MicroRNA-137-3p Protects PC12 Cells Against Oxidative Stress by Downregulation of Calpain-2 and nNOS.

The imbalance between excess reactive oxygen species (ROS) generation and insufficient antioxidant defenses contribute to a range of neurodegenerative diseases. High ROS levels damage cellular macromolecules such as DNA, proteins and lipids, leading to neuron vulnerability and eventual death. However, the underlying molecular mechanism of the ROS regulation is not fully elucidated. Recently, an increasing number of studies suggest that microRNAs (miRNAs) emerge as the targets in regulating oxidative stress. We recently reported the neuroprotective effect of miR-137-3p for brachial plexus avulsion-induced motoneuron death. The present study is sought to investigate whether miR-137-3p also could protect PC12 cells against hydrogen peroxide (H2O2) induced neurotoxicity. By using cell viability assay, ROS assay, gene and protein expression assay, we found that PC-12 cells exposed to H2O2 exhibited decreased cell viability, increased expression levels of calpain-2 and neuronal nitric oxide synthase (nNOS), whereas a decreased miR-137-3p expression. Importantly, restoring the miR-137-3p levels in H2O2 exposure robustly inhibited the elevated nNOS, calpain-2 and ROS expression levels, which subsequently improved the cell viability. Furthermore, the suppressive effect of miR-137-3p on the elevated ROS level under oxidative stress was considerably blunted when we mutated the binding site of calpain-2 targted by miR-137-3p, suggesting the critical role of calpain-2 involving the neuroprotective effect of miR-137-3p. Collectively, these findings highlight the neuroprotective role of miR-137-3p through down-regulating calpain and NOS activity, suggesting its potential role for combating oxidative stress insults in the neurodegenerative diseases.

Endothelial calpain systems orchestrate myofibroblast differentiation during wound healing.

The transformation of fibroblasts to myofibroblasts plays a major role in fibrogenic responses during dermal wound healing. We show a contribution of calpain systems (intracellular regulatory protease systems) in vascular endothelial cells (ECs) to myofibroblast differentiation in wound sites. Dermal wound healing experiments in mice found that calpastatin (an endogenous inhibitor of calpains) is enriched in preexisting vessels but not in newly formed capillaries. Transgenic overexpression of calpastatin in ECs delayed wound healing in mice as well as reducing the keratinocyte layer, extracellular matrix deposition, and myofibroblast accumulation in wound sites. EC and leukocyte markers, however, remain unchanged. Calpastatin overexpression reduced the expression of genes encoding platelet-derived growth factor-B and PDGF receptor-ß (PDGFR-ß). Topical application of platelet-derived growth factor-BB-containing ointment to wounds accelerated healing in control mice, but calpastatin overexpression prevented this acceleration. In cultured human dermal fibroblasts, α-smooth muscle actin and PDGFR-ß were up-regulated by coculturing with ECs, but this action was inhibited by suppression of EC calpain activity. EC-driven transformation of mouse dermal fibroblasts was also suppressed by calpastatin overexpression in ECs. These results suggest that endothelial calpain systems influence PDGFR-ß signaling in fibroblasts, EC-driven myofibroblast differentiation, and subsequent fibrogenic responses in wounds.-Miyazaki, T., Haraguchi, S., Kim-Kaneyama, J.-R., Miyazaki, A. Endothelial calpain systems orchestrate myofibroblast differentiation during wound healing.

Early CALP2 expression and microglial activation are potential inducers of spinal IL-6 up-regulation and bilateral pain following motor nerve injury.

Previous work from our laboratory showed that motor nerve injury by lumbar 5 ventral root transection (L5-VRT) led to interleukin-6 (IL-6) over-expression in bilateral spinal cord, and that intrathecal administration of IL-6 neutralizing antibody delayed the induction of mechanical allodynia in bilateral hind paws. However, early events and upstream mechanisms underlying spinal IL-6 expression following L5-VRT require elucidation. The model of L5-VRT was used to induce neuropathic pain, which was assessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Calpain-2 (CALP2, a calcium-dependent protease) knockdown or over-expression and microglia depletion were conducted intrathecally. Western blots and immunohistochemistry were performed to explore the possible mechanisms. Here, we provide the first evidence that both IL-6 and CALP2 levels are increased in lumbar spinal cord within 30 min following L5-VRT. IL-6 and CALP2 co-localized in both spinal dorsal horn (SDH) and spinal ventral horn. Post-operative (PO) increase in CALP2 in ipsilateral SDH was evident at 10 min PO, preceding increased IL-6 at 20 min PO. Knockdown of spinal CALP2 by intrathecal CALP2-shRNA administration prevented VRT-induced IL-6 overproduction in ipsilateral spinal cord and alleviated bilateral mechanical allodynia. Spinal microglia activation also played a role in early IL-6 up-regulation. Macrophage/microglia markers ED1/Iba1 were increased at 30 min PO, while glial fibrillary acidic protein (astrocyte) and CNPase (oligodendrocyte) markers were not. Increased Iba1 was detected as early as 20 min PO and peaked at 3 days. Morphology changed from a small soma with fine processes in resting cells to an activated ameboid shape. Depletion of microglia using Mac-1-saporin partially prevented IL-6 up-regulation and attenuated VRT-induced bilateral mechanical allodynia. Taken together, our findings provide evidence that increased spinal cord CALP2 and microglia cell activation may have early causative roles in IL-6 over-expression following motor nerve injury. Agents that inhibit CALP2 and/or microglia activation may therefore prove valuable for treating neuropathic pain.

Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy).

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.

Calpain-2 triggers prostate cancer metastasis via enhancing CRMP4 promoter methylation through NF-κB/DNMT1 signaling pathway.

BACKGROUND: Metastasis is the major cause of cancer-specific death in patients with prostate cancer (PCa). We previously reported that collapsing response mediator protein-4 (CRMP4) is a PCa metastasis-suppressor gene and the hypermethylation in CRMP4 promoter is responsible for the transcription repression in metastatic PCa. However, the underlying mechanisms remain unknown. In this study, we aimed to investigate the role of calpain-2 in CRMP4 promoter hypermethylation and its functional modulation in PCa metastasis. METHODS: Calpain-2 expression in PCa tissues (n = 87) and its specific mechanisms of functional modulation in CRMP4 expression via limited enzymatic cleavage was investigated. We then focused on the cooperative crosstalk of calpain-2 and NF-κB RelA/p65 in CRMP4 promoter methylation for the initiation of PCa metastasis. Statistical differences between groups were determined using a two-tailed Student's t-test. P < 0.05 indicated statistically significant. RESULTS: Calpain-2 was differentially upregulated in metastatic PCa compared with localized PCa. Moreover, calpain-2 cleaved CRMP4 into the N-terminally fragment which promoted migration and invasion in PCa cells via nuclear translocation and activation of E2F1-mediated DNA methyltransferase 1 (DNMT1) expression. NF-κB RelA/p65 recruited DNMT1 to bind to and methylate CRMP4 promoter in which Serine276 phosphorylation of p65 was essential. Furthermore, CRMP4 exhibited anti-metastatic function via inhibiting the expression of VEGFC through Semaphorin3B-Neuropilin2 signaling. CONCLUSION: Calpain-2 may contribute to the promoter methylation of CRMP4 to repress its transcription, leading to the metastasis of PCa via enhancing VEGFC expression.

Hypoxia reduces mature hERG channels through calpain up-regulation.

Human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium current (IKr) potassium channel, which is important for cardiac repolarization. Impairment of hERG function is the primary cause of acquired long QT syndrome, which predisposes individuals to cardiac arrhythmias and sudden death. Patients with hypoxia due to conditions such as cardiac ischemia or obstructive sleep apnea display increased incidence of cardiac arrhythmias and sudden death. We sought to understand the mechanisms that underlie hypoxia-associated cardiac arrhythmias. Using cell biology and electrophysiologic techniques, we found that hypoxic culture of hERG-expressing human embryonic kidney (HEK) cells and neonatal rat cardiomyocytes reduced hERG current/IKr and mature ERG channel expression with a concomitant increase in calpain expression. Calpain was actively released into the extracellular milieu and degraded cell-surface hERG. In contrast to hERG, the ether-a-go-go (EAG) channel was not reduced by hypoxic culture. By making chimeric channels between hERG and EAG, we identified that hypoxia-induced calpain degraded hERG by targeting its extracellular S5-pore linker. The scorpion toxin BeKm-1, which is known to selectively bind to the S5-pore linker of hERG, prevented hypoxia-induced hERG reduction. Our data provide novel information about hypoxia-mediated hERG dysfunction and may have biological and clinical implications in hypoxia-associated diseases.-Lamothe, S. M., Song, W., Guo, J., Li, W., Yang, T., Baranchuk, A., Graham, C. H., Zhang, S. Hypoxia reduces mature hERG channels through calpain up-regulation.

The Human Ether-a-go-go-related Gene (hERG) Potassium Channel Represents an Unusual Target for Protease-mediated Damage.

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation, up-regulation, and secretion of various proteolytic enzymes. Here, using whole-cell patch clamp and Western blotting analysis, we demonstrate that the hERG/IKr channel was selectively cleaved by the serine protease, proteinase K (PK). Using molecular biology techniques including making a chimeric channel between protease-sensitive hERG and insensitive human ether-a-go-go (hEAG), as well as application of the scorpion toxin BeKm-1, we identified that the S5-pore linker of hERG is the target domain for proteinase K cleavage. To investigate the physiological relevance of the unique susceptibility of hERG to proteases, we show that cardiac ischemia in a rabbit model was associated with a reduction in mature ERG expression and an increase in the expression of several proteases, including calpain. Using cell biology approaches, we found that calpain-1 was actively released into the extracellular milieu and cleaved hERG at the S5-pore linker. Using protease cleavage-predicting software and site-directed mutagenesis, we identified that calpain-1 cleaves hERG at position Gly-603 in the S5-pore linker of hERG. Clarification of protease-mediated damage of hERG extends our understanding of hERG regulation. Damage of hERG mediated by proteases such as calpain may contribute to ischemia-associated QT prolongation and sudden cardiac death.

miR-203 inhibits augmented proliferation and metastasis of hepatocellular carcinoma residual in the promoted regenerating liver.

Liver resection is still the most commonly used therapeutic treatment for hepatocellular carcinoma (HCC), and liver regeneration promotes HCC growth in the regenerating liver. The high recurrence/metastasis of HCC is the main cause of death for HCC patients after liver resection. However, how the augmented growth and metastasis of residual HCC induced by the promoted liver regeneration following liver resection can be abolished remains unclear. In this study, a rat model with liver cirrhosis and diffused HCC was established by administration of diethylnitrosamine. Recombinant miR-203 adenovirus was administered to induce hepatic miR-203 overexpression and 30% partial hepatectomy (PH) followed. The effect of miR-203 on the proliferation, invasion and metastasis of the residual HCC in the remnant cirrhotic liver with promoted regeneration was investigated. We found that the basic spontaneous regeneration of the non-tumorous liver by 30% PH promoted proliferation, invasion and lung metastasis of the hepatic residual HCC. miR-203 overexpression further promoted the regeneration of the non-tumorous liver by upregulating Ki67 expression and enhancing IL-6/SOCS3/STAT3 pro-proliferative signals. Importantly, miR-203 overexpression markedly inhibited the proliferation, invasion and metastasis of hepatic residual HCC through suppressing expression of Ki67, CAPNS1 and lung metastasis. Moreover, it was found that miR-203 overexpression reversed the epithelial-mesenchymal transition induced by hepatectomy through targeting IL-1ß, Snail1 and Twist1. In conclusion, our results suggested that miR-203 overexpression inhibited the augmented proliferation and lung metastasis of the residual HCC induced by the promoted liver regeneration following PH partly by regulating epithelial-mesenchymal transition.

Molecular and biochemical evidence on the protective effects of quercetin in isoproterenol-induced acute myocardial injury in rats.

Cardioprotection represents one of the most important and realistic aspects of preventive therapy today. Quercetin, a naturally occurring dietary flavone, has been studied extensively for its antioxidant properties. The objective of present study is to find out the cardioprotective activity and to explore the underlying mechanisms of quercetin pretreatment (50 mg/kg body weight, orally) for 14 days against isoproterenol (ISO; 100 mg/kg body weight, subcutaneously) induced myocardial infarction in Wistar rats. Cardiac diagnostic markers, oxidative stress, inflammatory cytokines, histopathology along with gene expression analysis of calpain 1 and 2 were carried out in experimental rats. Quercetin pretreatment showed protective effects on heart by significantly attenuating the ISO-induced oxidative stress, inflammation, protecting heart architecture, and by downregulation of the expression of calpain. Overall, these findings revealed the cardio-protective potential of quercetin and its mechanism of action against ISO-induced MI in rats.

Autolytic activation of calpain 3 proteinase is facilitated by calmodulin protein.

Calpains are broadly distributed, calcium-dependent enzymes that induce limited proteolysis in a wide range of substrates. Mutations in the gene encoding the muscle-specific family member calpain 3 (CAPN3) underlie limb-girdle muscular dystrophy 2A. We have shown previously that CAPN3 knockout muscles exhibit attenuated calcium release, reduced calmodulin kinase (CaMKII) signaling, and impaired muscle adaptation to exercise. However, neither the precise role of CAPN3 in these processes nor the mechanisms of CAPN3 activation in vivo have been fully elucidated. In this study, we identify calmodulin (CaM), a known transducer of the calcium signal, as the first positive regulator of CAPN3 autolytic activity. CaM was shown to bind CAPN3 at two sites located in the C2L domain. Biochemical studies using muscle extracts from transgenic mice overexpressing CAPN3 or its inactive mutant revealed that CaM binding enhanced CAPN3 autolytic activation. Furthermore, CaM facilitated CAPN3-mediated cleavage of its in vivo substrate titin in tissue extracts. Therefore, these studies reveal a novel interaction between CAPN3 and CaM and identify CaM as the first positive regulator of CAPN3 activity.

Calpain-6 deficiency promotes skeletal muscle development and regeneration.

Calpains are Ca(2+)-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.

Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis.

The protective antigen component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8, and ß1-integrin, and, with the assistance of other host proteins, enters targeted cells by receptor-mediated endocytosis. Using an antisense-based phenotypic screen, we discovered the role of calpains in this process. We show that functions of a ubiquitous Ca(2+)-dependent cysteine protease, calpain-2, and of the calpain substrate talin-1 are exploited for association of anthrax toxin and its principal receptor, CMG2, with higher-order actin filaments and consequently for toxin entry into host cells. Down-regulated expression of calpain-2 or talin-1, or pharmacological interference with calpain action, did not affect toxin binding but reduced endocytosis and increased the survival of cells exposed to anthrax lethal toxin. Adventitious expression of wild-type talin-1 promoted toxin endocytosis and lethality, whereas expression of a talin-1 mutant (L432G) that is insensitive to calpain cleavage did not. Disruption of talin-1, which links integrin-containing focal adhesion complexes to the actin cytoskeleton, facilitated association of toxin bound to its principal cell-surface receptor, CMG2, with higher-order actin filaments undergoing dynamic disassembly and reassembly during endocytosis. Our results reveal a mechanism by which a bacterial toxin uses constitutively occurring calpain-mediated cytoskeletal rearrangement for internalization.

[EFFECT OF SHORT-TERM DRY IMMERSION ON PROTEOLYTIC SIGNALING IN HUMAN SOLEUS MUSCLE].

The signaling processes initiating proteolytic events in m. soleus of humans during short-term exposure in the non-weight bearing conditions were analyzed. Dry immersion (DI) was used to induce weight deprivation over 3 days. Western blotting was used to define the IRS-1 content, total and phosphorylated neuronal NO-synthase (nNOS), AMP-activated protein kinase (AMPK) that control the anabolic and catabolic pathways, and concentrations of cytoskeletal protein desmin and Ca²âº-activated protease calpin. Already on day-3 of DI calpain-dependent proteolysis manifests itself by reductions in both the total content and level of nNOS phosphorilation. Moreover, AMPK phosphorilation was decreased drastically.

Calpain 5 is highly expressed in the central nervous system (CNS), carries dual nuclear localization signals, and is associated with nuclear promyelocytic leukemia protein bodies.

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF hand motif characteristic of classical calpains but retains catalytic and Ca(2+) binding domains, and it contains a unique C-terminal domain. TRA-3, an ortholog of CAPN5, has been shown to be involved in necrotic cell death in Caenorhabditis elegans. CAPN5 is expressed throughout the CNS, but its expression relative to other calpains and subcellular distribution has not been investigated previously. Based on relative mRNA levels, Capn5 is the second most highly expressed calpain in the rat CNS, with Capn2 mRNA being the most abundant. Unlike classical calpains, CAPN5 is a non-cytosolic protein localized to the nucleus and extra-nuclear locations. CAPN5 possesses two nuclear localization signals (NLS): an N-terminal monopartite NLS and a unique bipartite NLS closer to the C terminus. The C-terminal NLS contains a SUMO-interacting motif that contributes to nuclear localization, and mutation or deletion of both NLS renders CAPN5 exclusively cytosolic. Dual NLS motifs are common among transcription factors. Interestingly, CAPN5 is found in punctate domains associated with promyelocytic leukemia (PML) protein within the nucleus. PML nuclear bodies are implicated in transcriptional regulation, cell differentiation, cellular response to stress, viral defense, apoptosis, and cell senescence as well as protein sequestration, modification, and degradation. The roles of nuclear CAPN5 remain to be determined.

ROS-Induced Nuclear Translocation of Calpain-2 Facilitates Cardiomyocyte Apoptosis in Tail-Suspended Rats.

Isoproterenol (ISO) induced nuclear translocation of calpain-2 which further increased susceptibility of cardiomyocyte apoptosis in tail-suspended rats. The underlying mechanisms remain elusive. In the present study, the results showed that ISO (10 nM) significantly elevated NADPH oxidases (NOXs) activity and NOXs-derived ROS productions which induced nuclear translocation of calpain-2 in cardiomyocytes of tail-suspended rats. In contrast, the inhibition of NADPH oxidase or cleavage of ROS not only reduced ROS productions, but also resisted nuclear translocation of calpain-2 and decreased ISO-induced apoptosis of cardiomyocyte in tail-suspended rats. ISO also increased the constitutive binding between calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMK II δB) in nuclei, concomitant with the promotion of CaMK II δB degradation and subsequent down-regulation of Bcl-2 mRNA expression and the ratio of Bcl-2 to Bax protein in tail-suspended rat cardiomyocytes. These effects of ISO on cardiomyocytes were abolished by a calpain inhibitor PD150606. Inhibition of calpain significantly reduced ISO-induced loss of the mitochondrial membrane potential, cytochrome c release into the cytoplasm, as well as the activation of caspase-3 and caspase-9 in mitochondrial apoptotic pathway. In summary, the above results suggest that ISO increased NOXs-derived ROS which activated nuclear translocation of calpain-2, subsequently nuclear calpain-2 degraded CaMK II δB which reduced the ratio of Bcl-2 to Bax, and finally the mitochondria apoptosis pathway was triggered in tail-suspended rat cardiomyocytes. Therefore, calpain-2 may represent a potentially therapeutic target for prevention of oxidative stress-associated cardiomyocyte apoptosis.

Expression of calpain-like proteins and effects of calpain inhibitors on the growth rate of Angomonas deanei wild type and aposymbiotic strains.

BACKGROUND: Angomonas deanei is a trypanosomatid parasite of insects that has a bacterial endosymbiont, which supplies amino acids and other nutrients to its host. Bacterium loss induced by antibiotic treatment of the protozoan leads to an aposymbiotic strain with increased need for amino acids and results in increased production of extracellular peptidases. In this work, a more detailed examination of A. deanei was conducted to determine the effects of endosymbiont loss on the host calpain-like proteins (CALPs), followed by testing of different calpain inhibitors on parasite proliferation. RESULTS: Western blotting showed the presence of different protein bands reactive to antibodies against calpain from Drosophila melanogaster (anti-Dm-calpain), lobster calpain (anti-CDPIIb) and cytoskeleton-associated calpain from Trypanosoma brucei (anti-CAP5.5), suggesting a possible modulation of CALPs influenced by the endosymbiont. In the cell-free culture supernatant of A. deanei wild type and aposymbiotic strains, a protein of 80 kDa cross-reacted with the anti-Dm-calpain antibody; however, no cross-reactivity was found with anti-CAP5.5 and anti-CDPIIb antibodies. A search in A. deanei genome for homologues of D. melanogaster calpain, T. brucei CAP5.5 and lobster CDPIIb calpain revealed the presence of hits with at least one calpain conserved domain and also with theoretical molecular mass consistent with the recognition by each antibody. No significant hit was observed in the endosymbiont genome, indicating that calpain molecules might be absent from the symbiont. Flow cytometry analysis of cells treated with the anti-calpain antibodies showed that a larger amount of reactive epitopes was located intracellularly. The reversible calpain inhibitor MDL28170 displayed a much higher efficacy in diminishing the growth of both strains compared to the non-competitive calpain inhibitor PD150606, while the irreversible calpain inhibitor V only marginally diminished the proliferation. CONCLUSIONS: Altogether, these results indicate that distinct calpain-like molecules are expressed by A. deanei, with a possible modulation in the expression influenced by the endosymbiont. In addition, treatment with MDL28170 affects the growth rate of both strains, as previously determined in the human pathogenic species Leishmania amazonensis and Trypanosoma cruzi, with whom A. deanei shares immunological and biochemical relationships.

Calpain in Breast Cancer: Role in Disease Progression and Treatment Response.

The calpains are a family of intracellular cysteine proteases that function in a wide array of cellular activities, including cytoskeletal remodelling, survival and apoptosis. The ubiquitously expressed micro (µ)-calpain and milli (m)-calpain are archetypal family members that require calcium for function and can be inhibited by their endogenous inhibitor calpastatin. This review describes the role of the calpain system in the prognosis of breast cancer and disease progression, in addition to the role of the calpain system in the response to breast cancer treatments, including chemotherapeutic, endocrine and targeted therapies.

Restriction of calpain3 expression to the skeletal muscle prevents cardiac toxicity and corrects pathology in a murine model of limb-girdle muscular dystrophy.

BACKGROUND: Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus-mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. METHODS AND RESULTS: Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle-restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. CONCLUSIONS: The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.

Capn4 is a marker of poor clinical outcomes and promotes nasopharyngeal carcinoma metastasis via nuclear factor-κB-induced matrix metalloproteinase 2 expression.

Calpain small subunit 1 (Capn4) plays a key role in tumor migration or invasion. In this study, expression and function of Capn4 was investigated in human nasopharyngeal carcinoma (NPC). Here we report that both mRNA and protein levels of Capn4 were elevated in NPC tissues when compared to normal NP tissues. Similarly, Capn4 was also highly expressed in multiple NPC cell lines, compared to immortalized human nasopharyngeal epithelial cell line NP69. Moreover, expression of Capn4 was significantly correlated with Epstein-Barr virus infection, advanced stages, and lymph node or distant metastasis (P < 0.001). The patients with NPC displaying higher Capn4 had a significantly shorter overall survival (P = 0.002) and progression-free survival (P = 0.003). Furthermore, siRNA knockdown of Capn4 suppressed cell migration and invasion in vitro and in vivo. These events resulted from Capn4 downregulation were associated with reduced expression of matrix metalloproteinase 2 (MMP2), Snail, and Vimentin. Finally, we demonstrated that Capn4 upregulated MMP2 via nuclear factor-κB (NF-κB) activation, manifested by increased phosphorylation of p65, a subunit of NF-κB. Together, these findings argue a novel function of Capn4 in invasion and metastasis of NPC, and thereby suggest that Capn4 may represent an independent prognostic factor and a potential therapeutic target in NPC.

cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response.

Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus-secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus-containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.