Publications by authors named "Peter A Greer"

75 Publications

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

Mol Ther 2021 06 10;29(6):1984-2000. Epub 2021 Feb 10.

Center for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, Canada. Electronic address:

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.
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http://dx.doi.org/10.1016/j.ymthe.2021.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872859PMC
June 2021

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways.

J Biol Chem 2020 12 28;295(49):16840-16851. Epub 2020 Sep 28.

Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China; Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; Lawson Health Research Institute of London Health Sciences Centre, London, Ontario, Canada; Department of Medicine, Western University, London, Ontario, Canada. Electronic address:

The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of , which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Notably, the protective effects of deletion were associated with the prevention of phosphorylation of Ser-345 on p47 and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47 , and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47 in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47 phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.
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http://dx.doi.org/10.1074/jbc.RA119.011890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864076PMC
December 2020

A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation.

Mol Cell Proteomics 2020 12 10;19(12):1968-1986. Epub 2020 Sep 10.

Departments of Cellular & Molecular Pharmacology, University of California San Francisco, San Francisco, California, USA; Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA; Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA; The Liver Center, University of California San Francisco, San Francisco, California, USA. Electronic address:

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.
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http://dx.doi.org/10.1074/mcp.RA120.002316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710137PMC
December 2020

PTEN dephosphorylates Abi1 to promote epithelial morphogenesis.

J Cell Biol 2020 09;219(9)

Department of Surgery, Rutgers University Robert Wood Johnson Medical School, New Brunswick, NJ.

The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study, we demonstrate that PTEN's protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN's lipid phosphatase activity or both PTEN's lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3-regulated actin cytoskeletal dynamics in epithelial morphogenesis.
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http://dx.doi.org/10.1083/jcb.201910041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480098PMC
September 2020

Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation.

Front Immunol 2019 22;10:2481. Epub 2019 Oct 22.

Department of Medicine 1, Friedrich-Alexander-Universität Erlangen-Nürnberg, Deutsches Zentrum Immuntherapie, Kussmaul Campus for Medical Research and Translational Research Center, Erlangen, Germany.

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.
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http://dx.doi.org/10.3389/fimmu.2019.02481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817590PMC
September 2020

Reactive Oxygen Species-Dependent Calpain Activation Contributes to Airway and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease.

Antioxid Redox Signal 2019 10 24;31(12):804-818. Epub 2019 Jun 24.

Department of Pharmacology & Toxicology, Medical College of Georgia, Augusta University, Augusta, Georgia.

Airway and pulmonary vascular remodeling is an important pathological feature in the pathogenesis of chronic obstructive pulmonary disease (COPD). Tobacco smoke (TS) induces the production of large amounts of reactive oxygen species (ROS) in COPD lungs. We investigated how ROS lead to airway and pulmonary vascular remodeling in COPD. We used bronchial and pulmonary artery smooth muscle cells (BSMCs and PASMCs), TS-induced COPD rodent models, and lung tissues of COPD patients. We found that HO and TS extract (TSE) induced calpain activation in BSMCs and PASMCs. Calpain activation was elevated in smooth muscle of bronchi and pulmonary arterioles in COPD patients and TS-induced COPD rodent models. Calpain inhibition attenuated HO- and TSE-induced collagen synthesis and proliferation of BSMCs and PASMCs. Exposure to TS causes increases in airway resistance, right ventricular systolic pressure (RVSP), and thickening of bronchi and pulmonary arteries. Calpain inhibition by smooth muscle-specific knockout of calpain and the calpain inhibitor MDL28170 attenuated increases in airway resistance, RVSP, and thickening of bronchi and pulmonary arteries. Moreover, smooth muscle-specific knockout of calpain did not reduce TS-induced emphysema in the mouse model, but MDL28170 did reduce TS-induced emphysema in the rat model. This study provides the first evidence that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling in TS-induced COPD. Calpain might be a novel therapeutic target for the treatment of COPD. These results indicate that ROS-induced calpain activation contributes to airway and pulmonary vascular remodeling and pulmonary hypertension in COPD.
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http://dx.doi.org/10.1089/ars.2018.7648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061305PMC
October 2019

Selective deletion of endothelial cell calpain in mice reduces diabetic cardiomyopathy by improving angiogenesis.

Diabetologia 2019 05 18;62(5):860-872. Epub 2019 Feb 18.

Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China.

Aims/hypothesis: The role of non-cardiomyocytes in diabetic cardiomyopathy has not been fully addressed. This study investigated whether endothelial cell calpain plays a role in myocardial endothelial injury and microvascular rarefaction in diabetes, thereby contributing to diabetic cardiomyopathy.

Methods: Endothelial cell-specific Capns1-knockout (KO) mice were generated. Conditions mimicking prediabetes and type 1 and type 2 diabetes were induced in these KO mice and their wild-type littermates. Myocardial function and coronary flow reserve were assessed by echocardiography. Histological analyses were performed to determine capillary density, cardiomyocyte size and fibrosis in the heart. Isolated aortas were assayed for neovascularisation. Cultured cardiac microvascular endothelial cells were stimulated with high palmitate. Angiogenesis and apoptosis were analysed.

Results: Endothelial cell-specific deletion of Capns1 disrupted calpain 1 and calpain 2 in endothelial cells, reduced cardiac fibrosis and hypertrophy, and alleviated myocardial dysfunction in mouse models of diabetes without significantly affecting systemic metabolic variables. These protective effects of calpain disruption in endothelial cells were associated with an increase in myocardial capillary density (wild-type vs Capns1-KO 3646.14 ± 423.51 vs 4708.7 ± 417.93 capillary number/high-power field in prediabetes, 2999.36 ± 854.77 vs 4579.22 ± 672.56 capillary number/high-power field in type 2 diabetes and 2364.87 ± 249.57 vs 3014.63 ± 215.46 capillary number/high-power field in type 1 diabetes) and coronary flow reserve. Ex vivo analysis of neovascularisation revealed more endothelial cell sprouts from aortic rings of prediabetic and diabetic Capns1-KO mice compared with their wild-type littermates. In cultured cardiac microvascular endothelial cells, inhibition of calpain improved angiogenesis and prevented apoptosis under metabolic stress. Mechanistically, deletion of Capns1 elevated the protein levels of β-catenin in endothelial cells of Capns1-KO mice and constitutive activity of calpain 2 suppressed β-catenin protein expression in cultured endothelial cells. Upregulation of β-catenin promoted angiogenesis and inhibited apoptosis whereas knockdown of β-catenin offset the protective effects of calpain inhibition in endothelial cells under metabolic stress.

Conclusions/interpretation: These results delineate a primary role of calpain in inducing cardiac endothelial cell injury and impairing neovascularisation via suppression of β-catenin, thereby promoting diabetic cardiomyopathy, and indicate that calpain is a promising therapeutic target to prevent diabetic cardiac complications.
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http://dx.doi.org/10.1007/s00125-019-4828-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702672PMC
May 2019

Intravital imaging reveals systemic ezrin inhibition impedes cancer cell migration and lymph node metastasis in breast cancer.

Breast Cancer Res 2019 01 24;21(1):12. Epub 2019 Jan 24.

Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada.

Background: Limited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients. The regional lymph nodes are the most common site of metastasis in most solid cancers and their involvement is a strong predictor of relapse in breast cancer (BC). We have previously shown that ezrin, a cytoskeletal-membrane linker protein, is associated with lymphovascular invasion and promotes metastatic progression in BC. However, the efficacy of pharmacological inhibition of ezrin in blocking cancer cell migration and metastasis remains unexplored in BC.

Methods: We quantified ezrin expression in a BC tissue microarray (n = 347) to assess its correlation with risk of relapse. Next, we developed a quantitative intravital microscopy (qIVM) approach, using a syngeneic lymphatic reporter mouse tumor model, to investigate the effect of systemic ezrin inhibition on cancer cell migration and metastasis.

Results: We show that ezrin is expressed at significantly higher levels in lymph node metastases compared to matched primary tumors, and that a high tumor ezrin level is associated with increased risk of relapse in BC patients with regional disease. Using qIVM, we observe a subset of cancer cells that retain their invasive and migratory phenotype at the tumor-draining lymph node. We further show that systemic inhibition of ezrin, using a small molecule compound (NSC668394), impedes the migration of cancer cells in vivo. Furthermore, systemic ezrin inhibition leads to reductions in metastatic burden at the distal axillary lymph node and lungs.

Conclusions: Our findings demonstrate that the tumor ezrin level act as an independent biomarker in predicting relapse and provide a rationale for therapeutic targeting of ezrin to reduce the metastatic capacity of cancer cells in high-risk BC patients with elevated ezrin expression.
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http://dx.doi.org/10.1186/s13058-018-1079-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345049PMC
January 2019

Genetic Models of Calpain Deficiency and Ectopic Expression.

Methods Mol Biol 2019 ;1915:261-274

Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada.

Genetic manipulation in cell and animal models can provide important insights into gene function and the relationships between gene mutation and disease. This chapter describes methods to generate models of calpain-1 and calpain-2 deficiency, or their recombinant ectopic expression in cultured cells, and to genotype a conditional transgenic mouse model of calpain-1/calpain-2 deficiency that can be used to explore physiologic roles for these calpains.
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http://dx.doi.org/10.1007/978-1-4939-8988-1_20DOI Listing
June 2019

Genetic disruption of calpain-1 and calpain-2 attenuates tumorigenesis in mouse models of HER2+ breast cancer and sensitizes cancer cells to doxorubicin and lapatinib.

Oncotarget 2018 Sep 7;9(70):33382-33395. Epub 2018 Sep 7.

Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada.

Calpains are a family of calcium activated cysteine proteases which participate in a wide range of cellular functions including migration, invasion, autophagy, programmed cell death, and gene expression. Calpain-1 and calpain-2 isoforms are ubiquitously expressed heterodimers composed of isoform specific catalytic subunits coupled with an obligate common regulatory subunit encoded by . Here, we report that conditional deletion of capns1 disrupted calpain-1 and calpain-2 expression and activity, and this was associated with delayed tumorigenesis and altered signaling in a transgenic mouse model of spontaneous HER2 breast cancer and effectively blocked tumorigenesis in an orthotopic engraftment model. Furthermore, knockout in a tumor derived cell line correlated with enhanced sensitivity to the chemotherapeutic doxorubicin and the HER2/EGFR tyrosine kinase inhibitor lapatinib. Collectively, these results indicate pro-tumorigenic roles for calpains-1/2 in HER2 breast cancer and provide evidence that calpain-1/2 inhibitors could have anti-tumor effects if used either alone or in combination with chemotherapeutics and targeted agents.
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http://dx.doi.org/10.18632/oncotarget.26078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161787PMC
September 2018

Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems.

Oncotarget 2017 Oct 24;8(46):80804-80819. Epub 2017 Aug 24.

Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, ON, Canada.

Aberrant Ras-MAPK signaling from receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2), is a hallmark of triple negative breast cancer (TNBC); thus providing rationale for targeting the Ras-MAPK pathway. Components of this EGFR/HER2-Ras-Raf-Mek-Erk pathway were co-targeted in the MDA-MB-231 and MDA-MB-468 human TNBC cell lines, and effects on signaling and cytotoxicity, as well as effects on xenograft tumor growth and metastasis were assessed. The dual EGFR/HER2 inhibitor lapatinib (LPN) displayed greater cytotoxic potency and MAPK signaling inhibition than the EGFR inhibitor erlotinib, suggesting both EGFR and HER2 contribute to MAPK signaling in this TNBC model. The Raf inhibitor sorafenib (SFN) or the Mek inhibitor U0126 suppressed MAPK signaling to a greater extent than LPN; which correlated with greater cytotoxic potency of SFN, but not U0126. However, U0126 potentiated the cytotoxic efficacy of LPN and SFN in an additive and synergistic manner, respectively. This in-series Raf-Mek co-targeting synergy was recapitulated in orthotopic mouse xenografts, where SFN and the Mek inhibitor selumitinib (AZD6244) inhibited primary tumor growth and pulmonary metastasis. Raf and Mek co-inhibition exhibits synergy in TNBC models and represent a promising combination therapy for this aggressive breast cancer type.
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http://dx.doi.org/10.18632/oncotarget.20534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655240PMC
October 2017

Inhibition of angiotensin II and calpain attenuates pleural fibrosis.

Pulm Pharmacol Ther 2018 02 4;48:46-52. Epub 2017 Nov 4.

Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; Key Laboratory of Respiratory Diseases, Ministry of Health of China, Wuhan 430030, China. Electronic address:

Pleural fibrosis is associated with various inflammatory processes such as tuberculous pleurisy and bacterial empyema. There is currently no ideal therapeutic to attenuate pleural fibrosis. Some pro-fibrogenic mediators induce fibrosis through inflammatory processes, suggesting that blockage of these mediators might prevent pleural fibrosis. The MeT-5A human pleural mesothelial cell line (PMC) was used in this study as an in vitro model of fibrosis; and intra-pleural injection of bleomycin with carbon particles was used as an in vivo mouse model of pleural fibrosis. Calpain knockout mice, calpain inhibitor (calpeptin), and angiotensin (Ang) II type 1 receptor (ATR) antagonist (losartan) were evaluated in prevention of experimental pleural fibrosis. We found that bleomycin and carbon particles induced calpain activation in cultured PMCs. This in vitro response was associated with increased collagen-I synthesis, and was blocked by calpain inhibitor or ATR antagonist. Calpain genetic or treatment with calpeptin or losartan prevented pleural fibrosis in a mouse model induced by bleomycin and carbon particles. Our findings indicate that Ang II signaling and calpain activation induce collagen-I synthesis and contribute to fibrotic alterations in pleural fibrosis. Inhibition of Ang II and calpain might therefore be a novel strategy in treatment of pleural fibrosis.
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http://dx.doi.org/10.1016/j.pupt.2017.10.012DOI Listing
February 2018

An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo.

Biochim Biophys Acta Mol Cell Res 2018 Feb 14;1865(2):221-230. Epub 2017 Nov 14.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address:

Calpain-1 and -2 are Ca-activated intracellular cysteine proteases that regulate a wide range of cellular functions through the cleavage of their protein substrates. Unlike degradative proteases, calpains make limited, transformative cleavages, typically in accessible sequences linking discrete subdomains, to irreversibly alter substrate functions. The biological roles of calpain and their interplay with calcium signaling are of significant biomedical interest as biomarkers and potential therapeutic targets in a growing number of diseases including Alzheimer's, cancer and fibrosis. Unfortunately, many of the colorimetric and fluorimetric assays that have been developed to study calpain activity suffer from low sensitivity and/or poor calpain specificity. To address the need for a highly sensitive and calpain-specific substrate suitable for in vitro and in vivo calpain activity analysis, we have developed a protein FRET probe. We inserted the optimized calpain cleavage sequence PLFAAR between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) and modulated its flanking sequences for optimal calpain cleavage. We demonstrate greater sensitivity and calpain-specificity of an optimal 16-residue PLFAAR-based FRET substrate compared to a standard α-spectrin-based probe. The 16-residue PLFAAR protein FRET substrate is not significantly cleaved by trypsin, chymotrypsin, cathepsin-L or caspase-3, and is highly sensitive to both calpain-1 and -2. After transfection of the substrate gene into breast cancer cells the PLFAAR protein FRET product was cut in lysed wild-type cells but not in those with a calpain knock-out phenotype. Blockage of substrate cleavage in the lysates by endogenous and exogenous calpastatin was observed, and was overcome by adding extra calpain.
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http://dx.doi.org/10.1016/j.bbamcr.2017.10.013DOI Listing
February 2018

Pathological significance and prognostic significance of FES expression in bladder cancer vary according to tumor grade.

J Cancer Res Clin Oncol 2018 Jan 26;144(1):21-31. Epub 2017 Sep 26.

Department of Urology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.

Purpose: The feline sarcoma oncogene protein (FES) is a non-receptor tyrosine kinase implicated in both oncogenesis and tumor suppression. Here, cancer cell lines and human tissues were employed to clarify the pathological and prognostic significance of FES in bladder cancer.

Methods: The relationship between FES expression and cancer aggressiveness was investigated using 3 cell lines (T24: corresponding to grade 3, 5637: corresponding to grade 2, and RT4: corresponding to grade 1) and 203 tissues derived from human bladder malignancies. Proliferation, invasion, and migration of cancer cells were assessed following the knockdown (KD) of FES expression by the siRNA method. Relationships between FES expression and pathological features, aggressiveness, and outcome were investigated.

Results: FES-KD inhibited the proliferation, migration, and invasion of T24 cells but not of RT4 cells and 5637 cells. Considering all patients, FES expression demonstrated a negative relationship with grade but no association with muscle invasion or cancer cell proliferation. However, it was positively correlated with pT stage and cell proliferation in high-grade tumors (p = 0.002); no such association was found for low-grade tumors. In addition, elevated FES expression was a negative prognostic indicator of metastasis after radical surgery for patients with high-grade tumors (p = 0.021) but not for those with low-grade malignancies.

Conclusions: FES appeared to act as a suppressor of carcinogenesis, being associated with low tumor grade in the overall patient group. However, its expression correlated with cancer aggressiveness and poor outcome in high-grade bladder cancer. FES, therefore, represents a potential therapeutic target and useful prognostic factor for such patients.
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http://dx.doi.org/10.1007/s00432-017-2524-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5756570PMC
January 2018

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.

Cell Signal 2017 05 10;33:30-40. Epub 2017 Feb 10.

Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia; Discipline of Child and Adolescent Health, Faculty of Medicine, University of Sydney, Sydney, Australia. Electronic address:

Myoferlin and dysferlin are closely related members of the ferlin family of Ca-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2 domain; and a second cleavage site in the linker adjacent to C2, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.
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http://dx.doi.org/10.1016/j.cellsig.2017.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995151PMC
May 2017

Disruption of calpain reduces lipotoxicity-induced cardiac injury by preventing endoplasmic reticulum stress.

Biochim Biophys Acta 2016 Nov 12;1862(11):2023-2033. Epub 2016 Aug 12.

Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province 215123, China; Critical Illness Research, Lawson Health Research Institute, Western University, London, Ontario N6A 4G5, Canada; Department of Medicine, Western University, London, Ontario N6A 4G5, Canada; Department of Pathology and Laboratory Medicine, Western University, London, Ontario N6A 4G5, Canada. Electronic address:

Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4weeks) were fed a high fat diet (HFD) or normal diet for 20weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mice. Mechanistically, deletion of capns1 in HFD-fed mouse hearts and disruption of calpain with calpain inhibitor-III, silencing of capn1, or deletion of capns1 in palmitate-stimulated cardiomyocytes prevented endoplasmic reticulum stress, apoptosis, cleavage of caspase-12 and junctophilin-2, and pro-inflammatory cytokine expression. Pharmacological inhibition of endoplasmic reticulum stress diminished palmitate-induced apoptosis and pro-inflammatory cytokine expression in cardiomyocytes. In summary, disruption of calpain prevents lipotoxicity-induced apoptosis in cardiomyocytes and cardiac injury in mice fed a HFD. The role of calpain is mediated, at least partially, through endoplasmic reticulum stress. Thus, calpain/endoplasmic reticulum stress may represent a new mechanism and potential therapeutic targets for cardiac lipotoxicity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066874PMC
http://dx.doi.org/10.1016/j.bbadis.2016.08.005DOI Listing
November 2016

HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.

Genes Dev 2016 07;30(13):1542-57

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;

Ovarian cancer cells disseminate readily within the peritoneal cavity, which promotes metastasis, and are often resistant to chemotherapy. Ovarian cancer patients tend to present with advanced disease, which also limits treatment options; consequently, new therapies are required. The oncoprotein tyrosine kinase MET, which is the receptor for hepatocyte growth factor (HGF), has been implicated in ovarian tumorigenesis and has been the subject of extensive drug development efforts. Here, we report a novel ligand- and autophosphorylation-independent activation of MET through the nonreceptor tyrosine kinase feline sarcoma-related (FER). We demonstrated that the levels of FER were elevated in ovarian cancer cell lines relative to those in immortalized normal surface epithelial cells and that suppression of FER attenuated the motility and invasive properties of these cancer cells. Furthermore, loss of FER impaired the metastasis of ovarian cancer cells in vivo. Mechanistically, we demonstrated that FER phosphorylated a signaling site in MET: Tyr1349. This enhanced activation of RAC1/PAK1 and promoted a kinase-independent scaffolding function that led to recruitment and phosphorylation of GAB1 and the specific activation of the SHP2-ERK signaling pathway. Overall, this analysis provides new insights into signaling events that underlie metastasis in ovarian cancer cells, consistent with a prometastatic role of FER and highlighting its potential as a novel therapeutic target for metastatic ovarian cancer.
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http://dx.doi.org/10.1101/gad.284166.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949327PMC
July 2016

Activation of calpain by renin-angiotensin system in pleural mesothelial cells mediates tuberculous pleural fibrosis.

Am J Physiol Lung Cell Mol Physiol 2016 07 3;311(1):L145-53. Epub 2016 Jun 3.

Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Key Laboratory of Respiratory Diseases, Ministry of Health of China, Wuhan, Hubei, China;

Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) components that results in destruction of the normal pleural tissue architecture. It can result from diverse inflammatory conditions, especially tuberculous pleurisy. Pleural mesothelial cells (PMCs) play a pivotal role in pleural fibrosis. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in pleural fibrosis remains unknown. In the present study, we found that tuberculous pleural effusion (TPE) induced calpain activation in PMCs and that inhibition of calpain prevented TPE-induced collagen-I synthesis and cell proliferation of PMCs. Moreover, our data revealed that the levels of angiotensin (ANG)-converting enzyme (ACE) were significantly higher in pleural fluid of patients with TPE than those with malignant pleural effusion, and ACE-ANG II in TPE resulted in activation of calpain and subsequent triggering of the phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB signaling pathway in PMCs. Finally, calpain activation in PMCs and collagen depositions were confirmed in pleural biopsy specimens from patients with tuberculous pleurisy. Together, these studies demonstrated that calpain is activated by renin-angiotensin system in pleural fibrosis and mediates TPE-induced collagen-I synthesis and proliferation of PMCs via the PI3K/Akt/NF-κB signaling pathway. Calpain in PMCs might be a novel target for intervention in tuberculous pleural fibrosis.
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http://dx.doi.org/10.1152/ajplung.00348.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967195PMC
July 2016

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm.

Development 2016 07 25;143(13):2325-33. Epub 2016 May 25.

Department of Veterinary and Animal Science, Integrated Sciences Building, University of Massachusetts, Amherst, MA 01003, USA

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro.
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http://dx.doi.org/10.1242/dev.136499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958327PMC
July 2016

Calpain Genetic Disruption and HSP90 Inhibition Combine To Attenuate Mammary Tumorigenesis.

Mol Cell Biol 2016 08 14;36(15):2078-88. Epub 2016 Jul 14.

Division of Cancer Biology and Genetics, Queen's University, Kingston, ON, Canada Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada

Calpain is an intracellular Ca(2+)-regulated protease system whose substrates include proteins involved in proliferation, survival, migration, invasion, and sensitivity to therapeutic drugs. Genetic disruption of calpain attenuated the tumorigenic potential of breast cancer cells and hypersensitized cells to 17AAG, an inhibitor of the molecular chaperone HSP90. Calpain-1 or -2 overexpression rendered cells resistant to 17AAG, whereas downregulation or inhibition of calpain-1/2 led to increased cell death in multiple breast cancer cell lines, including models of HER2(+) (SKBR3) and triple-negative basal-cell-like (MDA-MB-231) breast cancer. In an MDA-MB-231 orthotopic xenograft model, calpain knockdown or 17AAG treatment independently attenuated tumor growth and metastasis, while the combination was most effective. Calpain knockdown was associated with increased 17AAG-induced degradation of the HSP90 clients cyclin D1 and AKT and multidrug resistance protein 2, which correlated with increased expression of antimitogenic p27(KIP1) and proapoptotic BIM proteins. Like other therapeutics, 17AAG can be effluxed by specific ABC transporters. Calpain expression positively correlated with the expression of P glycoprotein in mouse embryonic fibroblasts. Importantly, we show that calpain affects ABC transporter function and efflux of clinically relevant doxorubicin. These observations provide a compelling rationale for exploring the combination of calpain inhibition with new or existing cancer therapeutics.
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http://dx.doi.org/10.1128/MCB.01062-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946432PMC
August 2016

Mitochondrial Calpain-1 Disrupts ATP Synthase and Induces Superoxide Generation in Type 1 Diabetic Hearts: A Novel Mechanism Contributing to Diabetic Cardiomyopathy.

Diabetes 2016 Jan 15;65(1):255-68. Epub 2015 Oct 15.

Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province, China Department of Medicine, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada Department of Pathology, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada

Calpain plays a critical role in cardiomyopathic changes in type 1 diabetes (T1D). This study investigated how calpain regulates mitochondrial reactive oxygen species (ROS) generation in the development of diabetic cardiomyopathy. T1D was induced in transgenic mice overexpressing calpastatin, in mice with cardiomyocyte-specific capn4 deletion, or in their wild-type littermates by injection of streptozotocin. Calpain-1 protein and activity in mitochondria were elevated in diabetic mouse hearts. The increased mitochondrial calpain-1 was associated with an increase in mitochondrial ROS generation and oxidative damage and a reduction in ATP synthase-α (ATP5A1) protein and ATP synthase activity. Genetic inhibition of calpain or upregulation of ATP5A1 increased ATP5A1 and ATP synthase activity, prevented mitochondrial ROS generation and oxidative damage, and reduced cardiomyopathic changes in diabetic mice. High glucose concentration induced ATP synthase disruption, mitochondrial superoxide generation, and cell death in cardiomyocytes, all of which were prevented by overexpression of mitochondria-targeted calpastatin or ATP5A1. Moreover, upregulation of calpain-1 specifically in mitochondria induced the cleavage of ATP5A1, superoxide generation, and apoptosis in cardiomyocytes. In summary, calpain-1 accumulation in mitochondria disrupts ATP synthase and induces ROS generation, which promotes diabetic cardiomyopathy. These findings suggest a novel mechanism for and may have significant implications in diabetic cardiac complications.
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http://dx.doi.org/10.2337/db15-0963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686953PMC
January 2016

Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion.

Mol Biol Cell 2015 Oct 5;26(19):3464-79. Epub 2015 Aug 5.

Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, ON K7L 3N6, Canada Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON K7L 3N6, Canada

Up-regulation of the cytoskeleton linker protein ezrin frequently occurs in aggressive cancer types and is closely linked with metastatic progression. However, the underlying molecular mechanisms detailing how ezrin is involved in the invasive and metastatic phenotype remain unclear. Here we report a novel function of ezrin in regulating focal adhesion (FA) and invadopodia dynamics, two key processes required for efficient invasion to occur. We show that depletion of ezrin expression in invasive breast cancer cells impairs both FA and invadopodia turnover. We also demonstrate that ezrin-depleted cells display reduced calpain-mediated cleavage of the FA and invadopodia-associated proteins talin, focal adhesion kinase (FAK), and cortactin and reduced calpain-1-specific membrane localization, suggesting a requirement for ezrin in maintaining proper localization and activity of calpain-1. Furthermore, we show that ezrin is required for cell directionality, early lung seeding, and distant organ colonization but not primary tumor growth. Collectively our results unveil a novel mechanism by which ezrin regulates breast cancer cell invasion and metastasis.
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http://dx.doi.org/10.1091/mbc.E14-12-1584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591691PMC
October 2015

Deletion of capn4 Protects the Heart Against Endotoxemic Injury by Preventing ATP Synthase Disruption and Inhibiting Mitochondrial Superoxide Generation.

Circ Heart Fail 2015 Sep 5;8(5):988-96. Epub 2015 Aug 5.

From the Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu, China (R.N., D.Z., T.P.); Lawson Health Research Institute, Departments of Medicine (R.N., D.Z., T.S., T.P.), Pathology (R.N., D.Z., T.P.) and Biology (R.B.G.), University of Western Ontario, London, Ontario, Canada; Department of Dermatology (Q.W.) and Institute of Cardiovascular Diseases (Y.Y., R.C.), Zhongshan Hospital, Fudan University, Shanghai, China; Mitochondria and Metabolism Center, Departments of Anesthesiology and Pain Medicine, University of Washington, Seattle (W.W.); Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, OH (G.-C.F.); and Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada (P.A.G.).

Background: Our recent study has demonstrated that inhibition of calpain by transgenic overexpression of calpastatin reduces myocardial proinflammatory response and dysfunction in endotoxemia. However, the underlying mechanisms remain to be determined. In this study, we used cardiomyocyte-specific capn4 knockout mice to investigate whether and how calpain disrupts ATP synthase and induces mitochondrial superoxide generation during endotoxemia.

Methods And Results: Cardiomyocyte-specific capn4 knockout mice and their wild-type littermates were injected with lipopolysaccharides. Four hours later, calpain-1 protein and activity were increased in mitochondria of endotoxemic mouse hearts. Mitochondrial calpain-1 colocalized with and cleaved ATP synthase-α (ATP5A1), leading to ATP synthase disruption and a concomitant increase in mitochondrial reactive oxygen species generation during lipopolysaccharide stimulation. Deletion of capn4 or upregulation of ATP5A1 increased ATP synthase activity, prevented mitochondrial reactive oxygen species generation, and reduced proinflammatory response and myocardial dysfunction in endotoxemic mice. In cultured cardiomyocytes, lipopolysaccharide induced mitochondrial superoxide generation that was prevented by overexpression of mitochondria-targeted calpastatin or ATP5A1. Upregulation of calpain-1 specifically in mitochondria sufficiently induced superoxide generation and proinflammatory response, both of which were attenuated by ATP5A1 overexpression or mitochondria-targeted superoxide dismutase mimetics.

Conclusions: Cardiomyocyte-specific capn4 knockout protects the heart against lipopolysaccharide-induced injury in endotoxemic mice. Lipopolysaccharides induce calpain-1 accumulation in mitochondria. Mitochondrial calpain-1 disrupts ATP synthase, leading to mitochondrial reactive oxygen species generation, which promotes proinflammatory response and myocardial dysfunction during endotoxemia. These findings uncover a novel mechanism by which calpain mediates myocardial dysfunction in sepsis.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.115.002383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573911PMC
September 2015

Crosstalk between calpain activation and TGF-β1 augments collagen-I synthesis in pulmonary fibrosis.

Biochim Biophys Acta 2015 Sep 10;1852(9):1796-804. Epub 2015 Jun 10.

Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Key Laboratory of Pulmonary Diseases, Ministry of Health of China, Wuhan, Hubei, China. Electronic address:

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown cause that typically leads to respiratory failure and death within 3-5years of diagnosis. TGF-β1 is considered a major profibrotic factor. However, TGF-β1 is necessary but not sufficient to the pathogenesis of fibrotic lesion of the lungs. Recent observations have revealed that calpain, a calcium dependent protease, plays a pivotal role in tissue remodeling and fibrosis. However, the mechanism of calpain mediating pulmonary fibrosis is not understood. Calpain conditional knockout (ER-Cre(+/-)capns1(flox/flox)) mice and primary human lung fibroblasts (HLFs) were used here to investigate the relationship between calpain and TGF-β1. Calpain knockout mice were protected from fibrotic effects of bleomycin. Bleomycin induced increases in TGF-β1 via calpain activation in HLFs. Moreover, TGF-β1 also activated calpain. This crosstalk between calpain activation and TGF-β1 triggered the downstream signaling pathway including TGF-β1 Smad2/3 and non-Smad (Akt) pathways, as well as collagen-I synthesis. Taken together, our data indicate that the crosstalk between calpain activation and TGF-β1 augments collagen-I synthesis in HLFs and in pulmonary fibrosis. Intervention in the crosstalk between calpain activation and TGF-β1 is a novel potential strategy to prevent pulmonary fibrosis.
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http://dx.doi.org/10.1016/j.bbadis.2015.06.008DOI Listing
September 2015

Interleukin-4 Expressed By Neoplastic Cells Provokes an Anti-Metastatic Myeloid Immune Response.

J Clin Cell Immunol 2015 May;6(6):1-9

Department of Pathology and Molecular Medicine, Queen's University, Division of Cancer Biology and Genetics, Cancer Research Institute, Kingston, Ontario, K7L 3N6, Canada.

Objective: Interleukin-4 (IL-4) can induce macrophages to undergo alternative activation and polarize toward an M2-like or wound healing phenotype. Tumor associated macrophages (TAMs) are thought to assume M2-like properties, and it has been suggested they promote tumor growth and metastasis through effects on the tumor stroma, including extracelluar matrix remodeling and angiogenesis. IL-4 also promotes macrophage survival and formation of multinucleated giant cells, which have enhanced phagocytic behavior. This study was designed to explore the effect of cancer cell derived IL-4 on the tumor immune stroma and metastasis.

Methods: The metastatic mouse mammary carcinoma cell line AC2M2 was transduced with control or IL-4 encoding retroviruses and employed in orthotopic engraftment models. Tumor growth and metastasis were assessed. The cellular composition and biomarker expression of tumors were examined by immunohistochemical staining and flow cytometry; the transcriptome of the immune stroma was analyzed by nanoString based transcript quantitation; and and interactions between cancer cells and macrophages were assessed by flow cytometry and co-culture with video-time lapse microscopy, respectively.

Results: Unexpectedly, tumors from IL-4 expressing AC2M2 engrafted cells grew at reduced rates, and most surprising, they lost all metastatic potential relative to tumors from control AC2M2 cells. Myeloid cell numbers were not increased in IL-4 expressing tumors, but their expression of the M2 marker arginase I was elevated. Transcriptome analysis revealed an immune signature consistent with IL-4 induced M2 polarization of the tumor microenvironment and a generalized increase in myeloid involvement in the tumor stroma. Flow cytometry analysis indicated enhanced cancer cell phagocytosis by TAMs from IL-4 expressing tumors, and co-culture studies showed that IL-4 expressing cancer cells supported the survival and promoted the phagocytic behavior of macrophages.

Conclusions: Although M2-like TAMs have been linked to enhanced tumorigenesis, this study shows that IL-4 production by cancer cells is associated with suppressed tumor growth and loss of metastatic potential as well as enhanced phagocytic behavior of TAMs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995104PMC
May 2015

Pathological axonal death through a MAPK cascade that triggers a local energy deficit.

Cell 2015 Jan;160(1-2):161-76

Laboratory of Brain Development and Repair, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, we systematically investigate mitogen-activated protein kinase (MAPK) families and delineate a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wld(s) protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. Our results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration.
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http://dx.doi.org/10.1016/j.cell.2014.11.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306654PMC
January 2015

Recurrent copy number alterations in prostate cancer: an in silico meta-analysis of publicly available genomic data.

Cancer Genet 2014 Oct-Dec;207(10-12):474-88. Epub 2014 Sep 16.

Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada; Departments of Genetics and Pathology, School of Medicine of Ribeirao Preto, University of Sao Paulo at Ribeirao Preto, Sao Paulo, Brazil. Electronic address:

We present a meta-analysis of somatic copy number alterations (CNAs) from 11 publications that examined 662 prostate cancer patient samples, which were derived from 546 primary and 116 advanced tumors. Normalization, segmentation, and identification of corresponding CNAs for meta-analysis was achieved using established commercial software. Unsupervised analysis identified five genomic subgroups in which approximately 90% of the samples were characterized by abnormal profiles with gains of 8q. The most common loss was 8p (NKX3.1). The CNA distribution in other genomic subgroups was characterized by losses at 2q, 3p, 5q, 6q, 13q, 16q, 17p, 18q, and PTEN (10q), and acquisition of 21q deletions associated with the TMPRSS2-ERG fusion rearrangement. Parallel analysis of advanced and primary tumors in the cohort indicated that genomic deletions of PTEN and the gene fusion were enriched in advanced disease. A supervised analysis of the PTEN deletion and the fusion gene showed that PTEN deletion was sufficient to impose higher levels of CNA. Moreover, the overall percentage of the genome altered was significantly higher when PTEN was deleted, suggesting that this important genomic subgroup was likely characterized by intrinsic chromosomal instability. Predicted alterations in expression levels of candidate genes in each of the recurrent CNA regions characteristic of each subgroup showed that signaling networks associated with cancer progression and genome stability were likely to be perturbed at the highest level in the PTEN deleted genomic subgroup.
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http://dx.doi.org/10.1016/j.cancergen.2014.09.003DOI Listing
February 2015

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome.

Proc Natl Acad Sci U S A 2014 Dec 24;111(49):E5292-301. Epub 2014 Nov 24.

Department of Medicine, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, St. Louis, MO 63110; Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and β cells has not been elucidated. Here we implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. Our results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.
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http://dx.doi.org/10.1073/pnas.1421055111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267371PMC
December 2014

Calpains promote neutrophil recruitment and bacterial clearance in an acute bacterial peritonitis model.

Eur J Immunol 2014 Mar 27;44(3):831-41. Epub 2013 Dec 27.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.

Activation of the innate immune system is critical for clearance of bacterial pathogens to limit systemic infections and host tissue damage. Here, we report a key role for calpain proteases in bacterial clearance in mice with acute peritonitis. Using transgenic mice expressing Cre recombinase primarily in innate immune cells (fes-Cre), we generated conditional capns1 knockout mice. Consistent with capns1 being essential for stability and function of the ubiquitous calpains (calpain-1, calpain-2), peritoneal cells from these mice had reduced levels of calpain-2/capns1, and reduced proteolysis of their substrate selenoprotein K. Using an acute bacterial peritonitis model, we observed impaired bacterial killing within the peritoneum and development of bacteremia in calpain knockout mice. These defects correlated with significant reductions in IL-1α release, neutrophil recruitment, and generation of reactive oxygen species in calpain knockout mice with acute bacterial peritonitis. Peritoneal macrophages from calpain knockout mice infected with enterobacteria ex vivo, were competent in phagocytosis of bacteria, but showed impaired clearance of intracellular bacteria compared with control macrophages. Together, these results implicate calpains as key mediators of effective innate immune responses to acute bacterial infections, to prevent systemic dissemination of bacteria that can lead to sepsis.
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http://dx.doi.org/10.1002/eji.201343757DOI Listing
March 2014

Calpain 2 activation of P-TEFb drives megakaryocyte morphogenesis and is disrupted by leukemogenic GATA1 mutation.

Dev Cell 2013 Dec;27(6):607-20

Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. Electronic address:

Megakaryocyte morphogenesis employs a "hypertrophy-like" developmental program that is dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback-regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large, inactive 7SK small nuclear ribonucleoprotein particle (snRNP) complexes. Here, we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis that is mediated by dissolution of the 7SK snRNP complex. In this pathway, calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here, we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.
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http://dx.doi.org/10.1016/j.devcel.2013.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3892434PMC
December 2013