY5Y cultured cells. Conversely, increase in phospho-c-Jun and Bax/Bcl-2 ratio was observed in METH-treated cells. Calpastatin reversed the toxic effect of METH by increasing cell viability, reducing phospho-c-Jun and Bax/Bcl-2 ratio in METH-treated cells. These results indicated that calpastatin has the capacity to reverse an activation of the death process in METH-treated dopaminergic cell lines.

Topics: Analysis of Variance; Animals; bcl-2-Associated X Protein; Calcium-Binding Proteins; Cell Death; Cell Line, Tumor; Central Nervous System Stimulants; Cysteine Proteinase Inhibitors; Drug Interactions; Gene Expression Regulation, Neoplastic; Methamphetamine; Neuroblastoma; Phosphorylation; Proto-Oncogene Proteins c-jun; Rats

Methamphetamine (METH) is an abused psychostimulant drug that can cause neurotoxicity to dopaminergic cells. It has been demonstrated that METH can induce caspase- and calpain-dependent death cascades. The purpose of the present study was to investigate the functional role of calpastatin, a specific endogenous calpain inhibitor protein, on caspase and calpain activation in METH-induced degeneration in neuroblastoma SH-SY5Y cell cultures. In this study, we found that METH significantly decreased cell viability, tyrosine hydroxylase phosphorylation and calpastatin levels. Supplementation of cells with exogenous calpastatin was able to reverse the toxic effect of METH on reduction in cell viability and tyrosine hydroxylase phosphorylation. METH also significantly increased calpain levels, the formation of calpain-specific breakdown products and cleaved caspase-3 levels; once again, these effects were diminished by pretreating the cells with calpastatin. These data suggest the contribution of calpastatin as a potential regulatory factor for calpain- and caspase-dependent death processes.

Topics: Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Central Nervous System Stimulants; Enzyme Activation; Humans; Methamphetamine; Neuroblastoma; Neuroprotective Agents; Phosphorylation; Tyrosine 3-Monooxygenase

Time-lapse imaging analysis was previously used to show that spontaneous proteolysis of PrP(C), which is fluorescence-labeled at both NH(2)- and COOH-termini, occurred in mouse neuroblastoma neuro2a (N2a) cells susceptible to PrP(Sc). We demonstrated that, unlike other protease inhibitors, a calpain inhibitor, calpastatin, drastically inhibited endoproteolysis of PrP(C), as observed with time-lapse imaging in living cells, suggesting calpain-like activity. Calpastatin also inhibited cleavage of endogenous PrP(C), and unprocessed molecules and the double-labeled PrP(C) accumulated around the perinuclear region. The molecular weight of PrP(C) fragments generated by spontaneous proteolysis was identical to those produced when PrP(C) synthesized in vitro was exposed to exogenous calpain. These results suggest that a calpain-like activity mediates normal processing of PrP(C) in N2a cells.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Enzyme Activation; Mice; Neuroblastoma; Peptide Hydrolases; Protein Processing, Post-Translational; PrPC Proteins

Methamphetamine (METH) is a potent psychostimulant drug that may cause neuronal cell degeneration. The underlying mechanisms of METH-induced neuronal toxicity remains poorly understood. In this study, we investigated an important role of calpain-dependent cascades in methamphetamine-induced toxicity in human dopaminergic neuroblastoma SH-SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH-induced calpain-dependent death pathway was also investigated. The results of this study show that METH significantly decreased cell viability and tyrosine hydroxylase phosphorylation in SH-SY5Y cultured cells. Melatonin reversed the toxic effect of METH by inducing cell viability. In addition, melatonin was able to restore the reduction in mitochondrial function and phosphorylation of tyrosine hydroxylase in SH-SY5Y treated cells. An induction of calpain expression and activity but a reduction of calpain inhibitor (calpastatin) protein levels were observed in SH-SY5Y cells treated with METH but these effects were diminished by melatonin. These results implicated calpain-dependent death pathways in the processes of METH-induced toxicity and also indicated that melatonin has the capacity to reverse this toxic effect in SH-SY5Y cultured cells.

Topics: Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Cell Survival; Humans; Melatonin; Methamphetamine; Neuroblastoma

Mycoplasmas often contaminate cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction and metabolic pathways. Mycoplasmal contamination is often unnoticed, so that mycoplasma-induced alterations in cell functions may not be appreciated, unless specifically studied. Here, we show for the first time that contamination of SH-SY5Y cells by Mycoplasma hyorhinis leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease calpain), resulting in inhibition of Ca(2+)-induced calpain activation and inhibition of calpain-promoted proteolysis in the mycoplasmal-infected cells. Calpain activity is recovered upon calpastatin removal from extracts of contaminated cells. The calpain-calpastatin system has been implicated in a variety of physiological and pathological processes (signal transduction, motility, cell cycle, cell differentiation, membrane damage and apoptosis). Because the ratio of calpastatin to calpain is an important factor in the control of calpain activity within the cell, the elevated calpastatin may protect the mycoplasma-infected cells against certain types of damage (e.g. caused by high Ca(2+)). Thus, our results are important for studies on the modulation of host cells by mycoplasmas, and relevant to the pathobiology of processes involving mycoplasmal infections. The mycoplasma-infected cells provide a system for identifying factors that participate in the regulation of cellular calpastatin.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Equipment Contamination; Host-Pathogen Interactions; Mycoplasma hyorhinis; Neuroblastoma; Neurons; Proteins; Tumor Cells, Cultured; Up-Regulation

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

Topics: Alstrom Syndrome; Animals; Calcium-Binding Proteins; Calpain; Caspase 8; Cell Line, Tumor; Cell Movement; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Neoplasm Metastasis; Neuroblastoma; Talin

In neuroblastoma LAN-5 cells during calpain activation, in addition to the two expressed 70 kDa and 30 kDa calpastatin forms, other inhibitory species are produced, having molecular masses of 50 kDa and 15 kDa. At longer times of incubation, both native and new calpastatin species disappear. The formation of these new calpastatins as well as the decrease in intracellular total calpastatin activity are mediated by calpain itself, as indicated by the effect of the synthetic calpain inhibitor I, which prevents both degradative processes. Analysis of the calcium concentrations required for the two processes indicates that the first conservative proteolytic event is mediated by micro-calpain, whereas the second one is preferentially carried out by m-calpain. The appearance of the 15 kDa form, containing only the calpastatin repetitive inhibitory domain and identified also in red cells of hypertensive rats as the major inhibitor form, can be considered a marker of intracellular calpain activation, and it can be used for the monitoring of the involvement of calpain in pathological situations.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Humans; Neuroblastoma; Rats; Tumor Cells, Cultured

Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137*L, LSSD203*F and ALAD404*S), while only a single site (ALDD137*L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.

Topics: Apoptosis; Binding Sites; Calcium-Binding Proteins; Calpain; Caspase 1; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Hydrolysis; Jurkat Cells; Molecular Weight; Neuroblastoma; Recombinant Proteins; Tumor Cells, Cultured

Calpains have importance in human neurodegenerative disease pathogenesis, but these mechanisms are difficult to study in postmortem tissues. To establish a cellular model of the human calpain and calpastatin system, we characterized calpain I, calpain II, and calpastatin in SH-SY5Y human neuroblastoma cells in relation to their counterparts in human brain and investigated their expression and activity after inducing cellular differentiation with retinoic acid (RA), a physiological effector of normal brain development. Calpain I in both SH-SY5Y cells and human brain existed in the cytosolic and particulate fractions as three isoforms (80, 78, and 76 kDa) and exhibited atypical isoelectric focusing behavior. Calpain II in SH-SY5Y cells, as in human brain, migrated as a single predominantly cytosolic 76-kDa protein with an isoelectric point ranging from 5.9 to 6.3. Calpastatin from both sources was also 90% cytosolic. In the cells it was composed of four discrete bands, ranging in molecular weight from 110 to 127 kDa. Levels of activated (76 and 78 kDa) and precursor (80 kDa) calpain I isoforms rose 54% (P < 0.0001) in the particulate fraction and 26% (P < 0.0001) in the soluble fraction after 3 days of RA exposure. Because levels and activity of calpastatin remain unchanged during the first 7 days of RA exposure, the increased abundance of calpain I implies a net activation of the calpain system during differentiation. Calpain I activation may contribute to the remodeling of cell shape and neurite extension/retraction associated with neuronal differentiation.

Topics: Brain; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cysteine Proteinase Inhibitors; Humans; Isomerism; Molecular Weight; Neurites; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Changes in expression of calpains and calpastatin during differentiation in GOTO cells were examined using antibodies specific to calpains and calpastatin. Neuronal differentiation induced by dibutyryl cyclic AMP elicited a remarkable decrease in m-calpain. No marked changes in the levels of calpains were found in bromodeoxyuridine-induced Schwannian differentiation. Calpastatin was detected as a single band of 110k in undifferentiated and in neuronally differentiated cells by Western blot analysis. However, the appearance of a 120k species was detected in Schwannian differentiation associated with morphological change. The data show that marked changes in m-calpain and calpastatin occur in a differentiation-specific manner.

Topics: Bromodeoxyuridine; Bucladesine; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cytosol; Humans; Neuroblastoma; Neurons; Schwann Cells; Tumor Cells, Cultured