Intracellular Calpain Activity Research Articles

N-acetyl cysteine inhibits IL-1α release from murine keratinocytes induced by 2-hydroxyethyl methacrylate.

The hydrophilic compound 2-hydroxyethyl methacrylate (HEMA) is a major component of dental bonding materials, and it enhances the binding of resin-composites to biomolecules. However, HEMA is a well-known contact sensitizer. We reported previously that intradermal injection of HEMA induces the production of IL-1 locally in the skin. Keratinocytes are the first barrier against chemical insults and constitutively express IL-1α. In this study, we analyzed whether HEMA induces the production of inflammatory cytokines from murine keratinocyte cell line Pam212 cells. We demonstrated that HEMA induced the release of 17-kDa mature IL-1α and caused cytotoxicity. The activity of calpain, an IL-1α processing enzyme, was significantly higher in HEMA-treated cells. The thiol-containing antioxidant N-acetyl cysteine (NAC) inhibited HEMA-induced IL-1α release but not cytotoxicity. NAC inhibited intracellular calpain activity and reactive oxygen species (ROS) production induced by HEMA. NAC post-treatment also inhibited IL-1α release and intracellular ROS production induced by HEMA. Furthermore, HEMA-induced in vivo inflammation also inhibited by NAC. NAC inhibited polymerization of HEMA through adduct formation via sulfide bonds between the thiol group of NAC and the reactive double bond of HEMA. HEMA-induced IL-1α release and cytotoxicity were also inhibited if HEMA and NAC were pre-incubated before adding to the cells. These results suggested that NAC inhibited IL-1α release through decreases in intracellular ROS and the adduct formation with HEMA. We concluded that HEMA induces IL-1α release from skin keratinocytes, and NAC may be a promising candidate as a therapeutic agent against inflammation induced by HEMA.

401 Neutrophil elastase increases intracellular calpain activity and calcium in cystic fibrosis and non-cystic fibrosis macrophages

401 Neutrophil elastase increases intracellular calpain activity and calcium in cystic fibrosis and non-cystic fibrosis macrophages

Sodium Lauryl Sulfate Stimulates the Generation of Reactive Oxygen Species through Interactions with Cell Membranes.

Sodium lauryl sulfate (SLS), a representative anionic surfactant, is well-known to induce rough skin following single or multiple topical applications. The mechanism by which SLS induces rough skin is thought to result from the disruption of skin moisture function consisting of NMF and epidermal lipids. However, a recent study demonstrated that topically applied SLS easily penetrates into the living cell layers of the epidermis, which suggests that physiological alterations of keratinocytes might cause the SLS-induced rough skin. This study was conducted to clarify the effects of SLS on keratinocytes to demonstrate the contribution of SLS to the induction of rough skin. In addition, the potentials of other widely used anionic surfactants to induce rough skin were evaluated. HaCaT keratinocytes treated with SLS had increased levels of intracellular ROS and IL-1α secretion. Application of SLS on the surface of a reconstructed epidermal equivalent also showed the increased generation of ROS. Further, SLS-treated cells showed an increase of intracellular calpain activity associated with the increase of intracellular Ca2+ concentration. The increase of intracellular ROS was abolished by the addition of BAPTA-AM, a specific chelator of Ca2+. In addition, IL-1α also stimulated ROS generation by HaCaT keratinocytes. An ESR spin-labeling study demonstrated that SLS increased the fluidity of membranes of liposomes and cells. Together, those results indicate that SLS initially interacts with cell membranes, which results in the elevation of intracellular Ca2+ influx. Ca2+ stimulates the secretion of IL-1α due to the activation of calpain, and also increases ROS generation. IL-1α also stimulates ROS generation by HaCaT keratinocytes. We conclude from these results that the elevation of intracellular ROS levels is one of the causes of SLS-induced rough skin. Finally, among the other anionic surfactants tested, sodium lauryl phosphate has less potential to induce rough skin because of its lower generation of ROS.

Modulation by Syk of Bcl-2, calcium and the calpain–calpastatin proteolytic system in human breast cancer cells

Syk is a 72kDa non-receptor tyrosine kinase that is best characterized in hematopoietic cells. While Syk is pro-tumorigenic in some cancer cell types, it also has been reported as a negative regulator of metastatic cell growth in others. An examination of the RelA (p65) subunit of NF-κB expressed in MCF7 breast cancer cells indicated that either treatment with pervanadate or stable expression of Syk protected RelA from calpain-mediated proteolysis. Similar results were observed with the tyrosine phosphatase, PTP1B, another sensitive calpain substrate. The activity of calpain in MCF7 cell lysates was inhibited by both treatment with hydrogen peroxide and expression of Syk, the former due to oxidative inactivation of calpain and the latter to enhanced expression of calpastatin (CAST), the endogenous calpain inhibitor. The level of CAST was elevated in the cytosolic fraction of Syk-positive breast cancer cells resulting in more CAST present in complex with calpain in cell lysates. The high levels of CAST coincided with elevated basal levels of calcium–and of intracellular calpain activity–in Syk-expressing cells resulting from decreased levels of Bcl-2, an inhibitor of IP3-receptor-mediated calcium release. The inhibition of cellular calpain stimulated the Syk-mediated enhancement of NF-κB induced by TNF-α, enhanced tyrosine phosphorylation resulting from integrin crosslinking, and increased the localization of Syk to the plasma membrane.

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR

We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca 2+-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.

Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation

Background To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol. Methods To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging. Results and conclusions By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation. General significance The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.

Functional Role of HSP90 Complexes with Endothelial Nitric-oxide Synthase (eNOS) and Calpain on Nitric Oxide Generation in Endothelial Cells

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.

HIV protease inhibitor therapy reverses neutrophil apoptosis in AIDS patients by direct calpain inhibition

The reduction of neutrophils apoptosis is one of the main non-virological effects of protease inhibitor (PI) therapy. We explore here whether this may be due to the cross-inhibition of calpain, an important non-virological target of PI in vitro. We found that the high basal level of neutrophils apoptosis in AIDS patients is strictly related to an increased intracellular calpain activity. Both alterations disappear after PI treatment, with apoptosis and calpain going back to normal levels after 3 months of PI therapy, independently of a proficient antiviral effect. PI drugs exerted a similar antiapoptotic and anticalpain effects on neutrophils in ex vivo experiments: strikingly, the effects were mimicked by commercially available calpain inhibitors. This study shows, for the first time, that apoptosis of neutrophils in AIDS patients is mediated by calpain, and that neutrophil survival in PI treated AIDS patients is a non virological effect due to calpain inhibition.

Regulation of m-Calpain Activity by α-Synuclein and Its C-terminal Fragment (α-syn61-140)

The m-calpain activity hydrolyzing a fluorogenic substrate of N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcourmarin (LLVY-AMC) was significantly stimulated by more than two-fold in the presence of 5<TEX>$\mu$</TEX>M <TEX>$\alpha$</TEX>synuclein at <TEX>$15{^{\circ}C}$</TEX>. The stimulation was also confirmed with azocasein. The stimulation of the peptide hydrolyzing activity required structural intactness of <TEX>$\alpha$</TEX>-synuclein since the C-terminally or N-terminally modified proteins such as <TEX>$\beta$</TEX>-synuclein, <TEX>$\alpha$</TEX>-syn1-97, and <TEX>$\alpha$</TEX>-syn61-140 did not increase the proteolytic activity. Instead, however, the N-terminally truncated <TEX>$\alpha$</TEX>-syn61-140 was shown to drastically suppress the calpain activity. Since the N-terminal truncation was known to be the primary cleaving event of calpain-mediated proteolysis of <TEX>$\alpha$</TEX>-synuclein and the <TEX>$\alpha$</TEX>-syn61-140 has been demonstrated to be resistant against the calpain digestion, it has been proposed that the intracellular calpain activity could be regulated in a reciprocal manner by <TEX>$\alpha$</TEX>-synuclein and its proteolyzed C-terminal fragment. Based on the results, a possible physiological function of <TEX>$\alpha$</TEX>-synuclein has been suggested as a calpain regulator which contains both stimulatory and inhibitory activities.

Stimulation of β-amyloid precursor protein α-processing by phorbol ester involves calcium and calpain activation

Normal processing of Alzheimer’s β-amyloid precursor protein (APP) is markedly stimulated by phorbol esters, but the underlying mechanisms have yet to be fully understood. In this study, we observed that: (a) Phorbol 12,13-dibutyrate (PDBu)-stimulated APP secretion in cultured SH-SY5Y neuroblastoma and fibroblast cells was blocked by EGTA and calpain inhibitors in a concentration-dependent manner, but not by other protease inhibitors. (b) Secretion of fibronectin, another secretory protein tested for comparison, was enhanced by PDBu, but insensitive to calpain inhibitors. (c) PDBu stimulated intracellular calpain activity as measured by the hydrolysis of a fluorogenic calpain substrate. (d) PDBu also induced rapid proteolysis of two endogenous substrates of calpains, i.e., tau and microtubule-associated protein-2 (MAP-2) and the proteolysis was blocked by EGTA and calpain inhibitors. Taken together, these results suggest that stimulation of APP α-processing by PDBu is through a mechanism that involves the activation of Ca 2+ and, most notably, calpain. The implications of the findings are discussed in relation to the regulatory mechanism of APP α-processing.

Downregulation of the calpain inhibitor protein calpastatin by caspases during renal ischemia-reperfusion.

The interaction between the cysteine proteases calpain and caspases during renal ischemia-reperfusion (I/R) was investigated. An increase in the activity of calpain, as determined by 1) the appearance of calpain-mediated spectrin breakdown products and 2) the conversion of procalpain to active calpain, was demonstrated. Because intracellular calpain activity is regulated by calpastatin, the effect of I/R on calpastatin was determined. On immunoblot of renal cortex, there was a 50-100% decrease of a low molecular weight (LMW) form of calpastatin (41 kDa) after I/R. Calpastatin activity was also significantly decreased after I/R compared with sham-operated rats, indicating that the decreased protein expression had functional significance. In rats treated with the caspase inhibitor, z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB), the decrease in both calpastatin activity and protein expression was normalized, suggesting that caspases may be proteolyzing calpastatin. Caspase 3 activity increased significantly after I/R and was attenuated in ischemic kidneys from rats treated with the caspase inhibitor. In summary, during renal I/R injury, there is 1) calpain activation associated with downregulation of calpastatin protein and decreased calpastatin activity and 2) activation of caspase 3. In addition, in vivo caspase inhibition reverses the decrease in calpastatin activity. In conclusion, proteolysis of calpastatin by caspase 3 may regulate calpain activity during I/R injury. Although the protective effect of cysteine protease inhibition against hypoxic necrosis of proximal tubules has previously been demonstrated, the functional significance in ischemic acute renal failure in vivo merits further study.

Differential degradation of calpastatin by μ- and m-calpain in Ca 2+-enriched human neuroblastoma LAN-5 cells

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 μ-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.

Calpain activity in rat renal proximal tubules. An in situ assay.

AbstractAlthough considerable information about calpain has been obtained from studies using isolated enzyme preparations, this does not permit the dynamic assessment of intracellular protease activity during changes in intracellular Ca2+ and pH. Intracellular calpain activity is regulated by complex interactions with the specific inhibitory protein (calpastatin), Ca2+ and phospholipids, conditions which are difficult to duplicate in isolated systems. Thus a method to measure calpain activity over time in living cells would be helpful in studying its regulation and physiological roles.KeywordsProximal TubuleCalpain ActivityDynamic AssessmentWestern Blot ProcedurePage BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Molecular and Functional Properties of a Calpain Activator Protein Specific for μ-Isoforms

A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ from Haemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the mu-calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on mu-calpain independently from the source of the proteinase. The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca2+-dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation.

Different susceptibility of red cell membrane proteins to calpain degradation

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca 2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca 2+], degradation of anion transport protein (band 3) and Ca 2+-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10–50 μ m Ca 2+. In addition, when exposed to progressive and limited increases in [Ca 2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca 2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca 2+-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.

Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe

Stimulation of human neutrophils with phorbol myristate acetate or fMet-Leu-Phe results in translocation to the plasma membrane of approximately 25–40% of the cellular calpain activity. In the membrane-bound form the Ca 2+-requirement for proteolytic activity is substantially reduced. An anti-calpain monoclonal antibody that is internalized by stimulated neutrophils is recovered in the same subcellular fraction that contains the membrane-bound calpain, apparently in the form of pinocytotic vesicles. When both monoclonal antibody and calpain were present in these vesicles, a pronounced inhibition of the membrane bound proteinase activity was observed. These results provide an explanation for the previously observed inhibitory effect of the monoclonal antibody on intracellular calpain activity and on the concomitant inhibition of granule exocytosis. The activated calpain associated with the plasma membrane compartment is therefore identified as the form specifically involved in mediating the physiological responses.

An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.

An endogenous activator of the Ca2+-dependent proteinase (calpain) has been identified in human neutrophils. In the presence of the activator, the affinity of calpain for Ca2+ is increased by greater than 100-fold and maximum catalytic activity is observed with Ca2+ concentration below 1 microM. The activator is a heat-stable protein having an apparent molecular mass of approximately equal to 40 kDa. It appears to be associated with the cytoskeletal fraction of human neutrophils. Neutrophils also contain an endogenous cytosolic calpain inhibitor (calpastatin), which is readily separated from the activator by size-exclusion chromatography. The effects of the activator and inhibitor appear to be antagonistic and may constitute a physiological mechanism for modulating intracellular calpain activity.

Increased phosphorylation in red cell membranes of subjects affected by essential hypertension

In hemolysates of red cells from hypertensive patients the proteolytic activity of calpain is expressed at a rate approximately three fold higher than in red cells of normotensive subjects. Susceptibility to lysis upon exposure to ionophore A23187 and calcium, conditions that increase intracellular calpain activity, is also significantly enhanced in erythrocytes of hypertensive patients. In inside-out vesicles prepared from erythrocytes of these patients band 3 region undergoes a high extent of phosphorylation which is 1.5 fold higher than that occurring in control red cells from normotensive subjects. This increased phosphorylation can be reproduced in inside-out vesicles from erythrocytes of normal subjects following pretreatment with calpain. Taken together, these results suggest that the presence in erythrocytes of hypertensive subjects of an unregulated calpain dependent proteolytic activity may affect the structure of plasma membranes and determine an increased phosphorylation of intrinsic membrane proteins.

Characterization of the defective calpain-endogenous calpain inhibitor system in erythrocytes from Milan hypertensive rats

In mature red cells of rats from Milan normal (MNS) and hypertensive strains (MHS), the soluble Ca2+ dependent neutral proteinase (calpain) is present in similar amounts with identical Mr of 110 kDa and a dimeric structure composed of two unequal subunits of Mr of 84 and 26 kDa. Conversely, the amount of the endogenous inhibitor is now confirmed by analysis of the specific activity to be approximately 10 times less in red cells of MHS rats. The inhibitor is present in red cells of both strains in three different oligomeric forms of Mr of 240, 120 and 64 kDa. This last molecular species corresponds to the single basic constituent subunit which is the reacting inhibitor form. The apparent equilibrium between the three oligomeric structures is Ca2+-dependent. The high (0.1 mM) Ca2+ requirement for the activity of calpain from erythrocytes of both strains is reduced to 1-5 microM in the presence of plasma membrane phospholipids. Activation of the enzyme in these conditions is prevented by the natural inhibitor. These results strongly support and further emphasize the hypothesis that the structural and functional abnormalities in MHS rats red cells result from an impairment in the modulation of intracellular calpain activity by interaction with its endogenous inhibitor.